Citation
Axonal interactions between fetal spinal cord transplants and the adult rat spinal cord

Material Information

Title:
Axonal interactions between fetal spinal cord transplants and the adult rat spinal cord
Creator:
Jakeman, Lyn Burrell, 1961-
Publication Date:
Language:
English
Physical Description:
viii, 218 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Axons ( jstor )
Brain ( jstor )
Cells ( jstor )
Lesions ( jstor )
Neurons ( jstor )
Rats ( jstor )
Spinal cord ( jstor )
Tissue grafting ( jstor )
Tissue transplantation ( jstor )
Transplantation ( jstor )
Axons -- growth & development ( mesh )
Department of Neuroscience thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Neuroscience -- UF ( mesh )
Fetal Tissue Transplantation ( mesh )
Neuronal Plasticity ( mesh )
Rats ( mesh )
Research ( mesh )
Spinal Cord -- transplantation ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1990.
Bibliography:
Bibliography: leaves 194-217.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Lyn Burrell Jakeman.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
030487628 ( ALEPH )
22503288 ( OCLC )
AGZ6058 ( NOTIS )

Downloads

This item has the following downloads:

axonalinteractio00jake.pdf

axonalinteractio00jake_0023.txt

axonalinteractio00jake_0102.txt

axonalinteractio00jake_0114.txt

axonalinteractio00jake_0205.txt

axonalinteractio00jake_0061.txt

axonalinteractio00jake_0057.txt

axonalinteractio00jake_0181.txt

axonalinteractio00jake_0147.txt

axonalinteractio00jake_0195.txt

axonalinteractio00jake_0206.txt

axonalinteractio00jake_0017.txt

axonalinteractio00jake_0160.txt

axonalinteractio00jake_0178.txt

axonalinteractio00jake_0042.txt

axonalinteractio00jake_0179.txt

axonalinteractio00jake_0170.txt

axonalinteractio00jake_0140.txt

axonalinteractio00jake_0044.txt

axonalinteractio00jake_0221.txt

axonalinteractio00jake_0051.txt

axonalinteractio00jake_0041.txt

axonalinteractio00jake_0043.txt

axonalinteractio00jake_0209.txt

axonalinteractio00jake_0118.txt

axonalinteractio00jake_0148.txt

axonalinteractio00jake_0081.txt

axonalinteractio00jake_0016.txt

axonalinteractio00jake_0193.txt

axonalinteractio00jake_0133.txt

axonalinteractio00jake_0002.txt

axonalinteractio00jake_0092.txt

axonalinteractio00jake_0135.txt

axonalinteractio00jake_0121.txt

axonalinteractio00jake_0087.txt

axonalinteractio00jake_0132.txt

axonalinteractio00jake_0228.txt

axonalinteractio00jake_0188.txt

axonalinteractio00jake_0074.txt

axonalinteractio00jake_0009.txt

axonalinteractio00jake_0165.txt

axonalinteractio00jake_0227.txt

AA00009071_00001.pdf

axonalinteractio00jake_0036.txt

axonalinteractio00jake_0187.txt

axonalinteractio00jake_0168.txt

axonalinteractio00jake_0067.txt

axonalinteractio00jake_0049.txt

axonalinteractio00jake_0101.txt

axonalinteractio00jake_0164.txt

axonalinteractio00jake_0014.txt

axonalinteractio00jake_0029.txt

axonalinteractio00jake_0229.txt

axonalinteractio00jake_0138.txt

axonalinteractio00jake_0190.txt

axonalinteractio00jake_0075.txt

axonalinteractio00jake_0111.txt

axonalinteractio00jake_0171.txt

axonalinteractio00jake_0212.txt

axonalinteractio00jake_0003.txt

axonalinteractio00jake_0077.txt

axonalinteractio00jake_0013.txt

axonalinteractio00jake_0019.txt

axonalinteractio00jake_0096.txt

axonalinteractio00jake_0080.txt

axonalinteractio00jake_0034.txt

axonalinteractio00jake_0220.txt

axonalinteractio00jake_0001.txt

axonalinteractio00jake_0149.txt

axonalinteractio00jake_0021.txt

axonalinteractio00jake_0046.txt

AA00009071_00001_pdf.txt

axonalinteractio00jake_0137.txt

axonalinteractio00jake_0172.txt

axonalinteractio00jake_0004.txt

axonalinteractio00jake_0185.txt

axonalinteractio00jake_0027.txt

axonalinteractio00jake_0192.txt

axonalinteractio00jake_0119.txt

axonalinteractio00jake_0146.txt

axonalinteractio00jake_0153.txt

axonalinteractio00jake_0126.txt

axonalinteractio00jake_0177.txt

axonalinteractio00jake_0022.txt

axonalinteractio00jake_0071.txt

axonalinteractio00jake_0127.txt

axonalinteractio00jake_0155.txt

axonalinteractio00jake_0203.txt

axonalinteractio00jake_0037.txt

axonalinteractio00jake_0163.txt

axonalinteractio00jake_0200.txt

axonalinteractio00jake_0156.txt

axonalinteractio00jake_0026.txt

axonalinteractio00jake_0095.txt

axonalinteractio00jake_0005.txt

axonalinteractio00jake_0054.txt

axonalinteractio00jake_0197.txt

axonalinteractio00jake_0085.txt

axonalinteractio00jake_0117.txt

axonalinteractio00jake_0030.txt

axonalinteractio00jake_0218.txt

axonalinteractio00jake_0216.txt

axonalinteractio00jake_0215.txt

axonalinteractio00jake_0120.txt

axonalinteractio00jake_0078.txt

axonalinteractio00jake_0047.txt

axonalinteractio00jake_0134.txt

axonalinteractio00jake_0176.txt

axonalinteractio00jake_0213.txt

axonalinteractio00jake_0089.txt

axonalinteractio00jake_0116.txt

axonalinteractio00jake_0015.txt

axonalinteractio00jake_0210.txt

axonalinteractio00jake_0204.txt

axonalinteractio00jake_0050.txt

axonalinteractio00jake_0161.txt

axonalinteractio00jake_0143.txt

axonalinteractio00jake_0008.txt

axonalinteractio00jake_0145.txt

axonalinteractio00jake_0010.txt

axonalinteractio00jake_0136.txt

axonalinteractio00jake_0108.txt

axonalinteractio00jake_0186.txt

axonalinteractio00jake_0174.txt

axonalinteractio00jake_0159.txt

axonalinteractio00jake_0202.txt

axonalinteractio00jake_0069.txt

axonalinteractio00jake_0107.txt

axonalinteractio00jake_0151.txt

axonalinteractio00jake_0024.txt

axonalinteractio00jake_0189.txt

axonalinteractio00jake_0157.txt

axonalinteractio00jake_0099.txt

axonalinteractio00jake_0073.txt

axonalinteractio00jake_0045.txt

axonalinteractio00jake_0090.txt

axonalinteractio00jake_0129.txt

axonalinteractio00jake_0068.txt

axonalinteractio00jake_0125.txt

axonalinteractio00jake_0035.txt

axonalinteractio00jake_0122.txt

axonalinteractio00jake_0184.txt

axonalinteractio00jake_0115.txt

axonalinteractio00jake_0223.txt

axonalinteractio00jake_0167.txt

axonalinteractio00jake_0141.txt

axonalinteractio00jake_0062.txt

axonalinteractio00jake_0070.txt

axonalinteractio00jake_0028.txt

axonalinteractio00jake_0144.txt

axonalinteractio00jake_0088.txt

axonalinteractio00jake_0060.txt

axonalinteractio00jake_0052.txt

axonalinteractio00jake_0208.txt

axonalinteractio00jake_0040.txt

axonalinteractio00jake_0198.txt

axonalinteractio00jake_0173.txt

axonalinteractio00jake_0175.txt

axonalinteractio00jake_0191.txt

axonalinteractio00jake_0100.txt

axonalinteractio00jake_0076.txt

axonalinteractio00jake_0217.txt

axonalinteractio00jake_0150.txt

axonalinteractio00jake_0130.txt

axonalinteractio00jake_0094.txt

axonalinteractio00jake_0222.txt

axonalinteractio00jake_0103.txt

axonalinteractio00jake_0018.txt

axonalinteractio00jake_0084.txt

axonalinteractio00jake_0006.txt

axonalinteractio00jake_0064.txt

axonalinteractio00jake_0226.txt

axonalinteractio00jake_0158.txt

axonalinteractio00jake_0055.txt

axonalinteractio00jake_0032.txt

axonalinteractio00jake_0082.txt

axonalinteractio00jake_0011.txt

axonalinteractio00jake_0020.txt

axonalinteractio00jake_0007.txt

axonalinteractio00jake_0066.txt

axonalinteractio00jake_0098.txt

axonalinteractio00jake_0180.txt

axonalinteractio00jake_0038.txt

axonalinteractio00jake_0169.txt

axonalinteractio00jake_0199.txt

axonalinteractio00jake_0012.txt

axonalinteractio00jake_0000.txt

axonalinteractio00jake_0063.txt

axonalinteractio00jake_0065.txt

axonalinteractio00jake_0194.txt

axonalinteractio00jake_0056.txt

axonalinteractio00jake_0025.txt

axonalinteractio00jake_0033.txt

axonalinteractio00jake_0110.txt

axonalinteractio00jake_0104.txt

axonalinteractio00jake_pdf.txt

axonalinteractio00jake_0048.txt

axonalinteractio00jake_0139.txt

axonalinteractio00jake_0097.txt

axonalinteractio00jake_0162.txt

axonalinteractio00jake_0058.txt

axonalinteractio00jake_0079.txt

axonalinteractio00jake_0112.txt

axonalinteractio00jake_0201.txt

axonalinteractio00jake_0166.txt

axonalinteractio00jake_0053.txt

axonalinteractio00jake_0211.txt

axonalinteractio00jake_0091.txt

axonalinteractio00jake_0059.txt

axonalinteractio00jake_0123.txt

axonalinteractio00jake_0031.txt

axonalinteractio00jake_0113.txt

axonalinteractio00jake_0196.txt

axonalinteractio00jake_0142.txt

axonalinteractio00jake_0124.txt

axonalinteractio00jake_0225.txt

axonalinteractio00jake_0128.txt

axonalinteractio00jake_0154.txt

axonalinteractio00jake_0105.txt

axonalinteractio00jake_0109.txt

axonalinteractio00jake_0182.txt

axonalinteractio00jake_0039.txt

axonalinteractio00jake_0219.txt

axonalinteractio00jake_0106.txt

axonalinteractio00jake_0207.txt

axonalinteractio00jake_0093.txt

axonalinteractio00jake_0224.txt

axonalinteractio00jake_0152.txt

axonalinteractio00jake_0183.txt

axonalinteractio00jake_0072.txt

axonalinteractio00jake_0083.txt

axonalinteractio00jake_0214.txt

axonalinteractio00jake_0086.txt

axonalinteractio00jake_0131.txt


Full Text











AXONAL INTERACTIONS BETWEEN FETAL SPINAL CORD
TRANSPLANTS AND THE ADULT RAT SPINAL CORD
















By

LYN BURRELL JAKEMAN


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1990




AXONAL INTERACTIONS BETWEEN FETAL SPINAL CORD
TRANSPLANTS AND THE ADULT RAT SPINAL CORD
By
LYN BURRELL JAKEMAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1990


ACKNOWLE DGEMENTS
This undertaking is one which could not have been
completed if it were not for the help of several people.
First and foremost has been my mentor, Dr. Paul Reier, for
whom I have the utmost respect and admiration for his advice
and role as teacher and scientist. He contributed an
invaluable amount of time and patience to my training and
taught me the importance of maintaining a balance between
persistence and flexibility; a lesson that is vital in
research, writing, and communication in science. Additional
appreciation is extended to each member of my committee
Drs. Barbara Bregman, John Munson, Roger Reep, Lou Ritz, Don
Stehouwer, and Chuck Vierck for continued support and
constructive criticisms regarding my work.
The daily progress was made more pleasant with the superb
technical and organizational assistance of Barbara O'Steen,
Minnie Smith, and Regina Reier, who kept track of my loose
pieces of paper and saved me months of effort. Additional
help was provided by the secretarial and support personnel in
the Departments of Neuroscience and Neurosurgery. Gratitude
is also extended to fellow graduate students, especially
Denise and Greg, who taught me to believe in myself when the
game seemed lost. Oversight of animal care and use was kept
ii


by Dr. Dan Theele, D.V.M.. Finally, instruction in the
concepts of morphometric image analysis was made available by
Mr. Dan Williams.
An incredible amount of emotional support has been
extended over the past six years by family and friends;
especially my sister Barb, my parents, and my in-laws.
However, the greatest appreciation is extended to my husband,
David T. Lee, who provided both support and encouragement,
and also helped with editorial suggestions, technical
assistance, and scientific criticism.
The last personal acknowledgements go to Bob Yant and
Jim Sutherland, who provided needed reminders of the
importance of developing a progressive outlook with regard to
spinal cord research.
Financial support for equipment, supplies, and the much
needed student assistantship was provided by NIH grants 22316
and NS72300 to P.J. Reier, The Mark F. Overstreet Fund for
Spinal Cord Regeneration Research, The Center for
Neurobiological Sciences (NIMH grant MH15737), and the
Department of Neuroscience. Additional travel support was
provided by the American Paralysis Association.
iii


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF ABBREVIATIONS vi
ABSTRACT vii
CHAPTERS
1 INTRODUCTION AND BACKGROUND 1
Spinal Cord Injury 1
Treatments to Minimize Functional Loss 2
Promotion of Axonal Regeneration or
Sprouting 4
Fetal Neural Transplants and Spinal
Cord Repair 8
Development of a Neural Relay Across a
Spinal Injury Site 11
Experimental Goals 12
2 GENERAL METHODS 14
Experimental Animals 14
Preparation of Lesion Cavities 15
Preparation of Donor Tissue 16
Transplantation Procedure 16
Post-Operative Care 17
3 DIFFERENTIATION OF SUBSTANTIA GELATINOSA-
LIKE REGIONS IN INTRASPINAL TRANSPLANTS
OF EMBRYONIC SPINAL CORD TISSUE 18
Introduction 18
Materials and Methods 2 0
Results 23
Discussion 43
IV


CHAPTERS
4 AXONAL PROJECTIONS BETWEEN FETAL SPINAL CORD
TRANSPLANTS AND THE ADULT RAT SPINAL CORD:
A NEUROANATOMICAL TRACING AND IMMUNOCYTO-
CHEMICAL STUDY OF HOST-GRAFT INTERACTIONS 55
Introduction 55
Materials and Methods 57
Results 67
Discussion 116
5 INTERACTIONS BETWEEN INJURED CORTICOSPINAL
TRACT AXONS AND FETAL SPINAL CORD
TRANSPLANTS 129
Introduction 129
Materials and Methods 132
Results 139
Discussion 168
6 SUMMARY AND CONCLUSIONS 180
Construction of a Relay Across a FSC Graft 180
Specificity Issues 182
Possible Role of FSC Grafts in Segmental and
Long-Tract Functions 187
Future Directions 191
Conclusions 192
REFERENCES 194
BIOGRAPHICAL SKETCH 218
v


LIST OF ABBREVIATIONS
CGRP calcitonin gene-related peptide
CNS central nervous system
CST corticospinal tract
DAB diaminobenzidine
FG Fluoro-Gold
FI Fusion Index
FSC fetal spinal cord
GABA gamma-aminobutyric acid
GFAP glial fibrillary acidic protein
HRP horseradish peroxidase
MBP myelin basic protein
NT neurotensin
Ox oxytocin
PAP peroxidase anti-peroxidase
PHA-L Phaseolus vulgaris leucoagglutinin
PNS peripheral nervous system
TH tyrosine hydroxylase
WGA-HRP- wheat germ agglutinin, conjugated to HRP
5-HT serotonin (5-hydroxy-tryptamine)
vi


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
AXONAL INTERACTIONS BETWEEN FETAL SPINAL CORD
TRANSPLANTS AND THE ADULT RAT SPINAL CORD
By
Lyn Burrell Jakeman
May, 1990
Chairman: Dr. Paul J. Reier
Major Department: Neuroscience
One approach to spinal cord repair involves the
transplantation of homotopic fetal neural tissue into the
site of a spinal lesion. As a strategy with potential toward
eventual functional recovery, this approach includes two
primary objectives. The first goal is to replace intrinsic
spinal cord elements at the lesion site. The second is to
provide a means for reconstructing functional continuity
between the separated rostral and caudal ends of the spinal
cord. Accordingly, the following series of experiments
establish an anatomical setting for functional repair.
To evaluate the potential for replacement of intrinsic
spinal cord tissue, intraspinal transplants of fetal spinal
cord (FSC) tissue were examined using conventional light and
electron microscopic techniques and immunocytochemical
staining. The normal substantia gelatinosa was compared with
vii


distinct myelin-free regions of the grafts and the two were
found to contain similar cytological characteristics and
similar patterns of peptide staining.
Axonal projections between (FSC) grafts and the host
spinal cord were identified using a variety of neuro-
anatomical tracing and immunocytochemical techniques. The
presence of host fiber growth into the grafts, an extensive
pattern of intrinsic graft projections, and efferent growth
of axons into the host were consistent with the hypothesis
that fetal transplants may establish a neural relay across a
spinal cord lesion site.
Finally, interactions between the long myelinated fiber
tracts of the spinal cord and FSC grafts were examined using
the corticospinal tract (CST) as a model system. Injured CST
axons were observed in direct apposition to FSC tissue at the
host-graft interface, and CST axons were also seen extending
into the transplants.
Together, the results indicate that FSC transplants can
be used to restore anatomical continuity through 1) the
differentiation of intrinsic spinal cord regions at a lesion
site and 2) the development of axonal interactions between
host and graft tissues in the adult rat spinal cord. These
observations provide a basis for future studies to examine
the functional integration of FSC transplants, as well as a
model for investigating the biology of axonal elongation.
viii


CHAPTER 1
INTRODUCTION AND BACKGROUND
Spinal Cord Injury
Traumatic injury to the spinal cord begins a sequence of
events that result in the degeneration of spinal cord tissue
and subsequent loss of sensory and motor function. In order
to understand the clinical picture, the underlying
pathophysiology has been studied extensively in experimental
models of spinal cord injury (Dohrmann, '72; Sandler and
Tator, '76; Wagner et al., '78; Windle, 'SO; de la Torre,
'84; Beattie et al., '88; Fehlings and Tator, '88). The
progressive degeneration of spinal cord tissue is initiated
by a primary insult to neurons and vascular elements in the
region of the injury (Sandler and Tator, '76; Balentine and
Paris, '78a,b). The acute events then activate a cascade of
chemical reactions due to ischemia (Osterholm, '74), ionic
conductance changes (Young et al., '82; Stokes et al., '83),
and the accumulation of cytotoxic free oxygen radicals
(Demopoulos et al., '80) at the lesion site. Collectively,
these responses lead to the formation of a cavitated lesion
area within the spinal cord.
The chief functional consequences of this spinal lesion
are described in most basic neuroscience textbooks (e.g.,
1


2
Daube et al., '78). Briefly, the local degeneration of gray
matter leads to a loss of intrinsic and projection neurons at
the lesion site. This can result in the permanent
dysfunction of segmental actions, the extent of which is
dependent upon the size, type, and level of the resulting
lesion. The second outcome from the degeneration of spinal
cord tissue is the loss of functional continuity between
rostral and caudal levels of the spinal cord. Additional
compromise of autonomic and propriospinal systems underlie a
further level of complexity to the extent of functional
recovery (e.g. Cole, 788) .
Recently, some emphasis has also been placed upon the
recognition of long-term functional effects at distant
regions of the central nervous system, as a result of
denervation and retrograde changes after axotomy (Beattie et
al., '88). Specifically, compensatory changes such as
collateral sprouting, receptor super-sensitivity, and
behavioral substitution, are likely to contribute to chronic
adaptive and maladaptive functions after spinal cord injury.
Treatments to Minimize Functional Loss
To date, most therapeutic approaches designed to spare
function or improve recovery after spinal cord injury have
been based upon the progressive pathology described above.
One such strategy includes pharmacological treatments aimed
at reducing or reversing the pathological process by
interfering with the cascade of secondary biochemical events


3
(reviewed in de la Torre, '80; Beattie et al., '88). For
example, corticosteroids have been used to stabilize membrane
changes and reduce edema after injury. Antioxidants have
been reported to promote functional recovery by reducing the
consequences of lipid peroxidation (Saunders et al., '87;
Anderson et al., '88). Other drugs have been employed to
improve functional characteristics attributed to axonal
transmission of long-tract fibers by altering ionic channel
conductances (Blight and Gruner, '87).
In addition to pharmacological approaches, surgical
procedures, including the removal of external compression
sources and stabilization of the vertebral segments
surrounding the injury site, are used to reduce the extent of
functional loss after injury (Ransohoff, '80) .
Alternative efforts toward maximizing functional recovery
in later stages after injury have been directed at restoring
modulatory influences to spared regions of the spinal cord.
A number of animal experiments have suggested that the
circuitry below a lesion can be manipulated through the
application of pharmacological agents. Experiments using the
adrenergic agonist, clonidine, have demonstrated that
activation of catecholamine receptors can improve segmental
stepping behavior in cats in the first week after a spinal
injury (Forssberg and Grillner, '73). In addition,
application of bicuculline, a gamma-aminobutyric acid (GABA)
antagonist, has been associated with improvements in spinal


4
stepping after chronic spinal cord injuries. This latter
effect appears to be mediated by enhancing segmental
influences on the remaining circuits (Robinson and
Goldberger, '86). Conversely, the clinical use of the GABA
agonist, baclofen, has had beneficial effects in reducing
extreme spasticity in spinal cord injured patients by
enhancing the inhibitory influences upon segmental reflexes
(Bloch and Basbaum, '86). Thus, many proposed
pharmacological treatments for the chronic spinal injury
patient rely upon establishing a balance of receptor-mediated
neuronal activities at the segmental level.
Promotion of Axonal Regeneration or Sprouting
Over the past decade, advances in therapeutic strategies
have resulted in a progressive reduction of mortality with a
concurrent improvement in the quality of life following
spinal cord injury (e.g. Bloch and Basbaum, '86; Green and
Klose, '89). Nevertheless, a continued emphasis must be
placed upon research efforts to promote the repair of the
spinal cord and to restore functions normally mediated by
both segmental and long-tract systems.
Since neurogenesis is essentially lacking within the
adult mammalian central nervous system (CNS), there is no
mechanism for spontaneous replacement of neurons after
injury. Repair of the spinal cord is, therefore, dependent
upon the regeneration or sprouting of axons from existing
neurons. An emphasis in mammalian regeneration research has


5
been placed upon the evaluation of differences between the
peripheral nervous system (PNS), where injured axons
regenerate and are able to successfully reinnervate their
target tissues, and the CNS, where axons do not regenerate
such that they return to their original post-synaptic sites
(reviewed in Clemente, '64; Guth, '75; Bernstein et al., '78;
Kiernan, '79).
The aspects of these systems which have been contrasted
most often are the regenerative or sprouting capacity of the
axons and the permissive or inhibitory nature of their
environment. This interaction between axons and surrounding
cells received early attention by Ramon y Cajal ('28), who
observed small regenerative sprouts following experimental
injury to spinal cords of young kittens. The sprouts failed
to persist after two weeks, and no functional recovery was
observed. More recently, Aguayo and his colleagues confirmed
Ramon y Cajal's notion that the environment can profoundly
influence regeneration. By implanting pieces of peripheral
nerve into the brain and spinal cord, they demonstrated that
CNS neurons can extend and maintain long axonal sprouts
within the peripheral environment (David and Aguayo, 781;
Richardson et al., '82). Other researchers have provided
evidence of varying degrees of synaptic reorganization
following injury within the adult brain and spinal cord

(e.g., Cotman and Nieto-Sampedro, '85; Goldberger and Murray,
'88; Steward, '89b).
Together, this work has inspired


6
renewed encouragement in the field of CNS and spinal cord
repair.
Strategies aimed at promoting the regeneration of spinal
cord axons include changes to the CNS microenvironment as
well as methods that might stimulate axonal sprouting and
elongation. Guth et al. ('85a) demonstrated that axons
within the spinal cord will not extend into a vacant lesion
site; instead, they must encounter a cellular terrain for
successful elongation. In addition, the establishment of a
dense meshwork of glial and connective tissue elements at the
lesion site may also present a problem for growing axons.
The concept of fibro-glial scarring as an impenetrable
barrier to elongating axons was championed by Ramon y Cajal
(7 28) and has been a topic of debate for the several decades
(reviewed in Reier et al., '83b; Reier and Houle, '88). With
this in mind, several approaches have been taken to prevent
or reduce the extent of glial/fibroblastic scar formation
after injury and thus promote axonal elongation. The
invasion of fibroblasts can be minimized by using closed
spinal cord injury models such as the weight-drop contusion
or clip compression approaches. In addition, pharmacological
agents have been applied to prevent the formation of scar
tissue at a lesion site (e.g., Windle et al., '52; Guth et
al., '85b). These results have suggested that axons may
extend a short distance into a spinal lesion where such a
scar is reduced.


7
Some recent investigations have been directed at
promoting the elongation of injured spinal cord axons using
different methods. One such approach involves the
implantation of cultured cells into a lesion (Siegal et al.,
'88; Wrathall et al., '89). These studies reflect a desire
to apply substances known to induce axonal elongation in
vitro to the injured spinal cord. The results suggest that
the effects may be more complex in vivo because of many
uncontrolled variables. Finally, the application of
electrical fields has also been investigated as a means to
increase the distance of axonal elongation (Borgens et al.,
'86, '87). These results, however, are inconclusive and
await further confirmation.
It is important to note that throughout the history of
spinal cord regeneration research, one recurrent difficulty
has been the uneguivocal identification of regenerating axons
or collaterals into or across the site of a spinal cord
lesion. Using conventional histological procedures, such as
silver staining, the only way to verify that axons crossing
a lesion site represent true fiber growth has been to ensure
that the initial lesion constitutes a complete transection.
This injury model, however, neither provides the most
conducive environment for regeneration nor represents a
clinical injury. Furthermore, even in the case of a complete
transection, other important factors, such as the absolute
distance of fiber elongation, the origin and terminations of


8
axons, or patterns of reinnervation, cannot be verified using
these techniques.
Fetal Neural Transplants and Spinal Cord Repair
Reports of some degree of functional recovery following
transplantation of fetal CNS tissue into the brain (e.g.,
Gash et al., '80; Bjorklund and Stenevi, '84; Dunnett et al.,
'85; Buzsaki and Gage, '88) suggested that embryonic neural
tissue might promote neuronal repair following injury or
disease. These findings have led to the application of
similar transplantation strategies in the spinal cord. The
first intraspinal fetal grafting studies resulted in low
transplant survival rates as compared to similar experiments
in the brain (Nygren et al., '77; Patel and Bernstein, '83;
Das, '83; Commissiong, '84). Such difficulties served to
underscore the extreme pathological consequences of spinal
cord injury. It has been proposed that many of the grafts
failed to survive because they did not integrate with the
parenchyma of the injured spinal cord (Das, '83).
Following improvements in surgical procedures and careful
selection of donor tissue ages (Nornes et al., '83; Reier et
al., '83a; Reier, '85), more recent studies of intraspinal
transplantation have met with greater success. One approach
involves injecting suspensions of dissociated embryonic
brainstem cells caudal to the site of injury (Nygren et al.,
'77; Nornes et al., '83; Privat et al., '86). The focus of
this strategy is to restore supraspinal modulatory influences


9
to denervated regions below the level of a spinal lesion. An
emphasis has been placed upon the descending monoaminergic
systems which have been associated with the modulation of
segmental reflex and locomotor circuitries. These studies
have indicated that the injured spinal cord can be
reinnervated by grafted embryonic brainstem neurons.
Furthermore, such grafts can mediate some types of reflex
change after spinal injury or chemical denervation (Buchanan
and Nornes, '86; Moorman et al., '88; Privat et al., '86,
' 89) .
While transplants placed below a lesion site may
contribute to the replacement of modulatory influences,
recovery of sensation and voluntary motor capacities will
require applications that restore continuity at the lesion
site. Therefore, an alternative approach toward spinal cord
repair involves the transplantation of fetal tissue directly
into a lesion cavity (e.g. fetal spinal cord (FSC) grafts)
(Reier et al., '83a, '85,'86a; Houle and Reier, '88).
This approach differs from the transplantation of tissue
caudal to an injury, and it directly addresses three major
consequences of spinal cord injury. First, the presence of
embryonic tissue at the site of a lesion may provide trophic
influences to prevent degenerative changes after injury. For
example, fetal grafts placed into the injured spinal cord or
cortex of neonatal rats have been associated with a
significant reduction in the extent of cell death that is


10
characteristic of such lesions in the infant CNS (Bregman and
Reier, '86; Haun and Cunningham, '87). In addition, there
has been at least one suggestion that the presence of fetal
tissue in a spinal lesion cavity may prevent degeneration of
white matter fiber tracts in adult recipients (Das, 786).
Secondly, fetal tissue may serve to replace segmental
neurons at the level of the lesion. Interactions of
intrinsic and projection neurons may be important for the
repair of propriospinal influences after injury. This
approach for the replacement of damaged or diseased neurons
forms the basis for the transplantation of fetal neural
tissue into neurodegenerativo and excitotoxin-induced lesions
in the brain.
The main objective of the intralesion grafting paradigm,
however, is to provide a neuronal framework that could
ultimately subserve functional integration of the rostral and
caudal spinal cord segments. In this context, the hypothesis
has been advanced that embryonic CNS tissue might be used to
promote spinal cord repair by providing a bridge for axons to
extend across the lesion (Nornes et al., '84; Reier, '85).
While results from recent studies suggest that descending
axons can extend across a FSC graft in newborn rats, there
is no evidence to date to indicate that injured CNS axons in
the adult will bridge a fetal graft to reinnervate their
original spinal cord target regions. However, an alternative
possibility is that transplants may establish a neuronal


11
relay pathway across a spinal cord lesion (Johnson and Bunge,
'83; Nornes et al., '84,* Reier, '85, Reier et al.,'88;
Jakeman and Reier, 788).
Development of a Neural Relay Across a Spinal Injury Site
The concept of a neural relay has been discussed at its
most basic level by Shepard ('88). The three components of
the "synaptic triad" that form any neuronal circuit include
input neurons, intrinsic neurons, and projection neurons.
Complex variations of these components form the basis for
local circuits throughout the CNS (Rakic, '76). Several well
studied, integrative relays are found within the organization
of the dorsal and ventral horns of the normal spinal cord.
These circuits are responsible for the transmission and
integration of sensory and descending influences and
segmental reflex pathways.
The hypothesis of a relay with regard to FSC transplants
implies that the transmission of ascending and descending
information across a spinal cord lesion may be mediated
through interactions between host and graft tissues. These
interactions may take the form of afferent and efferent
projections between the surrounding host spinal gray matter
and fiber tracts and the intrinsic circuitry of the graft.
In the absence of axonal projections across the host-graft
interface, a relay circuit might be constructed by
interactions between axons which persist at the host-graft
interface and dendritic projections of host or grafted


12
neurons (Das, '83; Mahalik et al., '86; Clarke et al., '88b).
Alternatively, the relay may be more complex, as dictated by
differences in the relative growth and functions of different
host fibers. In the latter instance, the monoaminergic input
may serve to provide a modulatory influence upon the more
local host-graft interactions.
Experimental Goals
Despite efforts spanning over more than a decade of
research, the mechanisms that underlie various examples of
functional recovery following transplantation in the adult
brain are still unknown. Several recent review papers have
proposed a spectrum of possible mechanisms. In general, it
appears that functional changes following fetal neural
grafting may be obtained in a variety of ways in each model
system (Bjorklund et al., '87; Dunnett and Bjorklund, '87;
Gage and Buzsaki, '89). To better understand these models
and to test the capacity of the CNS for reorganization after
injury or disease, a recent emphasis has been placed upon
defining the anatomical correlates of host-graft
interactions. Through careful examination of the patterns of
axonal projections between transplants and the host CNS, the
strengths, potential mechanisms of behavioral improvement,
and the limitations of the grafting models can be assessed
more accurately.
Likewise, an important first step in determining a
potential functional role of FSC transplantation in the


13
spinal cord is to define the anatomical basis for integration
of host and graft tissues. Preliminary studies of
interactions between such transplants and the injured adult
rat spinal cord have indicated that some axonal projections
can form between the tissues (Reier et al., '85, '86a).
However, the purpose of these earlier studies was to identify
the general feasibility of transplantation and the
integration of such transplants into the adult spinal cord.
The objective of the following work is to identify, in
more detail, the anatomical basis for the role that FSC
transplants might play in the repair of the injured spinal
cord. The use of a variety of complementary neuroanatomical
methods will serve to define several aspects of host-graft
interactions, including the differentiation of regions in the
grafts and the development of axonal projections between
graft and host tissues. From these studies, information will
be obtained concerning the nature of neuronal relay
possibilities for transmission of information across the site
of a spinal cord lesion. In addition, the FSC transplant
model will be used to examine some of the biological issues
concerning axonal elongation within the adult spinal cord.


CHAPTER 2
GENERAL METHODS
A number of spinal cord injury models have been used to
evaluate potential strategies for intervention and repair.
These include discrete lesions of specific fiber tracts,
chemical axotomy of fiber types, blunt contusion or
compression injuries, and complete or partial transection
models (reviewed in de la Torre, '84; Beattie et al., '88;
Das, '89). The present investigations have employed a model
of transplantation into partial transection cavities prepared
by aspiration immediately before grafting (acute lesions).
The transplantation methods used throughout these studies are
similar to those detailed in previous reports (Reier et
al.,'83a,'86a). Each experimental design has employed only
minor modifications of the procedures described below.
Experimental Animals
Adult, female, inbred Sprague-Dawley rats were used
throughout these studies. All of the rats were obtained from
Zivic-Miller Laboratories (Allison Park, PA) and weighed 200-
3 00 grams at the start of the experiments. The rats were
housed two per cage in the University of Florida animal
resources facility (accredited by the American Association of
Laboratory Animal Caretakers), according to the guidelines
14


15
established by the National Institutes of Health (Publication
number 85-23). They were examined daily by a veterinarian
and/or veterinary technician for general health conditions
and for any post-operative complications due to the spinal
cord injury. All surgical procedures were performed with
instrument preparation in anti-microbial benzalkonium
chloride solution (Zepharin HC1, Winthrop Breon Laboratories)
and 95% Ethanol.
Preparation of Lesion Cavities
The transplant recipients were anesthetized with
intramuscular injections of a mixture of ketamine (Ketaset,
60 80 mg/kg; Aveco Co.) and xylazine (Rompun, 10 mg/kg;
Mobay Corp.). The spinous processes were then exposed by
longitudinal incisions of the overlying musculature.
Bleeding from the superficial muscles was controlled with
epinephrine-impregnated cotton pellets (Gingipak, Belport
Co.,Inc.). A laminectomy was then performed at the
appropriate vertebral level using fine-tipped rongeurs, and
the host spinal cord was exposed by a longitudinal incision
of the dorsal meninges just lateral to midline. Using
iridectomy scissors and a glass micropipette with light
aspirative pressure, a cavity of 3 6 mm in length was
created in the parenchyma of the spinal cord, and bleeding
was controlled with small pellets of gelatin sponge (Gelfoam,
Upjohn Co.) soaked in a saline solution containing bovine
thrombin (Thrombostat, Parke Davis).


16
Preparation of Donor Tissue
For each transplantation session, a pregnant rat (E.4,
embryonic day 14; E0 = day of insemination) was anesthetized
with 4.0% chloral hydrate (400 mg/kg, i.p.). A laparotomy
was performed and individual donor embryos (approximate
crown-rump length of 12 13 mm) were removed as needed and
placed into a standard tissue culture medium (Hank's Balanced
Salt Solution). The embryonic spinal cord tissue was then
dissected by removal of overlying skin layers and the
detachment of the spinal ganglia. The spinal cord was
stripped of embryonic meningeal layers and used for
transplantation within one hour of removal from the mother.
At the end of the transplantation session, the pregnant rat
was euthanized with an intracardiac injection of sodium
pentobarbital (Butler Co.).
Transplantation Procedure
Once hemostasis was achieved in the host, either one or
two pieces of donor spinal cord tissue were cut to
approximate the length of the prepared cavity. The donor
tissue and a small amount of tissue culture medium were
placed into the cavity with a flame-tipped micropipette.
After placement of the grafts, the excess tissue culture
medium was removed with light manual suction. A single
piece of hydrocephalus shunt film (Durafilm; Codman
Shurtleff, Inc.) was usually cut to a size just larger than
the lesion cavity and positioned directly above the graft.


17
The dural incision was closed with 10-0 interrupted sutures
and the spinal cord was then covered with a second piece of
synthetic dural covering. The overlying muscles were then
sutured in layers using 4-0 silk and the skin incision closed
with wound clips (Fisher).
Post-Operative Care
Following surgery, all rats received a subcutaneous
injection of long-acting penicillin (Dual-Pen; Tech America;
75,000 U.). They were carefully monitored and kept on a
heating pad or under a mild heat lamp until recovery from
anesthesia. Within 48 hours, the rats were returned to the
animal care facility where they were fed rat chow and water
ad libitum and maintained under a 12 hour light and 12 hour
dark schedule.
In the subsequent weeks after surgery, approximately 10%
of the animals initiated mild autotomy of the ipsilateral
forelimb or hindlimb (corresponding to lesion level) and were
treated with daily application of veterinary autophagic
repellent (Chewguard, Summit Hill Labs.). Those few rats
that failed to respond to such treatment within a few days
were sacrificed shortly thereafter and included in the data
analysis. This consideration contributed to the range of
post-graft survival times (post-graft intervals) referred to
throughout the work.


CHAPTER 3
DIFFERENTIATION OF SUBSTANTIA GELATINOSA-LIKE
REGIONS IN INTRASPINAL TRANSPLANTS OF
EMBRYONIC SPINAL CORD TISSUE
Introduction
Intracerebral grafts of fetal CNS tissue have been shown
to compensate for a variety of functional deficits in
experimental animal models. This may occur through the
replacement of neuronal circuitries or neurotransmitters or
by the production of neuronotrophic substances within the
host brain (reviewed in Bjorklund and Stenevi, '84; Sladek
and Gash, '84; Bjorklund et al., '87; Dunnett and Bjorklund,
'87) The degree to which such grafts can subserve the
functions of the damaged brain region may depend upon the
ability of the grafted tissue to differentiate and integrate
with the host neural circuitry. Thus, several investigators
have examined the extent to which fetal neural transplants
will differentiate and exhibit organotypic characteristics
when placed into homotopic and heterotopic sites within the
CNS (e.g., Jaeger and Lund, '80; Alvarado-Mallart and Sotelo,
'82; Kromer et al. '83; Eriksdotter-Nilsson et al., '86;
Harvey et al., '88; Sorensen and Zimmer, '88a,b). From these
studies, it is clear that grafted regions of the CNS exhibit
18


19
different degrees of differentiation depending upon the age
of the embryonic donor tissue and the region from which it
is obtained.
In recent years, there has been some enthusiasm for the
application of fetal CNS tissue transplantation techniques to
the problem of spinal cord injury (e.g. Das, '83;
Commissiong, '84; Privat et al., '86; Reier et al.,'85, '86a;
Houle and Reier, '88). Together, these reports indicate that
intraspinal transplantation results in the survival and
integration of embryonic donor tissue in both neonatal and
adult recipients. One approach involves the introduction of
fetal neurons into the lesion site (Reier, '85; Reier et al.,
'85). In particular, homotopic grafts placed into the site
of a spinal lesion may serve as a source of specific
intraspinal neuronal populations with an inherent potential
for integrating with synaptic circuits above and below the
injury. Some degree of homotypic differentiation has been
indicated by studies in which fetal spinal cord transplants
were placed into intracerebral or intraspinal cavities (Reier
and Bregman, '83; Reier et al., '85, '86a,b) In these
initial investigations, distinct myelin-free regions were
observed in matured grafts, leading to the hypothesis that
these unmyelinated areas corresponded to the superficial
dorsal horn especially the substantia gelatinosa (SG)
of the normal spinal cord. This region of the spinal cord is
easily identified in normal tissues based upon the paucity of


20
myelinated fibers, the small cells and compact nature of the
neuropil, and the density of peptide staining. Thus, the SG
provides a model system to determine whether grafts of FSC
tissue can differentiate and replace specific regions of the
normal spinal cord.
In the present study, the myelin-free regions of fetal
rat spinal cord grafts were examined in more depth to
determine the extent to which these areas develop cellular
and ultrastructural characteristics of the normal SG when
placed into lesion cavities in the adult rat spinal cord.
In addition, immunocytochemical methods were used to examine
the distribution of neurotensin-immunoreactive elements,
normally observed only within the superficial dorsal horn
(Portions of this study have been summarized in Reier et al,
'85; Jakeman et al., 789).
Materials and Methods
Animals and Surgical Procedures
Ten adult rats received intraspinal implants of Eu rat
spinal cord tissue. The surgical procedures were similar to
those described in previous reports (Reier et al., '83a,
'86a; Chapter 2, this volume). Each rat was anesthetized
with ketamine and xylazine and a laminectomy was performed at
the T13 vertebral (approximately I^- Lj spinal) level. An
aspirative lesion cavity, 3-4 mm in length, was created at
the exposed site. The lesion was then extended to include
either an extensive dorsal funiculotomy or lateral


21
hemisection. Whole segments of fetal spinal cord, 3-4 mm in
length, were dissected from donor embryos and introduced into
the lesion cavities (Reier et al., '83a).
Light and Electron Microscopy
At 1 to 2 months after transplantation, 5 recipients were
anesthetized with a lethal dose of sodium pentobarbital and
perfused through the heart with 0.9% NaCl followed by 5.0%
glutaraldehyde and 4.0% paraformaldehyde in 0.1M phosphate
buffer. Following the perfusion, segments of tissue
including the transplant and surrounding spinal cord were
then excised and divided into several transverse or
longitudinal slices. The specimens were subsequently
osmicated, dehydrated, and embedded in EM Bed 812 (Electron
Microscopy Sciences) for plastic thick sectioning. Regions
of the grafts classified as "SG-like" at the light
microscopic level were trimmed, and thin sections were
surveyed at the ultrastructural level using a Zeiss EM10C
electron microscope.
Immunocvtochemistrv
At similar intervals, the remaining intraspinal graft
recipients (n=5) were perfused with 4.0% paraformaldehyde in
0.1M Sorenson's phosphate buffer (pH 7.4). Tissue blocks
including the transplants were excised and prepared for
Vibratome (40 jum) sectioning. Adjacent 40 ¡im sections were
processed for the presence of myelin basic protein (MBP)- or
neurotensin (NT)- like immunoreactivity with the indirect


22
peroxidase anti-peroxidase (PAP) method (Sternberger, '76)
using primary antisera raised in rabbits. The antisera to
NT was obtained from Immunonuclear Corp. (Stillwater, MN.)
and the antiserum to MBP was provided by Dr. L. F. Eng (Palo
Alto, CA., VA Med Center). For PAP staining, tissue sections
were incubated overnight at room temperature in primary
antiserum diluted to 1:2000 with a solution of 0.3% Triton
X-100 in phosphate buffered saline (PBS). All incubations
were performed in the presence of 5% normal goat serum. The
sections were washed several times in PBS and then incubated
in goat anti-rabbit IgG (Cooper Biomedical or Sternberger-
Meyer) diluted 1:10 with the antiserum diluent for 45-60
minutes at room temperature. After a rinse with PBS, the
sections were incubated for 45 minutes in rabbit PAP (Cooper
Biomedical or Sternberger-Meyer) diluted 1:50. Following
several washes in PBS and 0.05M Tris buffer (pH 7.6), the
immunocytochemical reaction product was developed in a 0.05M
Tris buffer solution containing 0.05% 3,3'-diaminobenzidine
hydrochloride (DAB; Sigma) and 0.003% H202. Antibody
specificity was verified by the anatomical distribution of
immunoreactive elements in normal spinal cord tissue and by
the absence of immunoreactive elements when primary antibody
was replaced with normal serum alone.
Correspondence Between Plastic and Immunocvtochemistrv
In three specimens used for immunocytochemistry, 100 /im
vibratome sections were obtained to correspond to the


23
sections stained with antisera to MBP and NT. These sections
were osmicated and dehydrated as described above, and
embedded between vinyl slides with EM Bed 812. From these
sections, 2 ¡ira. sections were cut on an LKB ultramicrotome and
stained with 1.0 % Toluidine Blue. Similar 40 /m and 2 /urn
sections were also obtained from the thoracic spinal cord of
2 normal rats to illustrate normal characteristics and
staining patterns within the substantia gelatinosa.
Results
Light Microscopy and Cvtoloaical Characteristics
Sections of normal rat spinal cord, when stained with
antiserum directed against myelin basic protein (MBP),
exhibit a characteristic pattern of myelin distribution.
Specifically, the long fiber tracts appear densely stained,
while moderate staining reflects the presence of myelinated
fibers coursing throughout most of the central gray matter.
The most striking feature of this preparation, as also seen
with conventional myelin stains, is that region within the
dorsal horn which corresponds to the cytoarchitecturally
defined substantia gelatinosa (SG). This region stands out
against the background of intense myelin immunoreactivity due
to the paucity of myelinated axons (Fig. 3-1 a,b).
Fetal spinal cord transplants were stained with anti-MBP
to examine the differentiation of the grafts. All of the
transplants examined with this technique were heavily


Figure 3-1. Identification of myelin-free areas.
a) Pattern of PAP staining with antiserum against MBP in a transverse
section of a normal rat thoracic spinal cord, showing the myelin-free
regions in the superficial dorsal horn (arrows).
b) Higher magnification of Anti-MBP staining in a normal dorsal horn,
illustrating the large myelinated fibers which often traverse the
superficial laminae.
c) Anti-MBP staining of a 2 month intraspinal transplant (t) and
surrounding host intermediate gray (h]G) and lateral white matter (hg)
(Horizontal section). Three myelin free patches are seen at the edge of
the transplant (arrows).
d) Enlargement of boxed region in (c). Myelinated axons (arrow) cross
through this otherwise unmyelinated area. Dotted line highlights the
host/graft interface.
Scale in a,c = 200 /im; b,d = 50 /im.


25


26
myelinated as indicated by areas of very dense staining. In
addition, large regions exhibiting moderate immuno-
reactivity were evident. Finally, the transplants usually
contained one or more areas which were conspicuous due to the
marked absence of MBP-like staining (Fig. 3-1 c,d). These
myelin-free areas typically assumed a convoluted configu
ration within the grafts, and appeared as either single or
multiple patches or long strips of neuropil depending upon
the plane of section. In many cases, these regions were
located near the periphery of the grafts (Fig. 3-1 c) ;
however, some myelin-free areas were located more centrally.
Myelinated axons frequently curved along the surfaces of the
unstained regions, and often small bundles of anti-MBP
stained processes traversed the myelin-free zones in a radial
fashion, reminiscent of the pattern of myelinated primary
afferents projecting to deeper layers of the gray matter in
the normal spinal cord (Fig. 3-1 b,d).
With the perspective derived from MBP-stained grafts,
examination of toluidine blue-stained sections of FSC
transplants revealed areas that corresponded to patterns of
MBP- immunoreactivity (Fig. 3-2 a,c; Fig. 3-3 b) The 2 /xm
sections contained regions of extensive myelination, as well
as numerous myelin-deficient areas within the graft tissue.
To determine whether the myelin-free areas identified
within the grafts by immunohistochemistry or toluidine blue
staining were indeed equivalent, adjacent sections were


Figure 3-2. Comparison of MBP and toluidine blue stained
sections.
a) Toluidine blue stained semi-thick section within an
intraspinal FSC transplant showing myelinated regions (m)
containing larger neurons, and unmyelinated patches (outlined
in arrowheads) with smaller neurons and processes.
b) MBP- stained section reveals a myelin-free region
(arrowheads) near the host-graft interface in another
recipient. Several regions of the graft lack myelin, yet this
region corresponds to an area containing small, tightly
packed neurons and processes in (c) Transplant (t) and host
gray matter (hIG) are labeled.
c) Adjacent toluidine blue stained section from the same area
(within arrowheads) which is occupied by small cells and
processes.
Scale in a,c = 50 /im; b= 100 xm.


28


29
processed so that for each MBP-stained section there was a
corresponding 100 xm section embedded in plastic. In these
examples, zones that failed to show MBP immunoreactivity were
closely in register with homogenous unmyelinated areas in the
adjacent toluidine blue stained section (Fig. 3-2 b,c).
Further examination of these regions in plastic sections
revealed other similarities between the myelin-free areas of
FSC grafts and the normal SG laminae. As in the normal
substantia gelatinosa (Fig. 3-3 a,c), the unmyelinated zones
of matured transplants consisted of numerous small cells (7 -
15 /Ltm) characterized by a thin rim of cytoplasm surrounding
a prominent nucleus. The nuclei of these cells, as of those
in the normal substantia gelatinosa, were round or oval and
often exhibited large clefts (cf., Fig. 3-3 c,d). These
cells were qualitatively distinct from the larger neurons
(14-50 /xm diameter) that were found within the myelinated
regions of the transplants. In fact, the presence of larger
neurons within the unmyelinated areas was very rare. It was
interesting to note that some of the larger cells within the
myelinated regions were closely apposed to the border of the
unmyelinated zones. These general cellular relationships
were similar to the approximation of laminae III and IV
neurons with the normal SG.
In addition to features common to both the normal SG and
the myelin-free graft regions, there were some differences


Figure 3-3. Cytology of the normal SG and SG-like regions in FSC grafts.
a) A 2 p toluidine blue stained transverse section from the normal adult rat dorsal
horn. Laminae are designated in roman numerals as laminae I-V and the SG as lamina II.
b) An unmyelinated region from an Eu intraspinal graft (t) adjacent to the host white
matter (h) Several aspects of these regions resemble those of the normal dorsal horn,
although the divisions between unmyelinated patches (see arrowheads) suggest that the
graft region has assumed a serpentine orientation. Note larger neurons (n) in close
approximation to these regions in this figure. The interface is indicated by the
dotted line.
c) SG neurons in the normal spinal cord at a higher magnification. Neurons within
the SG are tightly packed and contain large nuclei with prominent indentations
(arrowheads). Note the parallel orientation of axons in transverse section.
d) In myelin-free regions of the transplants, the neurons are similar, although the
processes often lack the longitudinal orientation characteristic of the normal dorsal
horn.
Scale in a,b=50 /im; c,d=10 /Ltm.


< *
31


32
between the two regions. This was particularly evident with
regard to the distribution of cells and neuritic processes.
In the intact spinal cord, SG neurons can be differentiated
into two layers (i.e., Hi and IIo; Ralston, '79;
Szentagothai, '64a). However, no obvious cytoarchitectural
lamination was seen in these regions of the transplants. In
addition, many small, circular neuritic profiles were seen in
transverse sections of the normal SG, thus reflecting their
orientation parallel to the longitudinal axis of the spinal
cord (Fig. 3-3 c) In contrast, the neuritic processes in
the myelin-free areas of the grafts seemed more randomly
organized, and sectioned profiles assumed many orientations.
Electron Microscopy
Neurons within the normal substantia gelatinosa were
generally spheroid (Fig. 3-4 a) or fusiform (Fig. 3-4 b) in
shape. The perikarya ranged from 8 2 0 /m in diameter, and,
as seen with the light microscope, they usually contained a
large nucleus within a narrow rim of cytoplasm. These cells
were embedded in a neuropil that primarily consisted of
tightly packed, small unmyelinated axons and small to
intermediate-sized dendrites. The axons were often organized
into bundles or fascicles which were more evident when the
tissue sections were cut in the transverse plane. Many of
the synapses identified within the SG were axo-dendritic
in nature, although other synaptic types were found. In


Figure 3-4. Ultrastructure of the normal substantia
gelatinosa.
a) Transverse section contains a neuronal cell body (n) and
the compact neuropil containing abundant axo-dendritic
synapses (ad) interspersed with longitudinal bundles of
unmyelinated processes (arrows). Large glomerular axonal
processes were often observed (star).
b) Oblique section through the SG region of another normal
specimen. The fascicles are more difficult to discern than in
(a) .
Scale in a,b=2.5 /xm.


34


35
addition, large glomerular complexes were evident in the
substantia gelatinosa of these normal sections as well.
A survey of the unmyelinated regions within FSC
transplants in low power electron micrographs revealed many
characteristics similar to the normal SG (Fig. 3-5). The
neurons in these areas were also small and contained large
nuclei with prominent indentations. The cells were closely
spaced, and were surrounded by compact neuropil consisting of
small axons (0.1 0.3 /xm) and intermediate-sized dendrites
(0.4 1.6 nm). Occasionally, small bundles of unmyelinated
processes were seen which resembled the fascicles in the
normal SG. However, many of the axons and dendrites were
more randomly oriented (Fig. 3-5, 3-6). Except for an
occasional swollen neuritic profile containing lysosomes and
degenerating mitochondria, axonal and dendritic processes
did not display irregular cytological characteristics. In
some transplants, hypertrophic astrocytic processes were
observed particularly near the periphery of the grafts (Fig.
3-5, 3-6) .
Many of synaptic contacts observed within the
unmyelinated graft regions were axo-dendritic, with axo
axonic synapses occasionally being present as well. In
addition, a rare axo-somatic synapse could be found in the
normal and graft myelin-free areas. The boutons within the
myelin-free regions usually contained aggregates of small,
agranular vesicles (Fig. 3-6). Usually, the vesicles were


Figure 3-5. Low power electron micrograph of an SG-like
region in an E14 intracerebral transplant.
The small neurons in these regions were similar to the normal
SG regions in size, shape, and the presence of nuclear
clefts. An axo-dendritic (ad) and axo-somatic (as) synapse
are shown. While some unmyelinated axons traveled in
fascicles (arrows), most axonal and dendritic processes
assumed a variety of orientations, and appeared to lack some
of the organization of the normal spinal cord. Large
filamentous glial processes (*) were sometimes seen in these
grafts.
Scale
2.0 /j.m.


37


Figure 3-6. Higher magnification of SG-like regions in an
intraspinal transplant.
The vast majority of synapses were axo-dendritic (ad), and
vesicles were usually clear (agranular) and round (arrows),
although flattened vesicles and dense-cored vesicles
(arrowheads) were also observed. Astrocytic processes were
sometimes present (*) .
Scale = 0.5 /m.


39


40
round, rather than flattened, and some terminals contained
small dense-cored vesicles. Similar presynaptic structures
were also seen in the normal SG. However, the scalloped
terminals and related glomeruli characteristic of normal
primary afferent innervation of the SG were not found within
the grafts examined in this study.
Neurotensin-like Immunoreactivitv
When sections of normal spinal cord were reacted with
antisera to neurotensin (NT), staining was restricted to the
lamina II region of the superficial dorsal horn, where it was
seen in two distinct layers of very fine processes (Fig. 3-
7 a; Seybold and Elde, '82) Labeled axonal profiles were
not found in any other region of the normal spinal cord, and
no immunoreactive cell bodies were found in these sections.
Staining of FSC transplants with antisera raised against
NT revealed regions throughout the grafts which contained
small immunoreactive processes. Many of these patches
corresponded with myelin-free regions in neighboring sections
stained with anti-MBP (Fig. 3-7 b,d). However, in contrast
to the normal spinal cord, NT staining within the transplants
did not form two distinct bands. Other differences were also
observed between the patterns of NT staining and the normal
SG. In addition to the patches of fibers that corresponded
to MBP-free regions of the grafts, some NT fibers were
distributed throughout the myelinated areas of the
transplants. Furthermore, NT-like cells were also found


Figure 3-7. Neurotensin immunoreactivity in normal spinal cord and intraspinal
transplants.
a) Transverse section through the substantia gelatinosa of a normal spinal cord. NT-
like immunoreactivity is restricted to small fibers in this region of the spinal cord.
Labeled axons are separated into two distinct bands (arrows).
b) Sagittal section through a FSC transplant (t) and the adjacent host spinal cord
(h) after staining with anti-MBP. A region of the graft near the interface (between
h and t) exhibits a lack of myelin staining.
c) Cells containing NT-like immunoreactivity were found throughout transplants, but
they were not observed in the normal adult spinal cord.
d) Sagittal section adjacent to that shown in (b), after staining with anti-NT. The
unmyelinated region contains a lightly stained patch of NT-like immunoreactivity
containing very fine NT-like fibers. Similar axonal profiles were never seen within the
deeper laminae of normal spinal cord sections, suggesting that the axons in this
section have extended a short distance into the host spinal cord from within the
transplant.
Scale in a,c = 50 nm; b,d = 100 xm.


42


43
within the grafts (Fig. 3-7 b). These cells were small and
multipolar in shape, and were often found in groups of two or
three.
Neurotensin immunoreactive fibers were often observed at
the periphery of the transplants. Interestingly, in some
examples, these labeled fibers could be followed across the
host-graft interface, where they appeared to innervate
ventral regions of the host gray matter. This region does
not normally contain neurotensin fiber ingrowth.
Discussion
In previous descriptions of matured intracerebral and
intraspinal transplants of FSC tissue, regions of graft
neuropil were identified based upon the absence of myelinated
fibers (Reier et al., '85, '86b). The indication that these
regions might reflect some organotypic differentiation was
examined further in an experiment in which pregnant rats were
injected with tritiated thymidine on either day E12 or Eu.
Labeled donor tissue was removed from fetuses in one uterine
horn and transplanted; fetuses in the contralateral horn were
left to complete gestation (Reier et al., '83a) At one
month post-transplantation, autoradiography indicated that
the nuclei of neurons in the myelin-free regions of these
transplants exhibited the same relative degree of labeling as
did the nuclei of those cells present in the superficial
laminae of the intact spinal cords of the littermates of the
donor fetuses (Reier et al.,
'83a,'86b).


44
It thus appeared that the myelin-deficient regions in the
fetal spinal cord grafts were the counterparts of the normal
substantia gelatinosa. These general criteria, though
intriguing, did not provide sufficient proof of the exact
nature of the myelin-free areas in the grafts. In this
context, it is well recognized that other characteristics
make this region distinct from the rest of the gray matter in
the intact spinal cord. In particular, the abundance of
small cells led Rexed ('52) to make the distinction of lamina
II of the cat spinal cord, and similar studies have shown
this region within the rat spinal cord as well (Molander et
al., '89) Examination of the ultrastructure of this region
has also served to define the types of processes and synaptic
profiles that distinguish the substantia gelatinosa (lamina
II) from the surrounding marginal layer (lamina I) and lamina
III (Ralston, '79? Szentagothai, '64a). Additional
identifying features have been noted through the use of
immunocytochemical techniques, which have demonstrated a
variety of peptide-containing cells and fibers (reviewed in
Seybold and Elde, '80; Gibson et al., '81; Hunt, '83;
LaMotte, '86). With these characteristic cytological and
immunocytochemical features as a basis for comparison, the
present study has provided additional evidence in support of
the organotypic differentiation of substantia gelatinosa-like
regions in transplants of fetal spinal cord tissue.


45
Mvelin-free Areas and Peptidergic Elements
Previous studies have demonstrated regions of dense
immunoreactivity obtained with antibodies to several peptides
which are normally associated with the substantia gelatinosa
(Reier and Bregman, '83; Reier et al., '85). Patches of
tissue within the transplants stained heavily with antibodies
to met- and leu-enkephalin, somatostatin, and substance P.
These patches have been identified within FSC transplants
placed into the adult brain or neonatal and adult spinal
cord. In addition, similar findings have been reported
regarding the differentiation of regions of dense peptide
staining in FSC transplants that develop in oculo (Henschen
et al., '88).
In this study, a comparison has been made between the
myelin-free regions of FSC transplants and the SG of the
normal spinal cord. Standard light and electron microscopic
observations revealed many similarities between these areas,
especially with regard to cell and process sizes and the
compact nature of the neuropil. With regard to peptide
staining patterns, an emphasis was placed upon the
distribution of NT- containing fibers within the transplants.
As described for other peptides, regions within the FSC
transplants contained distinct patches of NT-like fibers that
corresponded to unmyelinated regions of the graft. In the
same spinal cord tissue rostral and caudal to the grafts,


46
however, NT-containing axons were found only within the
substantia gelatinosa region of the spinal cord.
Developmental Implications
The observation that fetal spinal cord tissue can exhibit
some degree of organotypic development is consistent with
reports that transplants of tissue from various embryonic
brain regions can achieve cytoarchitectural and
ultrastructural characteristics corresponding to those of the
homologous areas in the intact CNS (e.g., Kromer et al., '79,
'83; Lund and Harvey, '81). Fetal spinal cord tissue has
also been shown to exhibit some cytoarchitectural or
immunocytochemical characteristics resembling the normal
dorsal horn when grown in tissue culture (Naftchi et al.,
'81; Sobkowicz et al., '68) or in oculo (Henschen et al.,
'85). The present findings show that the differentiation of
the myelin-free areas also occurs when FSC tissue grafts
develop within the adult spinal cord.
In related studies currently in progress, similar
unmyelinated regions have been observed in cell suspension
grafts of rat FSC within the contused rat spinal cord
(Winialski et al., '89) and in solid piece grafts of cat
fetal spinal cord tissue in the adult cat spinal cord
(Anderson et al., '89; Reier et al., in preparation). These
regions have thus far been identified on the basis of 2 iim
thick plastic sections and anti-MBP stained sections of the
grafts. Additional observations suggest that the myelin-free


47
areas may also be analogous to small fiber-free areas
revealed with immunocytochemical staining using polyclonal
antibodies raised against the phosphorylated form of the
heavy neurofilament protein (see silver staining patterns in
Lund and Harvey, 781). However, one recent report has
indicated that SG-like regions are not seen following
injections of cell suspensions from E12 E13 fetal rat spinal
cord into the ibotenic acid lesions of the lumbar spinal cord
(Nothias et al. '89). This difference may reflect the
different donor ages used (Kromer et al., '83), however, SG-
like areas have been identified in grafts of E12 rat spinal
cord tissue placed into the rat brain (Reier et al., '83a).
Thus, while the procedures used in this study were not
applied by Nothias et al., it may be possible to observe SG-
like regions within their grafts placed into ibotenic acid
lesions by utilizing specific markers such as the antisera
raised against dorsal horn peptides or MBP.
The presence of a dorsal horn component in FSC grafts is
likely to be related to the developmental timing of this
region of the spinal cord. Evaluations of spinal cord
histogenesis in the rat (Alvardo-Mallart and Sotelo, '82;
Nornes et al., '74) have indicated that there is a peak at
approximately E15-E16 in the generation of neurons which
ultimately comprise the dorsal horn. These cells then
migrate with the majority of neuroblasts reaching the
presumptive dorsal horn region two days later. The


48
maturation of the SG-like areas in these fetal spinal cord
grafts must therefore occur after transplantation since donor
tissue in these experiments was obtained at Eu-E15. These
considerations pertaining to cell birthdates and onset of
migration also suggest that the clustering of small neurons
into the SG-like regions may be due to the persistence of
intrinsic recognition cues which influence the aggregation of
these cells during normal development (see also Kromer et
al., '83). Related studies have also shown that these
intrinsic cues are also retained if the graft is placed into
heterotopic sites, or when it is entirely isolated from host
afferent inputs by transplanting the fetal spinal cord with
the surrounding meninges attached (Jakeman et al., '89).
Anomalous Features of the Substantia Gelatinosa-Like Regions
While many features of the myelin-free regions of FSC
grafts reflect a homology with the normal SG, it is clear
that the correspondence was not perfect. Several aspects of
these areas represent a departure from the normal
organization of the mature superficial dorsal horn. For
example, the myelin-free graft regions lack the precise
orientation and formation of a dorsolateral cap shape with
cells organized in discreet layers. In addition, the
definition between outer and inner layers of lamina II
observed in the normal SG was not observed in either 2 /m
plastic sections or with by immunocytochemical staining with
antiserum to NT. Finally, while these graft regions


49
contained some bundles of unmyelinated axons, the parallel
longitudinal arrangement of neuronal processes characteristic
of the normal dorsal horn was absent from the myelin-free
regions of the grafts.
It is likely that some of these differences are related
to specific aspects of the grafting procedure, such as the
initial orientation of the graft tissue, donor age, and
changes in the precise timing of developmental cues (Kromer
et al., '83; Stenevi et al., '76). In addition, the
topography of some of these transplants may be distorted by
spatial restraints, which can contribute to the
organizational differences observed. The differentiation of
organotypic regions within an abnormal cytoarchitectural
framework has also been observed in other types of fetal CNS
transplants (e.g. Kromer et al., '83; Mufson et al., '87;
Sorensen and Zimmer, '88b).
Another factor that may contribute to the atypical
features observed here is the relatively deafferented state
of the transplant. As noted in our electron microscopic
results, the SG-like regions in these grafts lacked the
synaptic terminals characteristic of primary afferent
innervation. A qualitative analysis of the synaptic
composition of these grafts revealed similarities in
ultrastructure to the deafferented dorsal horn, specifically
in the absence of organized glomerular complexes (Rethelyi
and Szentagothai, '69; Coimbra et al.,
'74) .
While such


50
complexes have been found in dorsal regions of FSC grafts
intentionally innervated by primary afferent fibers (Itoh
and Tessler, '88) SG-like areas deeper within the
transplants receive few such afferents.
Innervation of FSC transplants from adult host fibers is
largely restricted to the periphery of the grafts (Chapter
4). Thus, the developing grafts may also be lacking much of
the descending modulation present in the normal spinal cord.
Therefore, rather than being indicative of aberrant
development, the atypical features recorded may instead
reflect the normal development of SG-regions in circumstances
of decreased afferent input. Such a situation would not only
alter the patterns of migration and lead to cytoarchitectural
differences, but might also prevent the normal expression of
neuropeptides and neurotransmitters.
In the present study, differences were observed between
the pattern of neurotensin-like immunoreactivity in the FSC
grafts and in the normal SG. Specifically, NT-positive cells
and fibers were observed throughout the grafts, while only
fibers were found in the normal spinal cord, and those were
restricted to the SG region. However, application of
intraventricular colchicine treatment and better fixation
methods have been used to identify NT-containing cells and
fibers in the normal spinal cord. These studies have
indicated that NT-like cells can be found in lamina I and V-
VII in addition to the SG in normal rats (Gibson et al., '81;


51
Seybold and Elde, '82; Miller and Seybold, '87). Thus, the
presence of these cells throughout FSC transplants in the
absence of such treatments may reflect alterations in peptide
expression or axonal transport mechanisms.
Similar discrepancies with regard to peptide expression
have been found recently in cortical and spinal grafts that
developed in oculo (Eriksdotter-Nilsson et al., 87; Henschen
et al., '88) Taken at face value, the results seen in oculo
suggested that the disturbed patterns of peptide staining
might be attributed to the isolation of these transplants
from the environment of the CNS. However, our present
findings indicate that some alterations in peptide expression
exist even in well integrated grafts that develop within
homotopic locations in the CNS. While these grafts contain
some afferent ingrowth from descending fibers, such input is
limited, and may not be sufficient to induce the normal
expression of such peptides. Further support for this
hypothesis comes from recent studies which have shown
differences in calcitonin gene-related peptide (CGRP)
immunoreactivity of hindlimb motoneurons after chronic spinal
cord transection (Arvidsson et al., '89).
Implications for Repair
The functional role of cells in the mature SG of the
spinal cord is a topic still under intense investigation
(reviewed in Willis and Coggeshall, '78; Cervero and Iggo,
'80). The termination of unmyelinated primary afferent


52
fibers in this region provides the basis for theories
concerning its role in the modulation and gating of pain and
reflex functions (Melzack and Wall, '65; Willis and
Coggeshall, '78). Szentagothai ('64a) used Golgi stains and
degeneration technigues to reveal the morphology and
projection patterns of SG neurons and proposed that the SG is
primarily a closed system, dominated by local projection
neurons that extend no more than 2-3 segments. More recent
evidence obtained with axonal tracing techniques has shown
that at least some of these SG neurons can project as far as
the medulla (Giesler et al., '78) and thalamus (Willis et
al., '78). It is now known that axonal projections from the
SG also extend into deeper laminae of the spinal cord as well
(Light and Kavookjian, '88). Therefore, the differentiation
of SG areas within FSC transplants suggests a source of
intrinsic modulatory cells as well as some projection neurons
that may play a role in the formation of a neural relay for
somatosensory information.
The availability of specific characteristics and peptide
markers to identify the substantia gelatinosa of the normal
spinal cord has allowed the identification of patches of SG-
like regions within FSC transplants. While these areas may
represent only a small portion of the total circuitry of the
spinal cord, it is likely that other regions of the embryonic
spinal tissue also differentiate and exhibit characteristics
of homotopic areas. Further anatomical markers for the


53
intermediate and ventral regions of the spinal cord may be
used to address this issue. However, one feature of the
normal spinal gray matter that is rarely seen in these
transplants obtained from Eu rat embryos is the development
of groups of large motoneurons within the grafts. Greater
numbers of motoneurons have been identified in grafts derived
from younger (E12) embryos (Reier et al., '83a). Thus,
selection of different aged donor tissue may be useful for
enriching transplants in either ventral or dorsal neuropil
(e.g. Nothias et al., '89).
It is not known which region of the embryonic neuraxis
is best suited to restore function in the injured spinal
cord. However, successful repair of damaged neural networks
may reguire the reconstruction of certain suprasegmental and
intraspinal circuits. Recent studies have shown that in some
instances, homotopic grafts are innervated in varying degrees
by host serotonergic and primary afferent fibers (Bregman
'87; Reier et al., '85, '86a; Tessler et al., '88). Both of
these axonal systems, as well as many other identifiable
fiber populations, normally project to the SG. Because these
SG-like areas are easily identified within FSC transplants,
and because the afferent innervation of the normal SG is well
characterized, this transplantation model should provide a
valuable opportunity for testing the ability of host axons to
recognize regions of these grafts with similar cytological
and peptidergic characteristics to the SG of the normal


54
spinal cord. Such information can be useful in further
understanding the potential of the grafts to reconstruct
specific circuitries in the injured spinal cord.
The differentiation of at least one region of the normal
spinal cord within FSC grafts suggests that these grafts may
replace populations of intrinsic spinal cord neurons. The
following studies are designed to determine whether these
neurons form projections both within the grafts and between
the transplant and host spinal cord.


CHAPTER 4
AXONAL PROJECTIONS BETWEEN FETAL SPINAL CORD TRANSPLANTS
AND THE ADULT RAT SPINAL CORD:
NEUROANATOMICAL TRACING AND IMMUNOCYTOCHEMICAL
STUDY OF HOST-GRAFT INTERACTIONS
Introduction
In neonatal rats, transplants of fetal spinal cord (FSC)
tissue have been shown to provide an environment conducive to
the elongation of some descending axons through a spinal
injury site (Bregman, '87). In addition, when placed into
hemisection lesions in these newborn rats, FSC transplants
have been shown to improve the development of specific
aspects of hindlimb function as compared with rats with
hemisections only (Kunkel-Bagden and Bregman, '89).
In adult rats, however, there is no evidence to support
the concept of axonal regeneration of descending axons across
fetal spinal cord transplants. Nevertheless, the propagation
of some aspects of ascending and descending information might
be achieved by fetal grafts through the establishment of a
neuronal relay between the rostral and caudal regions of the
recipient spinal cord (Reier et al., '85, '88).
To test this hypothesis, the present study was designed
to identify and characterize patterns of axonal interaction
established between FSC grafts and adjacent regions of the
55


56
host spinal cord. Therefore, the purpose of the first
experiment was to extend preliminary WGA-HRP tracing studies
which had suggested some axonal integration between host and
graft (Reier et al., '86a). A fluorescent retrograde tracer
(Fluoro-Gold) was then used to determine the distribution of
cells contributing to axonal interactions. To complete the
axonal tracing studies, the anterograde transport of the
plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L) was
included to reveal the patterns of the axonal projections
from local host and graft neurons and their relationship to
the host-graft interface. The combination of these three
contemporary axonal tracing techniques offers a unique
approach toward evaluating the local interactions between FSC
transplants and the adjacent regions of the spinal cord.
Complementary information about the ingrowth of specific
populations of host afferents into the transplants was then
obtained in a second group of animals by immunocytochemical
staining of transplant sections with antisera raised against
serotonin (5-HT), oxytocin (Ox), tyrosine hydroxylase (TH)
and calcitonin gene-related peptide (CGRP). Finally,
additional sections from both axonal tracing and
immunocytochemical specimens were stained with antiserum
against glial fibrillary acidic protein (GFAP) to examine the
relationship between host-graft projections across the
interface and the patterns of glial reactivity. Portions of


57
this study have been summarized previously (Jakeman and
Reier, '88) .
Materials and Methods
Animals and Transplantation Surcrerv
A total of 99 female, adult rats received transplants of
FSC tissue according to a modification of previously
described methods (Reier et al., '83a, '86a; Chapter 2).
Each transplant recipient was anesthetized with ketamine and
xylazine, a laminectomy was performed at the T13 vertebral
level, and a cavity of 3 6 mm in length was created in the
left half of the spinal cord. For this study, the lesion was
routinely extended to a full hemisection by removal of both
the lateral and ventral columns. The overlying dorsal roots
were reflected laterally during preparation of the cavity,
and replaced after grafting. Although no effort was made to
intentionally sever or remove the rootlets (Tessler et al.,
'88; Houle and Reier, '89), they were sometimes injured
during the surgical procedure. Once hemostasis was achieved
in the host, the donor tissue was placed into the cavity and
the dura and superficial tissues were closed in layers.
Axonal Tracer Application and Tissue Processing
At post-graft intervals ranging from 6 weeks to 14
months, transplant recipients were re-anesthetized with
ketamine and xylazine and prepared for tracer application.
The region containing the graft and surrounding host spinal
cord was exposed by removing new bone growth and extending


58
the original laminectomy rostrocaudally. After the tracer
was injected, the surface of the cord was washed with
physiological saline and a drop of mineral oil was placed
over the spinal cord to minimize diffusion of the tracer into
the surrounding tissues. The spinal cord was then covered
with a piece of Durafilm and the wound was closed as
described above.
Horseradish Peroxidase (HRP) and Wheat Germ Agglutinin HRP
conjugate (WGA-HRP)
Anterograde and retrograde labeling. A combination of
HRP (Type VI) and WGA-HRP (Sigma Chemical) were used for both
retrograde labeling of cells and anterograde filling of axons
(Mesulum, '82), as described in previous studies. In the
first part of the experiment, mixtures of the two tracers
were applied to transplants (n=16) using a variety of
techniques: (a) Seven rats received pressure injections of a
solution of 20% HRP and 1.0% WGA-HRP using a 1.0 jil Hamilton
syringe or a nitrogen burst picospritzer (Reier et al.,
'86a); (b) Two rats received iontophoretic injections of 2%
WGA-HRP; (c) one rat received a pledget of Gelfoam soaked in
20% HRP and 2.0% WGA-HRP; and (d) HRP and WGA-HRP were
applied to the remaining six rats using crystals dissolved
onto the end of a tungsten wire (Houle and Reier, '88).
For the reciprocal study, HRP and WGA-HRP were applied
to the host spinal cord with a tungsten wire ((d) above,
n=ll). In order to examine the HRP transport characteristics


59
using this method, a similar tungsten wire was placed into
one normal rat at the T13 vertebral level.
HRP histochemistry. After allowing 48 72 hours for
transport of the tracer, the recipients containing HRP and/or
WGA-HRP injections were deeply anesthetized with sodium
pentobarbital and perfused transcardially with 150 ml
heparinized 0.9% NaCl followed by 250 ml fixative (1.0%
paraformaldehyde + 2.5% glutaraldehyde in 0.1 M Sorenson's
phosphate buffer). Tissue blocks, including the transplant
and 5.0 10.0 mm of host spinal cord rostral and caudal to
the graft, were removed. Vibratome sections (50 /urn) were cut
in the sagittal or horizontal plane. The sections were
reacted within 2-4 hours according to the tetramethyl-
benzidine (TMB) protocol of de Olmos et al. ('78). Sections
were then mounted onto gelatin-coated slides and selected
slides were counterstained with 1.0% Neutral Red to reveal
the cytoarchitecture of the host and graft tissues.
Fluoro-Gold (FG)
Retrograde labeling. For the second set of experiments,
a 2.0% solution of FG (Fluorochrome, Inc.; Englewood, CO) was
made in 0.9% NaCl and the solution was then drawn into glass
pipettes (40 50 /Ltm tip diameter) After a dural incision
was made with the beveled end of a 25 gauge needle, the FG
solution was injected into the transplants (n=12) or the host
spinal cord (n=19) using a rapid nitrogen burst (Pico-
spritzer) applied to the end of the glass micropipette. The


60
approximate injected volume of the FG solution was estimated
by measuring the diameter of the "hemisphere" ejected onto a
parafilm sheet and using the approximation (v= (27r(d/2)3)
/3) Volumes of 0.1-1.0 /il were injected into the
transplants and 0.5-1.5 /I into the host tissue. One normal
rat also received an injection of 0.5 nl of FG solution at
the T13 vertebral level for comparison.
Tissue processing. The FG- containing tissue was
processed as described by Schmued and Fallon ('86). At 4
days after the injection, the rats were perfused with saline
followed by fixative containing 4.0% paraformaldehyde and
0.25% glutaraldehyde in 0.1 M phosphate buffer. Spinal cord
blocks containing the transplant and 4.0 mm of the
surrounding rostral and caudal spinal cord were removed and
postfixed in the same fixative for 2 hrs to overnight at 4C.
Vibratome sections of 40 /m were cut in the sagittal plane.
In addition, every sixth section was saved in 0.1 M PBS for
subsequent immunocytochemical detection of GFAP (see below).
In 4 of the rats that received larger injections of FG
into the transplants, six additional tissue blocks were also
sectioned. These included cross sections of the host spinal
cord 4 6 mm rostral and caudal to the transplant,
horizontal sections from cervical and thoracic spinal cord,
and sections of host brainstem and brain. The dorsal root
ganglia from these recipients were embedded in paraffin and
sectioned at 15 /m. The Vibratome and paraffin sections were


61
mounted directly onto gelatin-coated slides. Slides from
paraffin blocks were heated to 37C for 12 hours, and
deparaffinized. All FG slides were cleared in xylene,
coverslipped with Fluoromount (Gurr Bio/medical Specialties;
Santa Monica, CA), and viewed on a Zeiss Axiophot microscope
with fluorescent UV illumination.
Phaseolus vulgaris leucoaqqlutinin (PHA-L)
Anterograde labeling and tissue sectioning. To examine
the patterns of axonal elongation into graft and host
tissues, anterogradely-filled axons were identified by
immunocytochemical detection of PHA-L (Vector Laboratories
Inc.; Burlingham, CA). The tracer was applied by a
modification of the methods of Gerfen and Sawchenko ('84).
The PHA-L was dissolved to 2.5% in 10 mM phosphate buffer (pH
8.0). Glass micropipettes were cleaned with acetone and 100%
ethanol and broken to a tip diameter of 10 15/im. After
exposing the transplant (n= 13) or host spinal cord (n=8) ,
the tracer was applied to the appropriate site by
iontophoresis for 20 minutes using a 5 /A interrupted
positive current (7 sec on, 7 sec off).
After allowing 7-17 days for transport of the PHA-L,
the recipients were perfused as above with fixative
containing 4.0% paraformaldehyde and 0.25% glutaraldehyde.
The cord blocks including the transplant and 4 7 mm of the
surrounding rostral and caudal spinal cord were removed and
postfixed overnight at 4C. Sagittal sections of these


62
blocks were cut at 40 nm on a Vibratome and stored in 0.02 M
potassium phosphate buffered saline (KPBS). Every sixth
section was saved for immunocytochemical staining with
antibodies to GFAP (see below).
Immunocytochemical detection of PHA-L. The remaining
free-floating Vibratome sections were processed for the
identification of cells and processes containing PHA-L. The
sections were first washed in 0.02 M KPBS and incubated for
2-4 hrs in a preblocking bath containing 2.0% normal rabbit
serum and 0.3% Triton X-100. All the sections were then
incubated in goat anti-PHA-L (Vector) diluted 1:5000 in KPBS
for 36 hours at 4C and 2 additional hours at room temper
ature. The sections were rewashed and then processed with
biotinylated rabbit anti-goat IgG (1:225) and Vector Avidin-
Biotin-peroxidase Complex (ABC) as per the supplier's
instructions. The final peroxidase conjugate was reacted
with H202 in the presence of 0.005% DAB. The DAB reaction
was done either with the addition of 0.125% nickel ammonium
sulfate (black reaction product) or in the absence of nickel
(brown reaction product). The nickel-enhanced sections were
counterstained with 0.1% Cresyl Violet or 1.0% Neutral Red
prior to coverslipping.
Histological analysis of anatomical tracers
Mounted serial sections containing the tracer injection
sites were examined to determine the location of the
injection site and the extent of tracer diffusion relative to


63
the host-graft interface. Each specimen was then accepted or
rejected from the study according to specific transport and
diffusion criteria as described in Results. Retrogradely-
filled cells were identified and manually counted in
successive 1.0 mm fields at 125x. Cells which demonstrated
non-specific fluorescence when exposed to rhodamine (510-560
nm) or fluorescein (450-490 nm) microscope filters were not
counted. Each field was counted 3 times and the median value
was accepted. All cell counts were corrected according to
classical methods (Abercrombie, '46). Total cell number was
obtained by assuming an average cell diameter of 40 /m for
graft cells and 50 /nm for host neurons.
The distribution of labeled cells and axons was
determined from drawing tube tracings of darkfield or
brightfield images (HRP and PHA-L) or from photographic
montages of fluorescence micrographs (FG). To determine the
distances of anterogradely labeled axonal projections, a
digitizing tablet and morphometry software (Videoplan;
Kontron, FRG) was calibrated for the appropriate
magnification. Measurements were taken from the drawing tube
illustrations or photomicrographs. Similar methods were used
to document the distances between the injection site and the
outermost zone of tracer diffusion as well as the
relationship of these regions to the host-graft interface.


64
Immunocvtochemical staining and analysis of GFAP
A series of sections (240 jum apart) from recipients with
FG or PHA-L injections was incubated in rabbit polyclonal
antiserum produced against GFAP (gift of Dr. Lawrence F. Eng,
VA Medical Center, Palo Alto, CA) The antiserum was diluted
1:1200, and sections were incubated overnight at 4C.
Detection of the primary antibody was performed according to
the peroxidase anti-peroxidase method (Sternberger, '76) as
described below.
Tracings of the rostral and caudal interface regions for
each section were made using a drawing tube, delineating the
regions containing dense GFAP staining between host and graft
tissue. For specimens with FG injections, the lengths of the
interface and the regions containing glial scar formation
were measured using a digitizing pad and Videoplan software.
The composite Fusion Index (FI) for each interface region was
defined as the average percentage of the interface which was
devoid of dense glial scarring (Houle and Reier, '88).
The density of glial staining was determined for both
graft and surrounding host tissues in 7 recipients. A
program was developed using the Zeiss IBAS image analysis
system (Kontron, FRG) and a high resolution video camera
(DAGE Inc., CCD71). At a viewing magnification of 125x, four
pairs of images from each GFAP stained section (each pair
including a graft region and host gray matter region) were
digitized and converted to binary images. The first field of


65
each pair was segmented interactively by the user to
distinguish glial processes from background as described by
(Bjorklund,H. et al., '83). Based upon the assumption that
non-specific staining was consistent within each section, the
segmentation settings used for this first image were retained
for the second image of the pair. The percent area occupied
by glial profiles was calculated for each field, and values
were obtained for the average glial density within the graft
and host gray matter regions as well as the ratio of
graft/host glial density for each section. Statistical
comparison of graft and host glial density was performed
using the direct-difference Student's t test for paired
samples (Spence et al., '83).
Immuno-staininq For Specific Populations of Host Fibers
Immunohistochemical procedures
Adjacent series of sections from 24 transplant recipients
were stained with polyclonal antisera raised against
neurotransmitters, synthetic enzymes, or peptides found in
specific populations of host fibers (See Table 4-1). Of
this group, 6 recipients were selected from tracer specimens
with unacceptable or failed injections. All of the
recipients were perfused as described above with fixative
containing 4.0% paraformaldehyde and 0.25% glutaraldehyde in
0.1 M Sorenson's phosphate buffer (pH 7.4). Sections
containing the graft and surrounding host spinal cord were


66
cut at 40 /m on a Vibratome and stored in 0.1 M phosphate
buffered saline prior to staining.
TABLE 4-1: LIST OF ANTISERA SOURCE AND DILUTION
Antisera
Source Antibody dilution
5-HT
OX
TH
CGRP
Incstar Corp. 1:3000 overnight
Incstar Corp. 1:5000 overnight
Eugenetech,Inc. 1:750 overnight
Peninsula Labs 1:12000 36 hours
Immunocytochemical staining was performed on adjacent
series of sections using antisera directed against
the following:
5HT-serotonin; OX-oxytocin; TH-tyrosine hydroxylase.
CGRP-Calcitonin gene-related peptide
Free floating sections were processed using the PAP
immunocytochemical procedure (Sternberger, '76). The primary
antisera used in this study were all raised in rabbit (Table
4-1) and diluted in high salt buffer containing 0.3% Triton
X-100 (THSB) After the sections were removed from the
primary antibody solution, they were washed 3 times in THSB,
incubated in rabbit anti-goat IgG at 1:20 for 45 min to 1 hr,
washed again in THSB, incubated in Rabbit PAP 1:50-1:200 for
30 min, and finally washed in phosphate buffered saline (PBS)
or ammonium phosphate buffer. The peroxidase was visualized
with 0.05% DAB and 0.003% H202. Staining of fibers with
anti-5HT and anti-TH were done in the presence of 0.001%
nickel ammonium phosphate to produce a black reaction
product. Sections were then mounted on gelatin coated
slides. Some slides were counterstained with 1.0% Neutral


67
Red or 0.1% Cresyl Violet to reveal nearby cytoarchitecture.
The specificity of antibody staining was evaluated by the
morphological distribution of labeled cells and fibers in the
normal spinal cord.
Analysis of immunocvtochemical results
Sections were first examined for labeled cells within the
graft. Selected sections were then photographed on an
Axiophot microscope or drawn using a Zeiss microscope with
2.5x or 4Ox objective and 12.5 x ocular and 1.0 mag drawing
tube. The distances of fiber ingrowth and gualitative
comparisons of the regions of graft area occupied by stained
fibers were determined from the drawings or photos.
Results
General Transplant Characteristics
Viable transplants were present in 92% of the recipient
rats. Nearly all of the grafts filled the lesion cavity and
showed gross apposition with the surrounding host tissues.
The cellular organization and variability within the host-
graft interface was similar to that described in previous
studies from this laboratory (Reier et al., '86a; Houle and
Reier, '88). With regard to the interface, those sections
counterstained with Cresyl Violet or Neutral Red often
contained regions between the host and graft tissue that were
occupied by small, densely packed cell nuclei resembling
glial cells. In contrast, other regions of the interface
were devoid of an obvious cellular boundary between the two


68
tissues. In these more integrated areas, the only
distinction between host and graft was a transition in the
general cytoarchitectural organization. A similar range of
host-graft fusion was observed in GFAP stained sections.
Neuroanatomical Tracing With HRP and WGA-HRP
Injections into transplants
Solutions of HRP and WGA-HRP were injected or applied to
16 transplants (Table 4-2). The surviving grafts were
classified based upon the histological analysis of the
injection and the extent of tracer diffusion. The injection
sites were examined under darkfield illumination, and the
extent of each was defined as the area containing a purple-
opaque core and the entire surrounding region of orange TMB
reaction product (Fig. 4-1 a,b).
The injection site was restricted solely to the
transplant in six of the recipients (Table 4-2; Groups A, B) .
The two specimens classified into Group A contained the
smallest injection sites, which extended less than 0.5 mm in
maximum diameter. While no labeled cells or axons were
observed in the host spinal cord, these small injections
illustrated the presence of intrinsic graft projections (Fig.
4-la). The majority of retrogradely labeled cells in these
specimens were located within 0.5 mm of the center of the
injection site; however, additional retrogradely filled
neurons were found throughout the grafts. In the four other
recipients (Table 4-2; Group B), the injection sites were


69
TABLE 4-2: HRP/WGA-HRP INJECTIONS INTO FSC TRANSPLANTS
Code
(Post-Graft)
(Interval)
Method3
1
Groupb
Intrin.
proj .
Eff .c
proj .
Aff .d
proj .
HGP6(6 wk.)
Picospritz
A
+
HGP7(5 wk.)
Iontophoresis
A
+
HGP2(6 wk.)
Picospritz
B
+
HGP8(6 wk.)
Tungsten wire
B
+
HGP3(6 wk.)
Tungsten wire
B
+
+
HGP11(6 wk)
Tungsten wire
B
+
+
HGP5(9 wk.)
Picospritz
C
+
HGP10(6 wk.)
Hamilton
C
+
HG3 (2 mo.)
Tungsten wire
C
+
+
+
HGP9(6 wk.)
Hamilton
C
+
+
+ Indicates
evidence of projections from
labeled
profiles
present in host or graft tissue.
a The tracers were applied using one of five procedures
(see Methods).
b Recipients included in analysis were classified according
to the extent of tracer diffusion as follows: A Injection
site < 0.5 mm in diameter and confined to graft; B -
Injection larger than 0.5 mm and confined to graft; C -
Injection site within graft, diffused into or slightly over
interface.
c Efferent projections of graft axons. Anterograde axon
label extended into host spinal cord.
d Afferent projections from host neurons. Retrogradely
labeled cells found in host spinal cord.


Figure 4-1. HRP and WGA-HRP tracing revealed intrinsic
interactions and some projections of graft and host axons.
a) Drawing tube tracings of sequential sagittal sections
through a graft with a small (Group A) injection. The center
of the injection site is shown as solid black and the area
containing dense reaction product with no discernable cells
and axons is represented by the hatched region. The area
outlined by a dotted line represents a high density of
labeled cells and axons, and each individual cell within the
graft is indicated by larger dots.
b) Labeled cells and axons were distributed throughout a
transplant (t; HGP11) to the host-graft interface
(arrowheads) following a larger HRP/WGA-HRP injection into
the dorsal region of a graft.
c)Labeled graft axons coursed parallel to this region of the
host-graft interface, but do not penetrate the host spinal
cord (h).
d-g) Axonal projections formed between host and graft
tissues. Graft efferent projections were identified by
retrograde transport into transplant neurons following
injections into the host spinal cord(d) and anterogradely
labeled axons (white arrows) extending into the host spinal
cord after an injection into the transplant (e). f) Example
of short-distance ingrowth of host axons into a transplant
following an injection made 1.9 mm rostral to a graft, g)
Illustration of the potential for greater axonal interactions
following an injection which diffused across the dorsal
region of the host-graft interface (specimen HH5). Note that
retrogradely filled neurons (*) may represent intrinsic
projections labeled by tracer diffusion. However, labeled
fibers can be seen in this ventral section where the
diffusion does not confuse the host-graft border. Axons
extended across the interface region (i.e. arrowhead) between
host (heavily labeled) and graft (lightly labeled) tissues.
Scale in a = 1.0 mm; b,c = 200 nm; d-g = 100 /m.


71


72
larger than 0.5 mm in diameter but were still confined to the
transplants. An extensive network of intrinsic graft
projections was again evident. Both labeled cells and axonal
profiles were observed throughout the transplants and up to
the interface in all directions (Fig. 4-1 b,c,e).
The transplants in Group B also suggested the presence
of axonal projections between host and graft tissues.
Similar to findings from preliminary studies (Reier et al.,
'86a), retrogradely filled neurons were occasionally found
within the host spinal cord. Efferent projections from graft
neurons were also observed, as anterogradely-filled axons
could be followed across the interface into the host in two
recipients (Fig. 4-le). Unfortunately, the possibility of
additional labeled axons oriented perpendicular to the plane
of section could not be assessed. In contrast, there were
some regions of each host-graft interface where the two
tissues appeared to be separated by a glial partition. In
these regions axons coursed parallel to the interface, but
they did not extend into the host spinal cord (Fig. 4-1 c).
The recipients in Group C had injection sites which were
not confined to the graft. In each case, however, the outer
zone of TMB reaction product extended beyond either the
rostral or caudal border of the graft, while the opaque
center was confined to the graft. At those regions where the
injection extended over the interface, labeled axons and host
neurons were found within 1.0 mm of the host-graft interface,


73
but few labeled cells were observed farther away. This
labeling pattern differed from the pattern observed following
placement of HRP and WGA-HRP into the normal spinal cord (see
Menetrey et al., '85).
Injections into the host spinal cord
In the reciprocal experiment, HRP and WGA-HRP injections
were made both rostral and caudal to the transplants. The
details of the post-graft intervals, injection site location,
and distances between the injections and the host-graft
interface are summarized in Table 4-3. The injections were
completely confined to the host spinal cord in seven animals.
Evidence for axonal interactions between host and graft
tissues was obtained from specimens which contained large (>
1.5 mm radius) injections that extended to within 2 mm of the
interface region. Of the seven specimens, four contained
retrogradely- filled neurons within the transplants (Fig. 4-
1 d) The number of labeled neurons in these grafts ranged
from a single cell to over 30 cells, with most of these
located within 1.0 mm of the host-graft interface.
In addition to efferent projections from transplant
neurons, the larger HRP/WGA-HRP applications also labeled
axons from host cells that had extended processes into the
transplants. Evidence of such axonal ingrowth was observed
in 3 of 7 of the recipients. In each case, the interface
between host and graft tissues was readily apparent as a
distinct border between the dense axonal labeling in the host


74
TABLE 4-3. HRP/WGA-HRP INJECTIONS INTO HOST SPINAL CORD
Code I.S.a
(Post-Graft)
(Interval)
Dist.b
c.-Int.
(mm)
Dist.c
e.-Int.
(mm)
Eff .d
proj .
Aff ,e
proj .
HH3(14 mo.)
rost.
7.0
2.1
+
+
caud.
10.6
6.8


HH4(5.5 mo.
)rost.
1.9
1.0
-
+
HH6(2 mo)
rost.
5.0
3.2


caud.
5.7
4.3

-
HH7(2 mo.)
rost.
2.9
1.4
+

caud.
3.9
2.8

-
HH8(2 mo.)
rost.
2.0
0.6
+
+
caud.
3.7
1.9

-
HH9(6.5 mo)
rost.
2.4
1.2


caud.
2.7
1.7

-
HH11(6 mo.)
rost.
3.0
0.0
+

caud.
6.2
0.0

+ Indicates retrogradely filled cells or anterogradely filled
axons present within the transplant.
a Injection Site. Injection placed either rostral (rost.)
or caudal (caud.) to host-graft interface.
b Measured distance from center of injection site to the
host-graft interface.
c Measured distance from the edge of HRP reaction product
or diffusion to the host-graft interface.
d Efferent projections of transplant neurons. Retrogradely
labeled cells within the graft.
e Afferent projections into the graft. Anterogradely
labeled axons from the host spinal cord.


75
and very sparse axonal profiles in the transplant (Fig. 4-1
f) These ingrowing host fibers were restricted to a
peripheral border of the graft and most terminated within a
half millimeter of the host-graft interface. In contrast,
labeled axons stopped abruptly at the interface in the
remaining recipients.
The possibility of more extensive axonal integration
within the interface region was illustrated in one case in
which an injection had extended over a dorsal region of the
interface. The labeled axons and cell bodies within this
graft were excluded from consideration of host-graft
projections. However, a region ventral to the area of
diffusion was marked by the presence of labeled axons that
could be followed between host and graft tissues (Fig. 4-1
g)
Fluoro-Gold Injections: Distribution of Cells
Injections into the host spinal cord
Fluoro-Gold injections were made into the host spinal
cord of 19 recipients at distances ranging from 1.8 5.0 mm
from the host-graft interface. Injections in seven of the
rats met three criteria: 1) the injections were confined to
the host spinal cord, 2) they showed no evidence of diffusion
into the cerebral spinal fluid (as seen by a high degree of
non-specific fluorescence throughout the spinal cord) or
central canal region, and 3) they were large enough to label


76
a high percentage of host neurons adjacent to the graft.
Results from these animals are summarized in Table. 4-4.
TABLE 4-4: INJECTIONS OF FLUORO-GOLD INTO
THE HOST SPINAL CORD
Code I.S.
(Post-Graft)(mm)
(Interval)
(host)
# labeled3
cells
in graft
Fusion13
Index
(%+sd)
Glialc
Ratio
G/H
FGC-20(8 mo)
3.6
mm
rost.
0
47%(13)
2.3 *
FGC-17(9 mo)
4.3
mm
rost.
38
19%(11)
1.2
FGC-21(8 mo)
2.8
mm
rost.
28
40%(19)
4.7 *
FGC-23(8 mo)
1.5
mm
rost.
8
8%(13)
10.0 *
FGC-13(8 mo)
4.4
mm
caud.
697
51%(20)
3.1 *
FGC-14(5.5m)
3.6
mm
caud.
212
40%(16)
1.2
FGC-30(6 wk)
1.8
mm
caud.
439
27%(21)
1.1
a Total number of retrogradely filled cells within
the transplant. Cell counts corrected according to
the method of Abercrombie (/46).
b Composite Fusion Index: (FI) percentage of the
host-graft interface (closest to injection) which is
devoid of dense glial scar (average of 4 10
sections/transplant).
c Ratio of the percent area occupied by glial
elements. indicates that the glial density within
the graft was significantly higher than that in the
surrounding host tissue (p< 0.01).
The interface between the host gray matter and graft
tissue was characterized by a sharp decrease in the density
of labeled neurons between adjoining host and transplanted
tissue (Fig. 4-2 a) In all seven grafts, there were regions
of fusion between host and graft tissues as identified with
GFAP staining (see below; Fig. 4-2 b) Six of the grafts
contained retrogradely labeled cells. These included three


77
transplants (FGC-17,21,23), each with fewer than 50
retrogradely filled neurons within the graft and three others
(FGC-13,14,30) each with more than 200 labeled cells. In
this sample, the differences between the two groups did not
correspond with the post-grafting interval or the distance
between the injection site and the host-graft interface.
However, more labeled cells were found in the three
recipients with FG placed caudal to the graft than those with
injections placed at rostral levels.
Transplant neurons which projected into the host spinal
cord were distributed throughout the graft tissue (Fig. 4-2
e) Most of these retrogradely labeled neurons were
multipolar and small, measuring 8 20 [im in diameter (Fig.
4-2 c) although larger cells were observed occasionally
(Fig. 4-2 d) Histograms were made for each of these six
grafts to show the distribution of the labeled cells as a
function of distance from the host-graft interface (Fig. 4-
3). In the recipients with few labeled cells (top of Fig. 4-
3), there were more cells within the first millimeter of the
transplant and a decrease in the density of labeled cells
with distance from the host-graft interface. However,
labeled neurons were more evenly distributed in grafts
containing many fluorescent cells, regardless of the absolute
length of the transplants.


Figure 4-2. Identification of graft neurons projecting into
the host spinal cord by retrograde transport of Fluoro-Gold.
a) The interface between host (h) and transplant (t) is
characterized by a marked decrease in density of labeled
neurons.
b) Section adjacent to that shown in a), after staining with
anti-GFAP. A glial scar is present along the dorsal region
of this host-graft interface (bottom half of figure), while
the host and graft tissues are well fused in the ventral
region (between arrowheads).
c) Higher magnification of typical retrogradely labeled cells
within this graft.
d) Example of an occasional large graft neuron that projected
into the host spinal cord.
e) Photo-montage of a sagittal section from specimen FGC-30
to illustrate the distribution of labeled neurons throughout
a transplant following a FG injection into the host spinal
cord (h) The host-graft interface is marked by a white
dotted line.
Scale in a,b,e = 200 /m; c,d = 100 /xm.


79


Figure 4-3. Individual histograms show the distribution of labeled cells
within six transplants with respect to the host-graft interface closest
to the injection site. The corrected neuron total is in parentheses
above each graft. Each bar represents the number of neurons counted
within successive 1.0 mm segments of the graft. Note that the last bar
in each graph may represent less than a full millimeter of graft tissue.


Numbers of Labeled Cells
DISTRIBUTION OF RETROGRADELY LABELED TRANSPLANT
NEURONS FOLLOWING FLUORO-GOLD INJECTIONS
INTO THE HOST SPINAL CORD
FGC-17
(38)
0 to 11 VO *.1-3.0 3D
FGC-13
(697)
FGC-21
(28)
FGC-23
o-to u-1.0 *.1 u> ajo
FGC-30
(439)
0-10 11 LO
Distance from Host-Graft Interface (mm)


82
Injections of FG into transplants
In ten of the recipients with FG injections into the
grafts, the extent of tracer diffusion was confined to the
transplant. While seven grafts had some cells labeled in the
host spinal cord, the numbers of labeled cells ranged from
few host neurons to more than 70 host spinal cord cells and
200 dorsal root ganglion (DRG) neurons.
Figure 4-4 summarizes the location and extent of the
graft injections and the distribution of labeled host
neurons. Five of these FG injections were < 0.5 mm in
diameter (FGC- 2a, H6, H7, H8, H10; Fig. 4-5 a). In these
specimens, nearly all of the labeled neurons were found
within the graft itself. The intrinsic neurons were
concentrated in the region nearest the injection site.
However, some retrogradely labeled cells were found in all
regions of these transplants (Fig. 4-5 b) Labeled neurons
were also found in the adjacent host spinal cord in two of
these recipients. In both cases, the labeled cells were
located within 0.5 mm of the interface zone which was
adjacent to the injection site, and distributed within the
medial or lateral intermediate gray. The greatest number of
labeled host neurons were found in the recipient with an
injection located within 0.2 mm of the host-graft interface
(H10).
In the remaining transplants from this group (FGC-
H9,25,26,27,31) the injection site was larger than 0.5 mm in


Figure 4-4. Distribution of injection sites and labeled neurons
following injections of Fluoro-Gold into transplants. The center panel
contains freehand drawings of a representative section with individual
dots representing cells and their approximate locations within the graft.
Numbers of cells rostral and caudal to the graft are indicated according
to numbers within a range of distances from the interface. DRG= dorsal
root ganglia, totals Left and Right (all grafts were made on the
left).


Animal
(Post-g
Cel Is Rostral
I.) (Distance in
6-4.1 4-2.1 2-1.1
to Transplant
mm)
1.0-0.6 0.5-0
Glial
scar
(FI.Xj
FGC-2a
(2 mo.)
-
-
5
FGC-H6
(2 mo.)
-
-
-
35X
FGC-H7
(2 mo.)
-
-
-
33X
FGC-H8
(6.5 mo.
)
-
-
-
21X
FGC-H9
(6 mo.)
-
-
2
23X
FGC-M10
(5.5 mo.
)
-
-
2 30
42X
FGC-25
(2 mo.)
-
-
-
4
48X
FGC-26
(2 mo.)
-
-
-
-
32X
FGC-27
(14 mo.)
-
-
-
1 3
17X
FGC-31
_
.
.
_
30X
(6 wk.)
Glial
scar
FI.X)1
0-0.5
Cells Caudal
(Distance in
0.6-1.0 1.1-2
to Transplant
mm)
2.1-4 4.1-6
DRG
L R
-
1
-
-
-
35X
-
-
-
-
56X
-
-
-
-
1.0X
-
-
-
-
25X
-
-
-
-
21X
1
-
-
-
49X
58
5
10
13
27
219 2
47X
38
-
-
1
2
6 2
37X
3
-
-
-
-
- -
34X
4
_
_
_
_
7 1
j^nri
00
Numbers in each column represent the number of cells counted in each region. Scale corresponds to approx. 1.0 mm.
1 Fusion Index: X of linear host-graft interface which is deviod of glial scar (average of 4-10 sections per animal).


85
diameter, but was still confined to the graft. All of these
recipients contained some retrogradely labeled host neurons.
Again, the greatest numbers of host neurons were found when
the injection site was within 0.5 mm of either the rostral or
caudal interface. In addition, the majority of labeled host
neurons were found immediately adjacent to the transplants.
In the best case (FGC-25; Figs. 4-5 c-i) the FG injection
extended to 0.2 mm from the caudal interface. Retrogradely
labeled neurons were found throughout host spinal cord caudal
to the graft, while few neurons were found rostral to the
graft. Within the sacral spinal cord (Fig. 4-5 h; i.e. 4 -
6 mm away), labeled cells were distributed throughout the
dorsal and intermediate gray regions (laminae I-VII) as well
as regions of the ventral horn. In addition, more cells were
located ipsilateral than contralateral to the graft. In this
animal, a large number of ipsilateral DRG cells were found as
well (Fig. 4-5 i) However, sections from cervical and
thoracic spinal cord as well as brainstem and brain contained
no labeled cells in any of the recipients. The absence of
retrograde labeling of descending fibers was in contrast with
the pattern observed following a similar injection into a
normal rat.
Comparison of FG and HRP/WGA-HRP injections in normal rats
To compare the patterns of retrograde cell labeling using
the two tracers and the present injection technigues, each of
these tracers was also injected into a normal rat at the T13


Figure 4-5. Retrogradely labeled host neurons following FG
injections into transplants.
a,b) Intrinsic labeled neurons following a small FG injection
(< 0.5 mm diameter) into a transplant (t) a) The majority of
labeled neurons were located in the immediate vicinity of the
injection, which was adjacent to the rostral host-graft
interface (arrowheads). Cells were also found at the far end
of the graft (b) at a distance of 3 mm from the injection.
c-i) Distribution of retrogradely labeled cells following a
larger injection (dotted line in c) near the caudal border
(arrowheads) of specimen FGC-25. d) Most fluorescent labeled
cells (white arrows) were found immediately adjacent to the
host-graft interface. Inset: Verification of the graft
border was obtained in each case by viewing the interface
region with darkfield optics, using the location of blood
vessels (*) as landmark points, e) GFAP staining of one
section from this specimen illustrates a high degree of
fusion between host and graft (between arrowheads). f) A
patch of retrogradely labeled neurons found approximately 3
mm caudal to the transplant, g) Transverse section of the
sacral spinal cord contains four retrogradely labeled
neurons, h) Composite drawing of 40 sections from the host
sacral spinal cord 4 6 mm caudal to the transplant. Left
in the figure is ipsilateral to the graft, right is
contralateral. The photograph in g: was obtained from the
region enclosed in the box. i) Labeled dorsal root ganglion
neurons ipsilateral to the transplant.
Scale in a,c,d,e = 200 /urn; b,f,g,i = 100 m.


87


88
vertebral level. Histological analyses revealed neurons
within the cervical and thoracic spinal cord, brainstem
nuclei, DRG, and cortex of both animals. Similar to findings
from grafted animals, many more labeled cells were found in
each of these regions in the rat which received the pressure
injection of FG than the rat with the HRP/WGA-HRP injection.
PHA-L Injections: Patterns of Axonal Projections
Further definition of the axonal trajectories of host and
graft neurons was made with iontophoretic injections of PHA-
L placed either into transplants or the adjacent host spinal
cord (Table 4-5). The injection region was easily defined by
the presence of darkly filled perikarya. In some specimens,
the injection site contained only a small number of labeled
neurons (Fig. 4-6 a,b). Larger injection sites extended up
to 0.5 mm in diameter.
Projections of transplant neurons
Of the 13 recipients with PHA-L injections into the
transplants, five had acceptable axonal labeling (Table 4-5,
top). All of these injections revealed an extensive network
of axonal projections within the graft. Axons near the
injection site exhibited abundant branching and the nearby
neurons were surrounded by terminal enlargements. Survey
electron micrographs of one such graft showed labeled
terminal boutons throughout the graft neuropil (Jakeman and
Reier, unpublished observations). While the greatest density


89
TABLE 4-5: INJECTIONS OF PHA-L INTO FSC TRANSPLANTS
OR THE HOST SPINAL CORD
Code I.S.a Region6 Axonal Projections
(Post-Graft)
(Interval)
PHAL-8 (2 mo.)Graft Ventral Efferent axons can be traced
across interface. Injury filled
host axons also evident.
PHAL-10(10 wk)Graft dorsal-
medial
Efferent axons in rostral
dorsal horn and intermediate gray
regions of host.
PHAL-23(6 mo) Graft dorsal- Efferent axons present in
medial dorsal horn, dorsal tracts and
intermediate gray, and in rostral
intermediate gray regions.
PHAL-15(6
mo)
Graft
dorsal-
lateral
Efferent axons innervate
host lateral motoneurons.
PHAL-32(lOwk)
Graft
caudal
No Efferent axons.
PHAL-11(6
wk)
Host
0.5 mm
caudal
Afferent axons extending
< 0.2 mm into graft.
PHAL-21(6
mo)
Host
1.5 mm
caudal
Afferent axons extending
< 0.3 mm into graft.
PHAL-33(10wk)
Host
1.5 mm
rostral
All axons stop
interface.
a t
PHAL-27(12mo)
Host
0.5 mm
caudal
Many cells and
throughout graft*, most
1.0 mm of interface.
axons
within
PHAL-16(6
mo)
Host
2.0 mm
No afferent axons.
a Injection site. Iontophoretic injection into
graft or host spinal cord.
b Region of graft with injection or distance and
direction between injection in host spinal cord and
the host-graft interface.
* Evidence of both anterograde and retrograde
transport.


90
of fibers was found within the injection region, labeled
axons were found in all areas of the grafts.
Three general axonal projection patterns were seen within
the transplants. Within graft regions containing densely
packed neuronal cell bodies, the labeled axons branched
extensively (Figs. 4-6 b,c). In contrast, where few
perikarya were found, labeled axons remained mostly
unbranched and followed a relatively straight trajectory
(Fig. 4-6 b). Finally, at the interface regions, individual
axons often coursed parallel to the host-graft border, and
occasionally extended into the host neuropil (Fig. 4-6 d).
Labeled axons could be followed into the host spinal cord
in four of these grafts. The pattern of axonal outgrowth was
slightly different for each specimen. In one case (PHAL-8),
the injection was placed ventromedially within the transplant
and resulted in injury to fibers in the ventral white matter
of the host. The appearance of these injury-filled axons was
distinct. These axons were very heavily labeled, and they
exhibited bulbous terminal swellings. Similar profiles were
not found in any of the other animals in this group.
Following injections into two other grafts (PHAL-10, 23),
a high density of labeled axons were seen within the
transplants, especially near the dorsal region of the host-
graft interface. Labeled fibers extended across the
interface and into the adjacent rostral or caudal dorsal horn
(laminae I-III) of the host (Fig. 4-7a).


Figure 4-6. Intrinsic PHA-L labeled profiles following a small injection
into transplant PHAL-8.
a) Sagittal section providing orientation. The injection was placed in
the ventro-medial region of the transplant (t) (double arrowheads).
b) Enlargement from a nearby section of the same transplant. Labeled
axons within acellular regions exhibit little branching (arrows), while
extensive branching is observed around counterstained cell bodies
(arrowheads).
c) Within the transplant, but farther from the injection site, labeled
axons exhibit an extensive pattern of projections.
d) Enlargement of the interface region outlined in the box in (a) .
Labeled axons (arrowheads) within the transplant travel parallel to the
host graft interface. One axon then turns abruptly to enter the adjacent
host spinal cord (h).
Scale in a = 500 /m; b,c,d = 100 m.


Full Text
xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID ESB58HNF7_28ZJN4 INGEST_TIME 2012-03-13T14:33:42Z PACKAGE AA00009071_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


UNIVERSITY OF FLORIDA
3 1262 08554 3337


AXONAL INTERACTIONS BETWEEN FETAL SPINAL CORD
TRANSPLANTS AND THE ADULT RAT SPINAL CORD
By
LYN BURRELL JAKEMAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1990

ACKNOWLE DGEMENTS
This undertaking is one which could not have been
completed if it were not for the help of several people.
First and foremost has been my mentor, Dr. Paul Reier, for
whom I have the utmost respect and admiration for his advice
and role as teacher and scientist. He contributed an
invaluable amount of time and patience to my training and
taught me the importance of maintaining a balance between
persistence and flexibility; a lesson that is vital in
research, writing, and communication in science. Additional
appreciation is extended to each member of my committee —
Drs. Barbara Bregman, John Munson, Roger Reep, Lou Ritz, Don
Stehouwer, and Chuck Vierck — for continued support and
constructive criticisms regarding my work.
The daily progress was made more pleasant with the superb
technical and organizational assistance of Barbara 0'Steen,
Minnie Smith, and Regina Reier, who kept track of my loose
pieces of paper and saved me months of effort. Additional
help was provided by the secretarial and support personnel in
the Departments of Neuroscience and Neurosurgery. Gratitude
is also extended to fellow graduate students, especially
Denise and Greg, who taught me to believe in myself when the
game seemed lost. Oversight of animal care and use was kept
ii

by Dr. Dan Theele, D.V.M.. Finally, instruction in the
concepts of morphometric image analysis was made available by
Mr. Dan Williams.
An incredible amount of emotional support has been
extended over the past six years by family and friends;
especially my sister Barb, my parents, and my in-laws.
However, the greatest appreciation is extended to my husband,
David T. Lee, who provided both support and encouragement,
and also helped with editorial suggestions, technical
assistance, and scientific criticism.
The last personal acknowledgements go to Bob Yant and
Jim Sutherland, who provided needed reminders of the
importance of developing a progressive outlook with regard to
spinal cord research.
Financial support for equipment, supplies, and the much
needed student assistantship was provided by NIH grants 22316
and NS72300 to P.J. Reier, The Mark F. Overstreet Fund for
Spinal Cord Regeneration Research, The Center for
Neurobiological Sciences (NIMH grant MH15737), and the
Department of Neuroscience. Additional travel support was
provided by the American Paralysis Association.
iii

TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS ii
LIST OF ABBREVIATIONS vi
ABSTRACT vii
CHAPTERS
1 INTRODUCTION AND BACKGROUND 1
Spinal Cord Injury 1
Treatments to Minimize Functional Loss 2
Promotion of Axonal Regeneration or
Sprouting 4
Fetal Neural Transplants and Spinal
Cord Repair 8
Development of a Neural Relay Across a
Spinal Injury Site 11
Experimental Goals 12
2 GENERAL METHODS 14
Experimental Animals 14
Preparation of Lesion Cavities 15
Preparation of Donor Tissue 16
Transplantation Procedure 16
Post-Operative Care 17
3 DIFFERENTIATION OF SUBSTANTIA GELATINOSA-
LIKE REGIONS IN INTRASPINAL TRANSPLANTS
OF EMBRYONIC SPINAL CORD TISSUE 18
Introduction 18
Materials and Methods 2 0
Results 23
Discussion 43
IV

CHAPTERS
4 AXONAL PROJECTIONS BETWEEN FETAL SPINAL CORD
TRANSPLANTS AND THE ADULT RAT SPINAL CORD:
A NEUROANATOMICAL TRACING AND IMMUNOCYTO-
CHEMICAL STUDY OF HOST-GRAFT INTERACTIONS 55
Introduction 55
Materials and Methods 57
Results 67
Discussion 116
5 INTERACTIONS BETWEEN INJURED CORTICOSPINAL
TRACT AXONS AND FETAL SPINAL CORD
TRANSPLANTS 129
Introduction 129
Materials and Methods 132
Results 139
Discussion 168
6 SUMMARY AND CONCLUSIONS 180
Construction of a Relay Across a FSC Graft 180
Specificity Issues 182
Possible Role of FSC Grafts in Segmental and
Long-Tract Functions 187
Future Directions 191
Conclusions 192
REFERENCES 194
BIOGRAPHICAL SKETCH 218
v

LIST OF ABBREVIATIONS
CGRP - calcitonin gene-related peptide
CNS - central nervous system
CST - corticospinal tract
DAB - diaminobenzidine
FG - Fluoro-Gold
FI - Fusion Index
FSC - fetal spinal cord
GABA - gamma-aminobutyric acid
GFAP - glial fibrillary acidic protein
HRP - horseradish peroxidase
MBP - myelin basic protein
NT - neurotensin
Ox - oxytocin
PAP - peroxidase anti-peroxidase
PHA-L - Phaseolus vulgaris leucoagglutinin
PNS - peripheral nervous system
TH - tyrosine hydroxylase
WGA-HRP- wheat germ agglutinin, conjugated to HRP
5-HT - serotonin (5-hydroxy-tryptamine)
vi

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
AXONAL INTERACTIONS BETWEEN FETAL SPINAL CORD
TRANSPLANTS AND THE ADULT RAT SPINAL CORD
By
Lyn Burrell Jakeman
May, 1990
Chairman: Dr. Paul J. Reier
Major Department: Neuroscience
One approach to spinal cord repair involves the
transplantation of homotopic fetal neural tissue into the
site of a spinal lesion. As a strategy with potential toward
eventual functional recovery, this approach includes two
primary objectives. The first goal is to replace intrinsic
spinal cord elements at the lesion site. The second is to
provide a means for reconstructing functional continuity
between the separated rostral and caudal ends of the spinal
cord. Accordingly, the following series of experiments
establish an anatomical setting for functional repair.
To evaluate the potential for replacement of intrinsic
spinal cord tissue, intraspinal transplants of fetal spinal
cord (FSC) tissue were examined using conventional light and
electron microscopic techniques and immunocytochemical
staining. The normal substantia gelatinosa was compared with
vii

distinct myelin-free regions of the grafts and the two were
found to contain similar cytological characteristics and
similar patterns of peptide staining.
Axonal projections between (FSC) grafts and the host
spinal cord were identified using a variety of neuro-
anatomical tracing and immunocytochemical techniques. The
presence of host fiber growth into the grafts, an extensive
pattern of intrinsic graft projections, and efferent growth
of axons into the host were consistent with the hypothesis
that fetal transplants may establish a neural relay across a
spinal cord lesion site.
Finally, interactions between the long myelinated fiber
tracts of the spinal cord and FSC grafts were examined using
the corticospinal tract (CST) as a model system. Injured CST
axons were observed in direct apposition to FSC tissue at the
host-graft interface, and CST axons were also seen extending
into the transplants.
Together, the results indicate that FSC transplants can
be used to restore anatomical continuity through 1) the
differentiation of intrinsic spinal cord regions at a lesion
site and 2) the development of axonal interactions between
host and graft tissues in the adult rat spinal cord. These
observations provide a basis for future studies to examine
the functional integration of FSC transplants, as well as a
model for investigating the biology of axonal elongation.
viii

CHAPTER 1
INTRODUCTION AND BACKGROUND
Spinal Cord Injury
Traumatic injury to the spinal cord begins a sequence of
events that result in the degeneration of spinal cord tissue
and subsequent loss of sensory and motor function. In order
to understand the clinical picture, the underlying
pathophysiology has been studied extensively in experimental
models of spinal cord injury (Dohrmann, '72; Sandler and
Tator, '76; Wagner et al., '78; Windle, 'SO; de la Torre,
'84; Beattie et al., '88; Fehlings and Tator, '88). The
progressive degeneration of spinal cord tissue is initiated
by a primary insult to neurons and vascular elements in the
region of the injury (Sandler and Tator, '76; Balentine and
Paris, '78a,b). The acute events then activate a cascade of
chemical reactions due to ischemia (Osterholm, '74), ionic
conductance changes (Young et al., '82; Stokes et al., '83),
and the accumulation of cytotoxic free oxygen radicals
(Demopoulos et al., '80) at the lesion site. Collectively,
these responses lead to the formation of a cavitated lesion
area within the spinal cord.
The chief functional consequences of this spinal lesion
are described in most basic neuroscience textbooks (e.g.,
1

2
Daube et al., '78). Briefly, the local degeneration of gray
matter leads to a loss of intrinsic and projection neurons at
the lesion site. This can result in the permanent
dysfunction of segmental actions, the extent of which is
dependent upon the size, type, and level of the resulting
lesion. The second outcome from the degeneration of spinal
cord tissue is the loss of functional continuity between
rostral and caudal levels of the spinal cord. Additional
compromise of autonomic and propriospinal systems underlie a
further level of complexity to the extent of functional
recovery (e.g. Cole, 788) .
Recently, some emphasis has also been placed upon the
recognition of long-term functional effects at distant
regions of the central nervous system, as a result of
denervation and retrograde changes after axotomy (Beattie et
al., '88). Specifically, compensatory changes such as
collateral sprouting, receptor super-sensitivity, and
behavioral substitution, are likely to contribute to chronic
adaptive and maladaptive functions after spinal cord injury.
Treatments to Minimize Functional Loss
To date, most therapeutic approaches designed to spare
function or improve recovery after spinal cord injury have
been based upon the progressive pathology described above.
One such strategy includes pharmacological treatments aimed
at reducing or reversing the pathological process by
interfering with the cascade of secondary biochemical events

3
(reviewed in de la Torre, '80; Beattie et al., '88). For
example, corticosteroids have been used to stabilize membrane
changes and reduce edema after injury. Antioxidants have
been reported to promote functional recovery by reducing the
consequences of lipid peroxidation (Saunders et al., '87;
Anderson et al., '88). Other drugs have been employed to
improve functional characteristics attributed to axonal
transmission of long-tract fibers by altering ionic channel
conductances (Blight and Gruner, '87).
In addition to pharmacological approaches, surgical
procedures, including the removal of external compression
sources and stabilization of the vertebral segments
surrounding the injury site, are used to reduce the extent of
functional loss after injury (Ransohoff, '80) .
Alternative efforts toward maximizing functional recovery
in later stages after injury have been directed at restoring
modulatory influences to spared regions of the spinal cord.
A number of animal experiments have suggested that the
circuitry below a lesion can be manipulated through the
application of pharmacological agents. Experiments using the
adrenergic agonist, clonidine, have demonstrated that
activation of catecholamine receptors can improve segmental
stepping behavior in cats in the first week after a spinal
injury (Forssberg and Grillner, '73). In addition,
application of bicuculline, a gamma-aminobutyric acid (GABA)
antagonist, has been associated with improvements in spinal

4
stepping after chronic spinal cord injuries. This latter
effect appears to be mediated by enhancing segmental
influences on the remaining circuits (Robinson and
Goldberger, '86). Conversely, the clinical use of the GABA
agonist, baclofen, has had beneficial effects in reducing
extreme spasticity in spinal cord injured patients by
enhancing the inhibitory influences upon segmental reflexes
(Bloch and Basbaum, '86). Thus, many proposed
pharmacological treatments for the chronic spinal injury
patient rely upon establishing a balance of receptor-mediated
neuronal activities at the segmental level.
Promotion of Axonal Regeneration or Sprouting
Over the past decade, advances in therapeutic strategies
have resulted in a progressive reduction of mortality with a
concurrent improvement in the quality of life following
spinal cord injury (e.g. Bloch and Basbaum, '86; Green and
Klose, '89). Nevertheless, a continued emphasis must be
placed upon research efforts to promote the repair of the
spinal cord and to restore functions normally mediated by
both segmental and long-tract systems.
Since neurogenesis is essentially lacking within the
adult mammalian central nervous system (CNS), there is no
mechanism for spontaneous replacement of neurons after
injury. Repair of the spinal cord is, therefore, dependent
upon the regeneration or sprouting of axons from existing
neurons. An emphasis in mammalian regeneration research has

5
been placed upon the evaluation of differences between the
peripheral nervous system (PNS), where injured axons
regenerate and are able to successfully reinnervate their
target tissues, and the CNS, where axons do not regenerate
such that they return to their original post-synaptic sites
(reviewed in Clemente, '64; Guth, '75; Bernstein et al., '78;
Kiernan, '79).
The aspects of these systems which have been contrasted
most often are the regenerative or sprouting capacity of the
axons and the permissive or inhibitory nature of their
environment. This interaction between axons and surrounding
cells received early attention by Ramon y Cajal ('28), who
observed small regenerative sprouts following experimental
injury to spinal cords of young kittens. The sprouts failed
to persist after two weeks, and no functional recovery was
observed. More recently, Aguayo and his colleagues confirmed
Ramon y Cajal's notion that the environment can profoundly
influence regeneration. By implanting pieces of peripheral
nerve into the brain and spinal cord, they demonstrated that
CNS neurons can extend and maintain long axonal sprouts
within the peripheral environment (David and Aguayo, '81;
Richardson et al., '82). Other researchers have provided
evidence of varying degrees of synaptic reorganization
following injury within the adult brain and spinal cord
♦
(e.g., Cotman and Nieto-Sampedro, '85; Goldberger and Murray,
'88; Steward, '89b).
Together, this work has inspired

6
renewed encouragement in the field of CNS and spinal cord
repair.
Strategies aimed at promoting the regeneration of spinal
cord axons include changes to the CNS microenvironment as
well as methods that might stimulate axonal sprouting and
elongation. Guth et al. ('85a) demonstrated that axons
within the spinal cord will not extend into a vacant lesion
site; instead, they must encounter a cellular terrain for
successful elongation. In addition, the establishment of a
dense meshwork of glial and connective tissue elements at the
lesion site may also present a problem for growing axons.
The concept of fibro-glial scarring as an impenetrable
barrier to elongating axons was championed by Ramon y Cajal
(7 28) , and has been a topic of debate for the several decades
(reviewed in Reier et al., '83b; Reier and Houle, 788) . With
this in mind, several approaches have been taken to prevent
or reduce the extent of glial/fibroblastic scar formation
after injury and thus promote axonal elongation. The
invasion of fibroblasts can be minimized by using closed
spinal cord injury models such as the weight-drop contusion
or clip compression approaches. In addition, pharmacological
agents have been applied to prevent the formation of scar
tissue at a lesion site (e.g., Windle et al., '52; Guth et
al., '85b). These results have suggested that axons may
extend a short distance into a spinal lesion where such a
scar is reduced.

7
Some recent investigations have been directed at
promoting the elongation of injured spinal cord axons using
different methods. One such approach involves the
implantation of cultured cells into a lesion (Siegal et al.,
'88; Wrathall et al., '89). These studies reflect a desire
to apply substances known to induce axonal elongation in
vitro to the injured spinal cord. The results suggest that
the effects may be more complex in vivo because of many
uncontrolled variables. Finally, the application of
electrical fields has also been investigated as a means to
increase the distance of axonal elongation (Borgens et al.,
'86, '87). These results, however, are inconclusive and
await further confirmation.
It is important to note that throughout the history of
spinal cord regeneration research, one recurrent difficulty
has been the uneguivocal identification of regenerating axons
or collaterals into or across the site of a spinal cord
lesion. Using conventional histological procedures, such as
silver staining, the only way to verify that axons crossing
a lesion site represent true fiber growth has been to ensure
that the initial lesion constitutes a complete transection.
This injury model, however, neither provides the most
conducive environment for regeneration nor represents a
clinical injury. Furthermore, even in the case of a complete
transection, other important factors, such as the absolute
distance of fiber elongation, the origin and terminations of

8
axons, or patterns of reinnervation, cannot be verified using
these techniques.
Fetal Neural Transplants and Spinal Cord Repair
Reports of some degree of functional recovery following
transplantation of fetal CNS tissue into the brain (e.g.,
Gash et al., '80; Bjorklund and Stenevi, '84; Dunnett et al.,
'85; Buzsaki and Gage, '88) suggested that embryonic neural
tissue might promote neuronal repair following injury or
disease. These findings have led to the application of
similar transplantation strategies in the spinal cord. The
first intraspinal fetal grafting studies resulted in low
transplant survival rates as compared to similar experiments
in the brain (Nygren et al., '77; Patel and Bernstein, '83;
Das, '83; Commissiong, '84). Such difficulties served to
underscore the extreme pathological consequences of spinal
cord injury. It has been proposed that many of the grafts
failed to survive because they did not integrate with the
parenchyma of the injured spinal cord (Das, '83).
Following improvements in surgical procedures and careful
selection of donor tissue ages (Nornes et al., '83; Reier et
al., '83a; Reier, '85), more recent studies of intraspinal
transplantation have met with greater success. One approach
involves injecting suspensions of dissociated embryonic
brainstem cells caudal to the site of injury (Nygren et al.,
'77; Nornes et al., '83; Privat et al., '86). The focus of
this strategy is to restore supraspinal modulatory influences

9
to denervated regions below the level of a spinal lesion. An
emphasis has been placed upon the descending monoaminergic
systems which have been associated with the modulation of
segmental reflex and locomotor circuitries. These studies
have indicated that the injured spinal cord can be
reinnervated by grafted embryonic brainstem neurons.
Furthermore, such grafts can mediate some types of reflex
change after spinal injury or chemical denervation (Buchanan
and Nornes, '86; Moorman et al., '88; Privat et al., '86,
' 89) .
While transplants placed below a lesion site may
contribute to the replacement of modulatory influences,
recovery of sensation and voluntary motor capacities will
require applications that restore continuity at the lesion
site. Therefore, an alternative approach toward spinal cord
repair involves the transplantation of fetal tissue directly
into a lesion cavity (e.g. fetal spinal cord (FSC) grafts)
(Reier et al., '83a, '85,'86a; Houle and Reier, '88).
This approach differs from the transplantation of tissue
caudal to an injury, and it directly addresses three major
consequences of spinal cord injury. First, the presence of
embryonic tissue at the site of a lesion may provide trophic
influences to prevent degenerative changes after injury. For
example, fetal grafts placed into the injured spinal cord or
cortex of neonatal rats have been associated with a
significant reduction in the extent of cell death that is

10
characteristic of such lesions in the infant CNS (Bregman and
Reier, '86; Haun and Cunningham, '87). In addition, there
has been at least one suggestion that the presence of fetal
tissue in a spinal lesion cavity may prevent degeneration of
white matter fiber tracts in adult recipients (Das, 786).
Secondly, fetal tissue may serve to replace segmental
neurons at the level of the lesion. Interactions of
intrinsic and projection neurons may be important for the
repair of propriospinal influences after injury. This
approach for the replacement of damaged or diseased neurons
forms the basis for the transplantation of fetal neural
tissue into neurodegenerativo and excitotoxin-induced lesions
in the brain.
The main objective of the intralesion grafting paradigm,
however, is to provide a neuronal framework that could
ultimately subserve functional integration of the rostral and
caudal spinal cord segments. In this context, the hypothesis
has been advanced that embryonic CNS tissue might be used to
promote spinal cord repair by providing a bridge for axons to
extend across the lesion (Nornes et al., '84; Reier, '85).
While results from recent studies suggest that descending
axons can extend across a FSC graft in newborn rats, there
is no evidence to date to indicate that injured CNS axons in
the adult will bridge a fetal graft to reinnervate their
original spinal cord target regions. However, an alternative
possibility is that transplants may establish a neuronal

11
relay pathway across a spinal cord lesion (Johnson and Bunge,
'83; Nornes et al., '84,* Reier, '85, Reier et al.,'88;
Jakeman and Reier, 788).
Development of a Neural Relay Across a Spinal Injury Site
The concept of a neural relay has been discussed at its
most basic level by Shepard ('88). The three components of
the "synaptic triad" that form any neuronal circuit include
input neurons, intrinsic neurons, and projection neurons.
Complex variations of these components form the basis for
local circuits throughout the CNS (Rakic, '76). Several well
studied, integrative relays are found within the organization
of the dorsal and ventral horns of the normal spinal cord.
These circuits are responsible for the transmission and
integration of sensory and descending influences and
segmental reflex pathways.
The hypothesis of a relay with regard to FSC transplants
implies that the transmission of ascending and descending
information across a spinal cord lesion may be mediated
through interactions between host and graft tissues. These
interactions may take the form of afferent and efferent
projections between the surrounding host spinal gray matter
and fiber tracts and the intrinsic circuitry of the graft.
In the absence of axonal projections across the host-graft
interface, a relay circuit might be constructed by
interactions between axons which persist at the host-graft
interface and dendritic projections of host or grafted

12
neurons (Das, '83; Mahalik et al., '86; Clarke et al., '88b).
Alternatively, the relay may be more complex, as dictated by
differences in the relative growth and functions of different
host fibers. In the latter instance, the monoaminergic input
may serve to provide a modulatory influence upon the more
local host-graft interactions.
Experimental Goals
Despite efforts spanning over more than a decade of
research, the mechanisms that underlie various examples of
functional recovery following transplantation in the adult
brain are still unknown. Several recent review papers have
proposed a spectrum of possible mechanisms. In general, it
appears that functional changes following fetal neural
grafting may be obtained in a variety of ways in each model
system (Bjorklund et al., '87; Dunnett and Bjorklund, '87;
Gage and Buzsaki, '89). To better understand these models
and to test the capacity of the CNS for reorganization after
injury or disease, a recent emphasis has been placed upon
defining the anatomical correlates of host-graft
interactions. Through careful examination of the patterns of
axonal projections between transplants and the host CNS, the
strengths, potential mechanisms of behavioral improvement,
and the limitations of the grafting models can be assessed
more accurately.
Likewise, an important first step in determining a
potential functional role of FSC transplantation in the

13
spinal cord is to define the anatomical basis for integration
of host and graft tissues. Preliminary studies of
interactions between such transplants and the injured adult
rat spinal cord have indicated that some axonal projections
can form between the tissues (Reier et al., '85, '86a).
However, the purpose of these earlier studies was to identify
the general feasibility of transplantation and the
integration of such transplants into the adult spinal cord.
The objective of the following work is to identify, in
more detail, the anatomical basis for the role that FSC
transplants might play in the repair of the injured spinal
cord. The use of a variety of complementary neuroanatomical
methods will serve to define several aspects of host-graft
interactions, including the differentiation of regions in the
grafts and the development of axonal projections between
graft and host tissues. From these studies, information will
be obtained concerning the nature of neuronal relay
possibilities for transmission of information across the site
of a spinal cord lesion. In addition, the FSC transplant
model will be used to examine some of the biological issues
concerning axonal elongation within the adult spinal cord.

CHAPTER 2
GENERAL METHODS
A number of spinal cord injury models have been used to
evaluate potential strategies for intervention and repair.
These include discrete lesions of specific fiber tracts,
chemical axotomy of fiber types, blunt contusion or
compression injuries, and complete or partial transection
models (reviewed in de la Torre, '84; Beattie et al., '88;
Das, '89). The present investigations have employed a model
of transplantation into partial transection cavities prepared
by aspiration immediately before grafting (acute lesions).
The transplantation methods used throughout these studies are
similar to those detailed in previous reports (Reier et
al.,'83a,'86a). Each experimental design has employed only
minor modifications of the procedures described below.
Experimental Animals
Adult, female, inbred Sprague-Dawley rats were used
throughout these studies. All of the rats were obtained from
Zivic-Miller Laboratories (Allison Park, PA) and weighed 200-
3 00 grams at the start of the experiments. The rats were
housed two per cage in the University of Florida animal
resources facility (accredited by the American Association of
Laboratory Animal Caretakers), according to the guidelines
14

15
established by the National Institutes of Health (Publication
number 85-23). They were examined daily by a veterinarian
and/or veterinary technician for general health conditions
and for any post-operative complications due to the spinal
cord injury. All surgical procedures were performed with
instrument preparation in anti-microbial benzalkonium
chloride solution (Zepharin HC1, Winthrop Breon Laboratories)
and 95% Ethanol.
Preparation of Lesion Cavities
The transplant recipients were anesthetized with
intramuscular injections of a mixture of ketamine (Ketaset,
60 - 80 mg/kg; Aveco Co.) and xylazine (Rompun, 10 mg/kg;
Mobay Corp.). The spinous processes were then exposed by
longitudinal incisions of the overlying musculature.
Bleeding from the superficial muscles was controlled with
epinephrine-impregnated cotton pellets (Gingipak, Belport
Co.,Inc.). A laminectomy was then performed at the
appropriate vertebral level using fine-tipped rongeurs, and
the host spinal cord was exposed by a longitudinal incision
of the dorsal meninges just lateral to midline. Using
iridectomy scissors and a glass micropipette with light
aspirative pressure, a cavity of 3 - 6 mm in length was
created in the parenchyma of the spinal cord, and bleeding
was controlled with small pellets of gelatin sponge (Gelfoam,
Upjohn Co.) soaked in a saline solution containing bovine
thrombin (Thrombostat, Parke Davis).

16
Preparation of Donor Tissue
For each transplantation session, a pregnant rat (E.4,
embryonic day 14; E0 = day of insemination) was anesthetized
with 4.0% chloral hydrate (400 mg/kg, i.p.). A laparotomy
was performed and individual donor embryos (approximate
crown-rump length of 12 - 13 mm) were removed as needed and
placed into a standard tissue culture medium (Hank's Balanced
Salt Solution). The embryonic spinal cord tissue was then
dissected by removal of overlying skin layers and the
detachment of the spinal ganglia. The spinal cord was
stripped of embryonic meningeal layers and used for
transplantation within one hour of removal from the mother.
At the end of the transplantation session, the pregnant rat
was euthanized with an intracardiac injection of sodium
pentobarbital (Butler Co.).
Transplantation Procedure
Once hemostasis was achieved in the host, either one or
two pieces of donor spinal cord tissue were cut to
approximate the length of the prepared cavity. The donor
tissue and a small amount of tissue culture medium were
placed into the cavity with a flame-tipped micropipette.
After placement of the grafts, the excess tissue culture
medium was removed with light manual suction. A single
piece of hydrocephalus shunt film (Durafilm; Codman
Shurtleff, Inc.) was usually cut to a size just larger than
the lesion cavity and positioned directly above the graft.

17
The dural incision was closed with 10-0 interrupted sutures
and the spinal cord was then covered with a second piece of
synthetic dural covering. The overlying muscles were then
sutured in layers using 4-0 silk and the skin incision closed
with wound clips (Fisher) .
Post-Operative Care
Following surgery, all rats received a subcutaneous
injection of long-acting penicillin (Dual-Pen; Tech America;
75,000 U.). They were carefully monitored and kept on a
heating pad or under a mild heat lamp until recovery from
anesthesia. Within 48 hours, the rats were returned to the
animal care facility where they were fed rat chow and water
ad libitum and maintained under a 12 hour light and 12 hour
dark schedule.
In the subsequent weeks after surgery, approximately 10%
of the animals initiated mild autotomy of the ipsilateral
forelimb or hindlimb (corresponding to lesion level) and were
treated with daily application of veterinary autophagic
repellent (Chewguard, Summit Hill Labs.). Those few rats
that failed to respond to such treatment within a few days
were sacrificed shortly thereafter and included in the data
analysis. This consideration contributed to the range of
post-graft survival times (post-graft intervals) referred to
throughout the work.

CHAPTER 3
DIFFERENTIATION OF SUBSTANTIA GELATINOSA-LIKE
REGIONS IN INTRASPINAL TRANSPLANTS OF
EMBRYONIC SPINAL CORD TISSUE
Introduction
Intracerebral grafts of fetal CNS tissue have been shown
to compensate for a variety of functional deficits in
experimental animal models. This may occur through the
replacement of neuronal circuitries or neurotransmitters or
by the production of neuronotrophic substances within the
host brain (reviewed in Bjorklund and Stenevi, '84; Sladek
and Gash, '84; Bjorklund et al., '87; Dunnett and Bjorklund,
'87) . The degree to which such grafts can subserve the
functions of the damaged brain region may depend upon the
ability of the grafted tissue to differentiate and integrate
with the host neural circuitry. Thus, several investigators
have examined the extent to which fetal neural transplants
will differentiate and exhibit organotypic characteristics
when placed into homotopic and heterotopic sites within the
CNS (e.g., Jaeger and Lund, '80; Alvarado-Mallart and Sotelo,
'82; Kromer et al. , '83; Eriksdotter-Nilsson et al., '86;
Harvey et al., '88; Sorensen and Zimmer, '88a,b). From these
studies, it is clear that grafted regions of the CNS exhibit
18

19
different degrees of differentiation depending upon the age
of the embryonic donor tissue and the region from which it
is obtained.
In recent years, there has been some enthusiasm for the
application of fetal CNS tissue transplantation techniques to
the problem of spinal cord injury (e.g. Das, 'S3;
Commissiong, '84; Privat et al., '86; Reier et al.,'85, '86a;
Houle and Reier, '88). Together, these reports indicate that
intraspinal transplantation results in the survival and
integration of embryonic donor tissue in both neonatal and
adult recipients. One approach involves the introduction of
fetal neurons into the lesion site (Reier, '85; Reier et al.,
'85). In particular, homotopic grafts placed into the site
of a spinal lesion may serve as a source of specific
intraspinal neuronal populations with an inherent potential
for integrating with synaptic circuits above and below the
injury. Some degree of homotypic differentiation has been
indicated by studies in which fetal spinal cord transplants
were placed into intracerebral or intraspinal cavities (Reier
and Bregman, '83; Reier et al., '85, '86a,b) . In these
initial investigations, distinct myelin-free regions were
observed in matured grafts, leading to the hypothesis that
these unmyelinated areas corresponded to the superficial
dorsal horn — especially the substantia gelatinosa (SG) —
of the normal spinal cord. This region of the spinal cord is
easily identified in normal tissues based upon the paucity of

20
myelinated fibers, the small cells and compact nature of the
neuropil, and the density of peptide staining. Thus, the SG
provides a model system to determine whether grafts of FSC
tissue can differentiate and replace specific regions of the
normal spinal cord.
In the present study, the myelin-free regions of fetal
rat spinal cord grafts were examined in more depth to
determine the extent to which these areas develop cellular
and ultrastructural characteristics of the normal SG when
placed into lesion cavities in the adult rat spinal cord.
In addition, immunocytochemical methods were used to examine
the distribution of neurotensin-immunoreactive elements,
normally observed only within the superficial dorsal horn
(Portions of this study have been summarized in Reier et al,
'85; Jakeman et al., '89).
Materials and Methods
Animals and Surgical Procedures
Ten adult rats received intraspinal implants of Eu rat
spinal cord tissue. The surgical procedures were similar to
those described in previous reports (Reier et al., '83a,
'86a; Chapter 2, this volume). Each rat was anesthetized
with ketamine and xylazine and a laminectomy was performed at
the T13 vertebral (approximately I^- Lj spinal) level. An
aspirative lesion cavity, 3-4 mm in length, was created at
the exposed site. The lesion was then extended to include
either an extensive dorsal funiculotomy or lateral

21
hemisection. Whole segments of fetal spinal cord, 3-4 mm in
length, were dissected from donor embryos and introduced into
the lesion cavities (Reier et al., '83a).
Light and Electron Microscopy
At 1 to 2 months after transplantation, 5 recipients were
anesthetized with a lethal dose of sodium pentobarbital and
perfused through the heart with 0.9% NaCl followed by 5.0%
glutaraldehyde and 4.0% paraformaldehyde in 0.1M phosphate
buffer. Following the perfusion, segments of tissue
including the transplant and surrounding spinal cord were
then excised and divided into several transverse or
longitudinal slices. The specimens were subsequently
osmicated, dehydrated, and embedded in EM Bed 812 (Electron
Microscopy Sciences) for plastic thick sectioning. Regions
of the grafts classified as "SG-like" at the light
microscopic level were trimmed, and thin sections were
surveyed at the ultrastructural level using a Zeiss EM10C
electron microscope.
Immunocvtochemistrv
At similar intervals, the remaining intraspinal graft
recipients (n=5) were perfused with 4.0% paraformaldehyde in
0.1M Sorenson's phosphate buffer (pH 7.4). Tissue blocks
including the transplants were excised and prepared for
Vibratome (40 ¿¿m) sectioning. Adjacent 40 iim sections were
processed for the presence of myelin basic protein (MBP)- or
neurotensin (NT)- like immunoreactivity with the indirect

22
peroxidase anti-peroxidase (PAP) method (Sternberger, '76)
using primary antisera raised in rabbits. The antisera to
NT was obtained from Immunonuclear Corp. (Stillwater, MN.)
and the antiserum to MBP was provided by Dr. L. F. Eng (Palo
Alto, CA., VA Med Center). For PAP staining, tissue sections
were incubated overnight at room temperature in primary
antiserum diluted to 1:2000 with a solution of 0.3% Triton
X-100 in phosphate buffered saline (PBS). All incubations
were performed in the presence of 5% normal goat serum. The
sections were washed several times in PBS and then incubated
in goat anti-rabbit IgG (Cooper Biomedical or Sternberger-
Meyer) diluted 1:10 with the antiserum diluent for 45-60
minutes at room temperature. After a rinse with PBS, the
sections were incubated for 45 minutes in rabbit PAP (Cooper
Biomedical or Sternberger-Meyer) diluted 1:50. Following
several washes in PBS and 0.05M Tris buffer (pH 7.6), the
immunocytochemical reaction product was developed in a 0.05M
Tris buffer solution containing 0.05% 3,3'-diaminobenzidine
hydrochloride (DAB; Sigma) and 0.003% H202. Antibody
specificity was verified by the anatomical distribution of
immunoreactive elements in normal spinal cord tissue and by
the absence of immunoreactive elements when primary antibody
was replaced with normal serum alone.
Correspondence Between Plastic and Immunocvtochemistrv
In three specimens used for immunocytochemistry, 100 nm
vibratome sections were obtained to correspond to the

23
sections stained with antisera to MBP and NT. These sections
were osmicated and dehydrated as described above, and
embedded between vinyl slides with EM Bed 812. From these
sections, 2 ¡ira. sections were cut on an LKB ultramicrotome and
stained with 1.0 % Toluidine Blue. Similar 40 /¿m and 2 /urn
sections were also obtained from the thoracic spinal cord of
2 normal rats to illustrate normal characteristics and
staining patterns within the substantia gelatinosa.
Results
Light Microscopy and Cvtoloaical Characteristics
Sections of normal rat spinal cord, when stained with
antiserum directed against myelin basic protein (MBP),
exhibit a characteristic pattern of myelin distribution.
Specifically, the long fiber tracts appear densely stained,
while moderate staining reflects the presence of myelinated
fibers coursing throughout most of the central gray matter.
The most striking feature of this preparation, as also seen
with conventional myelin stains, is that region within the
dorsal horn which corresponds to the cytoarchitecturally
defined substantia gelatinosa (SG). This region stands out
against the background of intense myelin immunoreactivity due
to the paucity of myelinated axons (Fig. 3-1 a,b).
Fetal spinal cord transplants were stained with anti-MBP
to examine the differentiation of the grafts. All of the
transplants examined with this technique were heavily

Figure 3-1. Identification of myelin-free areas.
a) Pattern of PAP staining with antiserum against MBP in a transverse
section of a normal rat thoracic spinal cord, showing the myelin-free
regions in the superficial dorsal horn (arrows).
b) Higher magnification of Anti-MBP staining in a normal dorsal horn,
illustrating the large myelinated fibers which often traverse the
superficial laminae.
c) Anti-MBP staining of a 2 month intraspinal transplant (t) and
surrounding host intermediate gray (h]G) and lateral white matter (hg)
(Horizontal section). Three myelin free patches are seen at the edge of
the transplant (arrows).
d) Enlargement of boxed region in (c). Myelinated axons (arrow) cross
through this otherwise unmyelinated area. Dotted line highlights the
host/graft interface.
Scale in a,c = 200 /im; b,d = 50 /im.

25

26
myelinated as indicated by areas of very dense staining. In
addition, large regions exhibiting moderate immuno-
reactivity were evident. Finally, the transplants usually
contained one or more areas which were conspicuous due to the
marked absence of MBP-like staining (Fig. 3-1 c,d). These
myelin-free areas typically assumed a convoluted configu¬
ration within the grafts, and appeared as either single or
multiple patches or long strips of neuropil depending upon
the plane of section. In many cases, these regions were
located near the periphery of the grafts (Fig. 3-1 c) ;
however, some myelin-free areas were located more centrally.
Myelinated axons frequently curved along the surfaces of the
unstained regions, and often small bundles of anti-MBP
stained processes traversed the myelin-free zones in a radial
fashion, reminiscent of the pattern of myelinated primary
afferents projecting to deeper layers of the gray matter in
the normal spinal cord (Fig. 3-1 b,d).
With the perspective derived from MBP-stained grafts,
examination of toluidine blue-stained sections of FSC
transplants revealed areas that corresponded to patterns of
MBP- immunoreactivity (Fig. 3-2 a,c; Fig. 3-3 b) . The 2 /¿m
sections contained regions of extensive myelination, as well
as numerous myelin-deficient areas within the graft tissue.
To determine whether the myelin-free areas identified
within the grafts by immunohistochemistry or toluidine blue
staining were indeed equivalent, adjacent sections were

Figure 3-2. Comparison of MBP and toluidine blue stained
sections.
a) Toluidine blue stained semi-thick section within an
intraspinal FSC transplant showing myelinated regions (m)
containing larger neurons, and unmyelinated patches (outlined
in arrowheads) with smaller neurons and processes.
b) MBP- stained section reveals a myelin-free region
(arrowheads) near the host-graft interface in another
recipient. Several regions of the graft lack myelin, yet this
region corresponds to an area containing small, tightly
packed neurons and processes in (c) . Transplant (t) and host
gray matter (hIG) are labeled.
c) Adjacent toluidine blue stained section from the same area
(within arrowheads) which is occupied by small cells and
processes.
Scale in a,c = 50 /xm; b= 100 ¿xm.

28

29
processed so that for each MBP-stained section there was a
corresponding 100 ¿xm section embedded in plastic. In these
examples, zones that failed to show MBP immunoreactivity were
closely in register with homogenous unmyelinated areas in the
adjacent toluidine blue stained section (Fig. 3-2 b,c).
Further examination of these regions in plastic sections
revealed other similarities between the myelin-free areas of
FSC grafts and the normal SG laminae. As in the normal
substantia gelatinosa (Fig. 3-3 a,c), the unmyelinated zones
of matured transplants consisted of numerous small cells (7 -
15 iJ.m) characterized by a thin rim of cytoplasm surrounding
a prominent nucleus. The nuclei of these cells, as of those
in the normal substantia gelatinosa, were round or oval and
often exhibited large clefts (cf., Fig. 3-3 c,d). These
cells were qualitatively distinct from the larger neurons
(14-50 /xm diameter) that were found within the myelinated
regions of the transplants. In fact, the presence of larger
neurons within the unmyelinated areas was very rare. It was
interesting to note that some of the larger cells within the
myelinated regions were closely apposed to the border of the
unmyelinated zones. These general cellular relationships
were similar to the approximation of laminae III and IV
neurons with the normal SG.
In addition to features common to both the normal SG and
the myelin-free graft regions, there were some differences

Figure 3-3. Cytology of the normal SG and SG-like regions in FSC grafts.
a) A 2 fim toluidine blue stained transverse section from the normal adult rat dorsal
horn. Laminae are designated in roman numerals as laminae I-V and the SG as lamina II.
b) An unmyelinated region from an Eu intraspinal graft (t) adjacent to the host white
matter (h). Several aspects of these regions resemble those of the normal dorsal horn,
although the divisions between unmyelinated patches (see arrowheads) suggest that the
graft region has assumed a serpentine orientation. Note larger neurons (n) in close
approximation to these regions in this figure. The interface is indicated by the
dotted line.
c) SG neurons in the normal spinal cord at a higher magnification. Neurons within
the SG are tightly packed and contain large nuclei with prominent indentations
(arrowheads). Note the parallel orientation of axons in transverse section.
d) In myelin-free regions of the transplants, the neurons are similar, although the
processes often lack the longitudinal orientation characteristic of the normal dorsal
horn.
Scale in a,b=50 /im; c,d=10 /Ltm.

31

32
between the two regions. This was particularly evident with
regard to the distribution of cells and neuritic processes.
In the intact spinal cord, SG neurons can be differentiated
into two layers (i.e., Hi and IIo; Ralston, '79;
Szentagothai, '64a). However, no obvious cytoarchitectural
lamination was seen in these regions of the transplants. In
addition, many small, circular neuritic profiles were seen in
transverse sections of the normal SG, thus reflecting their
orientation parallel to the longitudinal axis of the spinal
cord (Fig. 3-3 c) . In contrast, the neuritic processes in
the myelin-free areas of the grafts seemed more randomly
organized, and sectioned profiles assumed many orientations.
Electron Microscopy
Neurons within the normal substantia gelatinosa were
generally spheroid (Fig. 3-4 a) or fusiform (Fig. 3-4 b) in
shape. The perikarya ranged from 8 - 2 0 /¿m in diameter, and,
as seen with the light microscope, they usually contained a
large nucleus within a narrow rim of cytoplasm. These cells
were embedded in a neuropil that primarily consisted of
tightly packed, small unmyelinated axons and small to
intermediate-sized dendrites. The axons were often organized
into bundles or fascicles which were more evident when the
tissue sections were cut in the transverse plane. Many of
the synapses identified within the SG were axo-dendritic
in nature, although other synaptic types were found. In

Figure 3-4. Ultrastructure of the normal substantia
gelatinosa.
a) Transverse section contains a neuronal cell body (n) and
the compact neuropil containing abundant axo-dendritic
synapses (ad) interspersed with longitudinal bundles of
unmyelinated processes (arrows). Large glomerular axonal
processes were often observed (star).
b) Oblique section through the SG region of another normal
specimen. The fascicles are more difficult to discern than in
(a) .
Scale in a,b=2.5 /xm.

34

35
addition, large glomerular complexes were evident in the
substantia gelatinosa of these normal sections as well.
A survey of the unmyelinated regions within FSC
transplants in low power electron micrographs revealed many
characteristics similar to the normal SG (Fig. 3-5). The
neurons in these areas were also small and contained large
nuclei with prominent indentations. The cells were closely
spaced, and were surrounded by compact neuropil consisting of
small axons (0.1 - 0.3 /¿m) and intermediate-sized dendrites
(0.4 - 1.6 /iin). Occasionally, small bundles of unmyelinated
processes were seen which resembled the fascicles in the
normal SG. However, many of the axons and dendrites were
more randomly oriented (Fig. 3-5, 3-6). Except for an
occasional swollen neuritic profile containing lysosomes and
degenerating mitochondria, axonal and dendritic processes
did not display irregular cytological characteristics. In
some transplants, hypertrophic astrocytic processes were
observed particularly near the periphery of the grafts (Fig.
3-5, 3-6) .
Many of synaptic contacts observed within the
unmyelinated graft regions were axo-dendritic, with axo¬
axonic synapses occasionally being present as well. In
addition, a rare axo-somatic synapse could be found in the
normal and graft myelin-free areas. The boutons within the
myelin-free regions usually contained aggregates of small,
agranular vesicles (Fig. 3-6). Usually, the vesicles were

Figure 3-5. Low power electron micrograph of an SG-like
region in an E14 intracerebral transplant.
The small neurons in these regions were similar to the normal
SG regions in size, shape, and the presence of nuclear
clefts. An axo-dendritic (ad) and axo-somatic (as) synapse
are shown. While some unmyelinated axons traveled in
fascicles (arrows), most axonal and dendritic processes
assumed a variety of orientations, and appeared to lack some
of the organization of the normal spinal cord. Large
filamentous glial processes (*) were sometimes seen in these
grafts.
Scale
2.0 /j.m.

37

Figure 3-6. Higher magnification of SG-like regions in an
intraspinal transplant.
The vast majority of synapses were axo-dendritic (ad), and
vesicles were usually clear (agranular) and round (arrows),
although flattened vesicles and dense-cored vesicles
(arrowheads) were also observed. Astrocytic processes were
sometimes present (*) .
Scale = 0.5 nm.

39

40
round, rather than flattened, and some terminals contained
small dense-cored vesicles. Similar presynaptic structures
were also seen in the normal SG. However, the scalloped
terminals and related glomeruli characteristic of normal
primary afferent innervation of the SG were not found within
the grafts examined in this study.
Neurotensin-like Immunoreactivitv
When sections of normal spinal cord were reacted with
antisera to neurotensin (NT), staining was restricted to the
lamina II region of the superficial dorsal horn, where it was
seen in two distinct layers of very fine processes (Fig. 3-
7 a; Seybold and Elde, '82). Labeled axonal profiles were
not found in any other region of the normal spinal cord, and
no immunoreactive cell bodies were found in these sections.
Staining of FSC transplants with antisera raised against
NT revealed regions throughout the grafts which contained
small immunoreactive processes. Many of these patches
corresponded with myelin-free regions in neighboring sections
stained with anti-MBP (Fig. 3-7 b,d). However, in contrast
to the normal spinal cord, NT staining within the transplants
did not form two distinct bands. Other differences were also
observed between the patterns of NT staining and the normal
SG. In addition to the patches of fibers that corresponded
to MBP-free regions of the grafts, some NT fibers were
distributed throughout the myelinated areas of the
transplants. Furthermore, NT-like cells were also found

Figure 3-7. Neurotensin immunoreactivity in normal spinal cord and intraspinal
transplants.
a) Transverse section through the substantia gelatinosa of a normal spinal cord. NT-
like immunoreactivity is restricted to small fibers in this region of the spinal cord.
Labeled axons are separated into two distinct bands (arrows).
b) Sagittal section through a FSC transplant (t) and the adjacent host spinal cord
(h) after staining with anti-MBP. A region of the graft near the interface (between
h and t) exhibits a lack of myelin staining.
c) Cells containing NT-like immunoreactivity were found throughout transplants, but
they were not observed in the normal adult spinal cord.
d) Sagittal section adjacent to that shown in (b), after staining with anti-NT. The
unmyelinated region contains a lightly stained patch of NT-like immunoreactivity
containing very fine NT-like fibers. Similar axonal profiles were never seen within the
deeper laminae of normal spinal cord sections, suggesting that the axons in this
section have extended a short distance into the host spinal cord from within the
transplant.
Scale in a,c = 50 nm; b,d = 100 ¿xm.

42

43
within the grafts (Fig. 3-7 b). These cells were small and
multipolar in shape, and were often found in groups of two or
three.
Neurotensin immunoreactive fibers were often observed at
the periphery of the transplants. Interestingly, in some
examples, these labeled fibers could be followed across the
host-graft interface, where they appeared to innervate
ventral regions of the host gray matter. This region does
not normally contain neurotensin fiber ingrowth.
Discussion
In previous descriptions of matured intracerebral and
intraspinal transplants of FSC tissue, regions of graft
neuropil were identified based upon the absence of myelinated
fibers (Reier et al., '85, '86b). The indication that these
regions might reflect some organotypic differentiation was
examined further in an experiment in which pregnant rats were
injected with tritiated thymidine on either day E12 or Eu.
Labeled donor tissue was removed from fetuses in one uterine
horn and transplanted; fetuses in the contralateral horn were
left to complete gestation (Reier et al., 783a) . At one
month post-transplantation, autoradiography indicated that
the nuclei of neurons in the myelin-free regions of these
transplants exhibited the same relative degree of labeling as
did the nuclei of those cells present in the superficial
laminae of the intact spinal cords of the littermates of the
donor fetuses (Reier et al.,
'83a,'86b).

44
It thus appeared that the myelin-deficient regions in the
fetal spinal cord grafts were the counterparts of the normal
substantia gelatinosa. These general criteria, though
intriguing, did not provide sufficient proof of the exact
nature of the myelin-free areas in the grafts. In this
context, it is well recognized that other characteristics
make this region distinct from the rest of the gray matter in
the intact spinal cord. In particular, the abundance of
small cells led Rexed ('52) to make the distinction of lamina
II of the cat spinal cord, and similar studies have shown
this region within the rat spinal cord as well (Molander et
al., '89) . Examination of the ultrastructure of this region
has also served to define the types of processes and synaptic
profiles that distinguish the substantia gelatinosa (lamina
II) from the surrounding marginal layer (lamina I) and lamina
III (Ralston, '79? Szentagothai, '64a). Additional
identifying features have been noted through the use of
immunocytochemical techniques, which have demonstrated a
variety of peptide-containing cells and fibers (reviewed in
Seybold and Elde, '80; Gibson et al., '81; Hunt, '83;
LaMotte, '86). With these characteristic cytological and
immunocytochemical features as a basis for comparison, the
present study has provided additional evidence in support of
the organotypic differentiation of substantia gelatinosa-like
regions in transplants of fetal spinal cord tissue.

45
Mvelin-free Areas and Peptidergic Elements
Previous studies have demonstrated regions of dense
immunoreactivity obtained with antibodies to several peptides
which are normally associated with the substantia gelatinosa
(Reier and Bregman, '83; Reier et al., '85). Patches of
tissue within the transplants stained heavily with antibodies
to met- and leu-enkephalin, somatostatin, and substance P.
These patches have been identified within FSC transplants
placed into the adult brain or neonatal and adult spinal
cord. In addition, similar findings have been reported
regarding the differentiation of regions of dense peptide
staining in FSC transplants that develop in oculo (Henschen
et al., '88).
In this study, a comparison has been made between the
myelin-free regions of FSC transplants and the SG of the
normal spinal cord. Standard light and electron microscopic
observations revealed many similarities between these areas,
especially with regard to cell and process sizes and the
compact nature of the neuropil. With regard to peptide
staining patterns, an emphasis was placed upon the
distribution of NT- containing fibers within the transplants.
As described for other peptides, regions within the FSC
transplants contained distinct patches of NT-like fibers that
corresponded to unmyelinated regions of the graft. In the
same spinal cord tissue rostral and caudal to the grafts,

46
however, NT-containing axons were found only within the
substantia gelatinosa region of the spinal cord.
Developmental Implications
The observation that fetal spinal cord tissue can exhibit
some degree of organotypic development is consistent with
reports that transplants of tissue from various embryonic
brain regions can achieve cytoarchitectural and
ultrastructural characteristics corresponding to those of the
homologous areas in the intact CNS (e.g., Kromer et al., '79,
'83; Lund and Harvey, '81). Fetal spinal cord tissue has
also been shown to exhibit some cytoarchitectural or
immunocytochemical characteristics resembling the normal
dorsal horn when grown in tissue culture (Naftchi et al.,
'81; Sobkowicz et al., '68) or in oculo (Henschen et al.,
'85). The present findings show that the differentiation of
the myelin-free areas also occurs when FSC tissue grafts
develop within the adult spinal cord.
In related studies currently in progress, similar
unmyelinated regions have been observed in cell suspension
grafts of rat FSC within the contused rat spinal cord
(Winialski et al., '89) and in solid piece grafts of cat
fetal spinal cord tissue in the adult cat spinal cord
(Anderson et al., '89; Reier et al., in preparation). These
regions have thus far been identified on the basis of 2 /urn
thick plastic sections and anti-MBP stained sections of the
grafts. Additional observations suggest that the myelin-free

47
areas may also be analogous to small fiber-free areas
revealed with immunocytochemical staining using polyclonal
antibodies raised against the phosphorylated form of the
heavy neurofilament protein (see silver staining patterns in
Lund and Harvey, 781). However, one recent report has
indicated that SG-like regions are not seen following
injections of cell suspensions from E12 - E13 fetal rat spinal
cord into the ibotenic acid lesions of the lumbar spinal cord
(Nothias et al. , '89). This difference may reflect the
different donor ages used (Kromer et al., '83), however, SG-
like areas have been identified in grafts of E12 rat spinal
cord tissue placed into the rat brain (Reier et al., '83a).
Thus, while the procedures used in this study were not
applied by Nothias et al., it may be possible to observe SG-
like regions within their grafts placed into ibotenic acid
lesions by utilizing specific markers such as the antisera
raised against dorsal horn peptides or MBP.
The presence of a dorsal horn component in FSC grafts is
likely to be related to the developmental timing of this
region of the spinal cord. Evaluations of spinal cord
histogenesis in the rat (Alvardo-Mallart and Sotelo, '82?
Nornes et al., '74) have indicated that there is a peak at
approximately E15-E16 in the generation of neurons which
ultimately comprise the dorsal horn. These cells then
migrate with the majority of neuroblasts reaching the
presumptive dorsal horn region two days later. The

48
maturation of the SG-like areas in these fetal spinal cord
grafts must therefore occur after transplantation since donor
tissue in these experiments was obtained at Eu-E15. These
considerations pertaining to cell birthdates and onset of
migration also suggest that the clustering of small neurons
into the SG-like regions may be due to the persistence of
intrinsic recognition cues which influence the aggregation of
these cells during normal development (see also Kromer et
al., '83). Related studies have also shown that these
intrinsic cues are also retained if the graft is placed into
heterotopic sites, or when it is entirely isolated from host
afferent inputs by transplanting the fetal spinal cord with
the surrounding meninges attached (Jakeman et al., '89).
Anomalous Features of the Substantia Gelatinosa-Like Regions
While many features of the myelin-free regions of FSC
grafts reflect a homology with the normal SG, it is clear
that the correspondence was not perfect. Several aspects of
these areas represent a departure from the normal
organization of the mature superficial dorsal horn. For
example, the myelin-free graft regions lack the precise
orientation and formation of a dorsolateral cap shape with
cells organized in discreet layers. In addition, the
definition between outer and inner layers of lamina II
observed in the normal SG was not observed in either 2 /¿m
plastic sections or with by immunocytochemical staining with
antiserum to NT. Finally, while these graft regions

49
contained some bundles of unmyelinated axons, the parallel
longitudinal arrangement of neuronal processes characteristic
of the normal dorsal horn was absent from the myelin-free
regions of the grafts.
It is likely that some of these differences are related
to specific aspects of the grafting procedure, such as the
initial orientation of the graft tissue, donor age, and
changes in the precise timing of developmental cues (Kromer
et al., '83; Stenevi et al., '76). In addition, the
topography of some of these transplants may be distorted by
spatial restraints, which can contribute to the
organizational differences observed. The differentiation of
organotypic regions within an abnormal cytoarchitectural
framework has also been observed in other types of fetal CNS
transplants (e.g. Kromer et al., '83; Mufson et al., '87;
Sorensen and Zimmer, '88b).
Another factor that may contribute to the atypical
features observed here is the relatively deafferented state
of the transplant. As noted in our electron microscopic
results, the SG-like regions in these grafts lacked the
synaptic terminals characteristic of primary afferent
innervation. A qualitative analysis of the synaptic
composition of these grafts revealed similarities in
ultrastructure to the deafferented dorsal horn, specifically
in the absence of organized glomerular complexes (Rethelyi
and Szentagothai, '69; Coimbra et al.,
'74) .
While such

50
complexes have been found in dorsal regions of FSC grafts
intentionally innervated by primary afferent fibers (Itoh
and Tessler, '88) , SG-like areas deeper within the
transplants receive few such afferents.
Innervation of FSC transplants from adult host fibers is
largely restricted to the periphery of the grafts (Chapter
4). Thus, the developing grafts may also be lacking much of
the descending modulation present in the normal spinal cord.
Therefore, rather than being indicative of aberrant
development, the atypical features recorded may instead
reflect the normal development of SG-regions in circumstances
of decreased afferent input. Such a situation would not only
alter the patterns of migration and lead to cytoarchitectural
differences, but might also prevent the normal expression of
neuropeptides and neurotransmitters.
In the present study, differences were observed between
the pattern of neurotensin-like immunoreactivity in the FSC
grafts and in the normal SG. Specifically, NT-positive cells
and fibers were observed throughout the grafts, while only
fibers were found in the normal spinal cord, and those were
restricted to the SG region. However, application of
intraventricular colchicine treatment and better fixation
methods have been used to identify NT-containing cells and
fibers in the normal spinal cord. These studies have
indicated that NT-like cells can be found in lamina I and V-
VII in addition to the SG in normal rats (Gibson et al., '81;

51
Seybold and Elde, '82; Miller and Seybold, '87) . Thus, the
presence of these cells throughout FSC transplants in the
absence of such treatments may reflect alterations in peptide
expression or axonal transport mechanisms.
Similar discrepancies with regard to peptide expression
have been found recently in cortical and spinal grafts that
developed in oculo (Eriksdotter-Nilsson et al., ' 87; Henschen
et al., '88) . Taken at face value, the results seen in oculo
suggested that the disturbed patterns of peptide staining
might be attributed to the isolation of these transplants
from the environment of the CNS. However, our present
findings indicate that some alterations in peptide expression
exist even in well integrated grafts that develop within
homotopic locations in the CNS. While these grafts contain
some afferent ingrowth from descending fibers, such input is
limited, and may not be sufficient to induce the normal
expression of such peptides. Further support for this
hypothesis comes from recent studies which have shown
differences in calcitonin gene-related peptide (CGRP)
immunoreactivity of hindlimb motoneurons after chronic spinal
cord transection (Arvidsson et al., '89).
Implications for Repair
The functional role of cells in the mature SG of the
spinal cord is a topic still under intense investigation
(reviewed in Willis and Coggeshall, '78; Cervero and Iggo,
'80). The termination of unmyelinated primary afferent

52
fibers in this region provides the basis for theories
concerning its role in the modulation and gating of pain and
reflex functions (Melzack and Wall, '65; Willis and
Coggeshall, '78). Szentagothai ('64a) used Golgi stains and
degeneration technigues to reveal the morphology and
projection patterns of SG neurons and proposed that the SG is
primarily a closed system, dominated by local projection
neurons that extend no more than 2-3 segments. More recent
evidence obtained with axonal tracing techniques has shown
that at least some of these SG neurons can project as far as
the medulla (Giesler et al., '78) and thalamus (Willis et
al., '78). It is now known that axonal projections from the
SG also extend into deeper laminae of the spinal cord as well
(Light and Kavookjian, '88). Therefore, the differentiation
of SG areas within FSC transplants suggests a source of
intrinsic modulatory cells as well as some projection neurons
that may play a role in the formation of a neural relay for
somatosensory information.
The availability of specific characteristics and peptide
markers to identify the substantia gelatinosa of the normal
spinal cord has allowed the identification of patches of SG-
like regions within FSC transplants. While these areas may
represent only a small portion of the total circuitry of the
spinal cord, it is likely that other regions of the embryonic
spinal tissue also differentiate and exhibit characteristics
of homotopic areas. Further anatomical markers for the

53
intermediate and ventral regions of the spinal cord may be
used to address this issue. However, one feature of the
normal spinal gray matter that is rarely seen in these
transplants obtained from Eu rat embryos is the development
of groups of large motoneurons within the grafts. Greater
numbers of motoneurons have been identified in grafts derived
from younger (E12) embryos (Reier et al., '83a). Thus,
selection of different aged donor tissue may be useful for
enriching transplants in either ventral or dorsal neuropil
(e.g. Nothias et al., '89).
It is not known which region of the embryonic neuraxis
is best suited to restore function in the injured spinal
cord. However, successful repair of damaged neural networks
may reguire the reconstruction of certain suprasegmental and
intraspinal circuits. Recent studies have shown that in some
instances, homotopic grafts are innervated in varying degrees
by host serotonergic and primary afferent fibers (Bregman
'87; Reier et al., '85, '86a; Tessler et al., '88). Both of
these axonal systems, as well as many other identifiable
fiber populations, normally project to the SG. Because these
SG-like areas are easily identified within FSC transplants,
and because the afferent innervation of the normal SG is well
characterized, this transplantation model should provide a
valuable opportunity for testing the ability of host axons to
recognize regions of these grafts with similar cytological
and peptidergic characteristics to the SG of the normal

54
spinal cord. Such information can be useful in further
understanding the potential of the grafts to reconstruct
specific circuitries in the injured spinal cord.
The differentiation of at least one region of the normal
spinal cord within FSC grafts suggests that these grafts may
replace populations of intrinsic spinal cord neurons. The
following studies are designed to determine whether these
neurons form projections both within the grafts and between
the transplant and host spinal cord.

CHAPTER 4
AXONAL PROJECTIONS BETWEEN FETAL SPINAL CORD TRANSPLANTS
AND THE ADULT RAT SPINAL CORD:
NEUROANATOMICAL TRACING AND IMMUNOCYTOCHEMICAL
STUDY OF HOST-GRAFT INTERACTIONS
Introduction
In neonatal rats, transplants of fetal spinal cord (FSC)
tissue have been shown to provide an environment conducive to
the elongation of some descending axons through a spinal
injury site (Bregman, '87). In addition, when placed into
hemisection lesions in these newborn rats, FSC transplants
have been shown to improve the development of specific
aspects of hindlimb function as compared with rats with
hemisections only (Kunkel-Bagden and Bregman, '89).
In adult rats, however, there is no evidence to support
the concept of axonal regeneration of descending axons across
fetal spinal cord transplants. Nevertheless, the propagation
of some aspects of ascending and descending information might
be achieved by fetal grafts through the establishment of a
neuronal relay between the rostral and caudal regions of the
recipient spinal cord (Reier et al., '85, '88).
To test this hypothesis, the present study was designed
to identify and characterize patterns of axonal interaction
established between FSC grafts and adjacent regions of the
55

56
host spinal cord. Therefore, the purpose of the first
experiment was to extend preliminary WGA-HRP tracing studies
which had suggested some axonal integration between host and
graft (Reier et al., '86a). A fluorescent retrograde tracer
(Fluoro-Gold) was then used to determine the distribution of
cells contributing to axonal interactions. To complete the
axonal tracing studies, the anterograde transport of the
plant lectin Phaseolus vulgaris leucoagglutinin (PHA-L) was
included to reveal the patterns of the axonal projections
from local host and graft neurons and their relationship to
the host-graft interface. The combination of these three
contemporary axonal tracing techniques offers a unique
approach toward evaluating the local interactions between FSC
transplants and the adjacent regions of the spinal cord.
Complementary information about the ingrowth of specific
populations of host afferents into the transplants was then
obtained in a second group of animals by immunocytochemical
staining of transplant sections with antisera raised against
serotonin (5-HT), oxytocin (Ox), tyrosine hydroxylase (TH)
and calcitonin gene-related peptide (CGRP). Finally,
additional sections from both axonal tracing and
immunocytochemical specimens were stained with antiserum
against glial fibrillary acidic protein (GFAP) to examine the
relationship between host-graft projections across the
interface and the patterns of glial reactivity. Portions of

57
this study have been summarized previously (Jakeman and
Reier, '88).
Materials and Methods
Animals and Transplantation Surcrerv
A total of 99 female, adult rats received transplants of
FSC tissue according to a modification of previously
described methods (Reier et al., '83a, '86a; Chapter 2).
Each transplant recipient was anesthetized with ketamine and
xylazine, a laminectomy was performed at the T13 vertebral
level, and a cavity of 3 - 6 mm in length was created in the
left half of the spinal cord. For this study, the lesion was
routinely extended to a full hemisection by removal of both
the lateral and ventral columns. The overlying dorsal roots
were reflected laterally during preparation of the cavity,
and replaced after grafting. Although no effort was made to
intentionally sever or remove the rootlets (Tessler et al.,
'88; Houle and Reier, '89), they were sometimes injured
during the surgical procedure. Once hemostasis was achieved
in the host, the donor tissue was placed into the cavity and
the dura and superficial tissues were closed in layers.
Axonal Tracer Application and Tissue Processing
At post-graft intervals ranging from 6 weeks to 14
months, transplant recipients were re-anesthetized with
ketamine and xylazine and prepared for tracer application.
The region containing the graft and surrounding host spinal
cord was exposed by removing new bone growth and extending

58
the original laminectomy rostrocaudally. After the tracer
was injected, the surface of the cord was washed with
physiological saline and a drop of mineral oil was placed
over the spinal cord to minimize diffusion of the tracer into
the surrounding tissues. The spinal cord was then covered
with a piece of Durafilm and the wound was closed as
described above.
Horseradish Peroxidase (HRP) and Wheat Germ Agglutinin - HRP
conjugate (WGA-HRP)
Anterograde and retrograde labeling. A combination of
HRP (Type VI) and WGA-HRP (Sigma Chemical) were used for both
retrograde labeling of cells and anterograde filling of axons
(Mesulum, '82), as described in previous studies. In the
first part of the experiment, mixtures of the two tracers
were applied to transplants (n=16) using a variety of
techniques: (a) Seven rats received pressure injections of a
solution of 20% HRP and 1.0% WGA-HRP using a 1.0 ¿il Hamilton
syringe or a nitrogen burst picospritzer (Reier et al.,
'86a); (b) Two rats received iontophoretic injections of 2%
WGA-HRP; (c) one rat received a pledget of Gelfoam soaked in
20% HRP and 2.0% WGA-HRP; and (d) HRP and WGA-HRP were
applied to the remaining six rats using crystals dissolved
onto the end of a tungsten wire (Houle and Reier, '88).
For the reciprocal study, HRP and WGA-HRP were applied
to the host spinal cord with a tungsten wire ((d) above,
n=ll). In order to examine the HRP transport characteristics

59
using this method, a similar tungsten wire was placed into
one normal rat at the T13 vertebral level.
HRP histochemistry. After allowing 48 - 72 hours for
transport of the tracer, the recipients containing HRP and/or
WGA-HRP injections were deeply anesthetized with sodium
pentobarbital and perfused transcardially with 150 ml
heparinized 0.9% NaCl followed by 250 ml fixative (1.0%
paraformaldehyde + 2.5% glutaraldehyde in 0.1 M Sorenson's
phosphate buffer). Tissue blocks, including the transplant
and 5.0 - 10.0 mm of host spinal cord rostral and caudal to
the graft, were removed. Vibratome sections (50 /nm) were cut
in the sagittal or horizontal plane. The sections were
reacted within 2-4 hours according to the tetramethyl-
benzidine (TMB) protocol of de Olmos et al. ('78). Sections
were then mounted onto gelatin-coated slides and selected
slides were counterstained with 1.0% Neutral Red to reveal
the cytoarchitecture of the host and graft tissues.
Fluoro-Gold (FG)
Retrograde labeling. For the second set of experiments,
a 2.0% solution of FG (Fluorochrome, Inc.; Englewood, CO) was
made in 0.9% NaCl and the solution was then drawn into glass
pipettes (40 - 50 /Ltm tip diameter) . After a dural incision
was made with the beveled end of a 25 gauge needle, the FG
solution was injected into the transplants (n=12) or the host
spinal cord (n=19) using a rapid nitrogen burst (Pico-
spritzer) applied to the end of the glass micropipette. The

60
approximate injected volume of the FG solution was estimated
by measuring the diameter of the "hemisphere" ejected onto a
parafilm sheet and using the approximation (v= (27r(d/2)3)
/3) . Volumes of 0.1-1.0 /il were injected into the
transplants and 0.5-1.5 /¿I into the host tissue. One normal
rat also received an injection of 0.5 nl of FG solution at
the T13 vertebral level for comparison.
Tissue processing. The FG- containing tissue was
processed as described by Schmued and Fallon ('86). At 4
days after the injection, the rats were perfused with saline
followed by fixative containing 4.0% paraformaldehyde and
0.25% glutaraldehyde in 0.1 M phosphate buffer. Spinal cord
blocks containing the transplant and 4.0 mm of the
surrounding rostral and caudal spinal cord were removed and
postfixed in the same fixative for 2 hrs to overnight at 4°C.
Vibratome sections of 40 /¿m were cut in the sagittal plane.
In addition, every sixth section was saved in 0.1 M PBS for
subsequent immunocytochemical detection of GFAP (see below).
In 4 of the rats that received larger injections of FG
into the transplants, six additional tissue blocks were also
sectioned. These included cross sections of the host spinal
cord 4 - 6 mm rostral and caudal to the transplant,
horizontal sections from cervical and thoracic spinal cord,
and sections of host brainstem and brain. The dorsal root
ganglia from these recipients were embedded in paraffin and
sectioned at 15 /¿m. The Vibratome and paraffin sections were

61
mounted directly onto gelatin-coated slides. Slides from
paraffin blocks were heated to 37°C for 12 hours, and
deparaffinized. All FG slides were cleared in xylene,
coverslipped with Fluoromount (Gurr Bio/medical Specialties;
Santa Monica, CA), and viewed on a Zeiss Axiophot microscope
with fluorescent UV illumination.
Phaseolus vulgaris leucoaqqlutinin (PHA-L)
Anterograde labeling and tissue sectioning. To examine
the patterns of axonal elongation into graft and host
tissues, anterogradely-filled axons were identified by
immunocytochemical detection of PHA-L (Vector Laboratories
Inc.; Burlingham, CA). The tracer was applied by a
modification of the methods of Gerfen and Sawchenko ('84).
The PHA-L was dissolved to 2.5% in 10 mM phosphate buffer (pH
8.0). Glass micropipettes were cleaned with acetone and 100%
ethanol and broken to a tip diameter of 10 - 15/im. After
exposing the transplant (n= 13) or host spinal cord (n=8) ,
the tracer was applied to the appropriate site by
iontophoresis for 20 minutes using a 5 /¿A interrupted
positive current (7 sec on, 7 sec off).
After allowing 7-17 days for transport of the PHA-L,
the recipients were perfused as above with fixative
containing 4.0% paraformaldehyde and 0.25% glutaraldehyde.
The cord blocks including the transplant and 4 - 7 mm of the
surrounding rostral and caudal spinal cord were removed and
postfixed overnight at 4°C. Sagittal sections of these

62
blocks were cut at 40 ¿¿m on a Vibratome and stored in 0.02 M
potassium phosphate buffered saline (KPBS). Every sixth
section was saved for immunocytochemical staining with
antibodies to GFAP (see below).
Immunocytochemical detection of PHA-L. The remaining
free-floating Vibratome sections were processed for the
identification of cells and processes containing PHA-L. The
sections were first washed in 0.02 M KPBS and incubated for
2-4 hrs in a preblocking bath containing 2.0% normal rabbit
serum and 0.3% Triton X-100. All the sections were then
incubated in goat anti-PHA-L (Vector) diluted 1:5000 in KPBS
for 36 hours at 4°C and 2 additional hours at room temper¬
ature. The sections were rewashed and then processed with
biotinylated rabbit anti-goat IgG (1:225) and Vector Avidin-
Biotin-peroxidase Complex (ABC) as per the supplier's
instructions. The final peroxidase conjugate was reacted
with H202 in the presence of 0.005% DAB. The DAB reaction
was done either with the addition of 0.125% nickel ammonium
sulfate (black reaction product) or in the absence of nickel
(brown reaction product). The nickel-enhanced sections were
counterstained with 0.1% Cresyl Violet or 1.0% Neutral Red
prior to coverslipping.
Histological analysis of anatomical tracers
Mounted serial sections containing the tracer injection
sites were examined to determine the location of the
injection site and the extent of tracer diffusion relative to

63
the host-graft interface. Each specimen was then accepted or
rejected from the study according to specific transport and
diffusion criteria as described in Results. Retrogradely-
filled cells were identified and manually counted in
successive 1.0 mm fields at 125x. Cells which demonstrated
non-specific fluorescence when exposed to rhodamine (510-560
nm) or fluorescein (450-490 nm) microscope filters were not
counted. Each field was counted 3 times and the median value
was accepted. All cell counts were corrected according to
classical methods (Abercrombie, '46). Total cell number was
obtained by assuming an average cell diameter of 40 /¿m for
graft cells and 50 /Lim for host neurons.
The distribution of labeled cells and axons was
determined from drawing tube tracings of darkfield or
brightfield images (HRP and PHA-L) or from photographic
montages of fluorescence micrographs (FG). To determine the
distances of anterogradely labeled axonal projections, a
digitizing tablet and morphometry software (Videoplan;
Kontron, FRG) was calibrated for the appropriate
magnification. Measurements were taken from the drawing tube
illustrations or photomicrographs. Similar methods were used
to document the distances between the injection site and the
outermost zone of tracer diffusion as well as the
relationship of these regions to the host-graft interface.

64
Immunocvtochemical staining and analysis of GFAP
A series of sections (240 jum apart) from recipients with
FG or PHA-L injections was incubated in rabbit polyclonal
antiserum produced against GFAP (gift of Dr. Lawrence F. Eng,
VA Medical Center, Palo Alto, CA) . The antiserum was diluted
1:1200, and sections were incubated overnight at 4°C.
Detection of the primary antibody was performed according to
the peroxidase anti-peroxidase method (Sternberger, '76) as
described below.
Tracings of the rostral and caudal interface regions for
each section were made using a drawing tube, delineating the
regions containing dense GFAP staining between host and graft
tissue. For specimens with FG injections, the lengths of the
interface and the regions containing glial scar formation
were measured using a digitizing pad and Videoplan software.
The composite Fusion Index (FI) for each interface region was
defined as the average percentage of the interface which was
devoid of dense glial scarring (Houle and Reier, '88).
The density of glial staining was determined for both
graft and surrounding host tissues in 7 recipients. A
program was developed using the Zeiss IBAS image analysis
system (Kontron, FRG) and a high resolution video camera
(DAGE Inc., CCD71). At a viewing magnification of 125x, four
pairs of images from each GFAP stained section (each pair
including a graft region and host gray matter region) were
digitized and converted to binary images. The first field of

65
each pair was segmented interactively by the user to
distinguish glial processes from background as described by
(Bjorklund,H. et al., '83). Based upon the assumption that
non-specific staining was consistent within each section, the
segmentation settings used for this first image were retained
for the second image of the pair. The percent area occupied
by glial profiles was calculated for each field, and values
were obtained for the average glial density within the graft
and host gray matter regions as well as the ratio of
graft/host glial density for each section. Statistical
comparison of graft and host glial density was performed
using the direct-difference Student's t test for paired
samples (Spence et al., '83).
Immuno-staininq For Specific Populations of Host Fibers
Immunohistochemical procedures
Adjacent series of sections from 24 transplant recipients
were stained with polyclonal antisera raised against
neurotransmitters, synthetic enzymes, or peptides found in
specific populations of host fibers (See Table 4-1). Of
this group, 6 recipients were selected from tracer specimens
with unacceptable or failed injections. All of the
recipients were perfused as described above with fixative
containing 4.0% paraformaldehyde and 0.25% glutaraldehyde in
0.1 M Sorenson's phosphate buffer (pH 7.4). Sections
containing the graft and surrounding host spinal cord were

66
cut at 40 /¿m on a Vibratome and stored in 0.1 M phosphate
buffered saline prior to staining.
TABLE 4-1: LIST OF ANTISERA SOURCE AND DILUTION
Antisera
Source Antibody dilution
5-HT
OX
TH
CGRP
Incstar Corp. 1:3000 overnight
Incstar Corp. 1:5000 overnight
Eugenetech,Inc. 1:750 overnight
Peninsula Labs 1:12000 36 hours
Immunocytochemical staining was performed on adjacent
series of sections using antisera directed against
the following:
5HT-serotonin; OX-oxytocin; TH-tyrosine hydroxylase.
CGRP-Calcitonin gene-related peptide
Free floating sections were processed using the PAP
immunocytochemical procedure (Sternberger, '76). The primary
antisera used in this study were all raised in rabbit (Table
4-1) and diluted in high salt buffer containing 0.3% Triton
X-100 (THSB) . After the sections were removed from the
primary antibody solution, they were washed 3 times in THSB,
incubated in rabbit anti-goat IgG at 1:20 for 45 min to 1 hr,
washed again in THSB, incubated in Rabbit PAP 1:50-1:200 for
30 min, and finally washed in phosphate buffered saline (PBS)
or ammonium phosphate buffer. The peroxidase was visualized
with 0.05% DAB and 0.003% H202. Staining of fibers with
anti-5HT and anti-TH were done in the presence of 0.001%
nickel ammonium phosphate to produce a black reaction
product. Sections were then mounted on gelatin coated
slides. Some slides were counterstained with 1.0% Neutral

67
Red or 0.1% Cresyl Violet to reveal nearby cytoarchitecture.
The specificity of antibody staining was evaluated by the
morphological distribution of labeled cells and fibers in the
normal spinal cord.
Analysis of immunocvtochemical results
Sections were first examined for labeled cells within the
graft. Selected sections were then photographed on an
Axiophot microscope or drawn using a Zeiss microscope with
2.5x or 4Ox objective and 12.5 x ocular and 1.0 mag drawing
tube. The distances of fiber ingrowth and gualitative
comparisons of the regions of graft area occupied by stained
fibers were determined from the drawings or photos.
Results
General Transplant Characteristics
Viable transplants were present in 92% of the recipient
rats. Nearly all of the grafts filled the lesion cavity and
showed gross apposition with the surrounding host tissues.
The cellular organization and variability within the host-
graft interface was similar to that described in previous
studies from this laboratory (Reier et al., '86a; Houle and
Reier, '88). With regard to the interface, those sections
counterstained with Cresyl Violet or Neutral Red often
contained regions between the host and graft tissue that were
occupied by small, densely packed cell nuclei resembling
glial cells. In contrast, other regions of the interface
were devoid of an obvious cellular boundary between the two

68
tissues. In these more integrated areas, the only
distinction between host and graft was a transition in the
general cytoarchitectural organization. A similar range of
host-graft fusion was observed in GFAP stained sections.
Neuroanatomical Tracing With HRP and WGA-HRP
Injections into transplants
Solutions of HRP and WGA-HRP were injected or applied to
16 transplants (Table 4-2). The surviving grafts were
classified based upon the histological analysis of the
injection and the extent of tracer diffusion. The injection
sites were examined under darkfield illumination, and the
extent of each was defined as the area containing a purple-
opaque core and the entire surrounding region of orange TMB
reaction product (Fig. 4-1 a,b).
The injection site was restricted solely to the
transplant in six of the recipients (Table 4-2; Groups A, B) .
The two specimens classified into Group A contained the
smallest injection sites, which extended less than 0.5 mm in
maximum diameter. While no labeled cells or axons were
observed in the host spinal cord, these small injections
illustrated the presence of intrinsic graft projections (Fig.
4-la). The majority of retrogradely labeled cells in these
specimens were located within 0.5 mm of the center of the
injection site; however, additional retrogradely filled
neurons were found throughout the grafts. In the four other
recipients (Table 4-2; Group B), the injection sites were

69
TABLE 4-2: HRP/WGA-HRP INJECTIONS INTO FSC TRANSPLANTS
Code
(Post-Graft)
(Interval)
Method3
1
Groupb
Intrin.
proj .
Eff .c
proj .
Aff .d
proj .
HGP6(6 wk.)
Picospritz
A
+
HGP7(5 wk.)
Iontophoresis
A
+
HGP2(6 wk.)
Picospritz
B
+
HGP8(6 wk.)
Tungsten wire
B
+
HGP3(6 wk.)
Tungsten wire
B
+
+
HGP11(6 wk)
Tungsten wire
B
+
+
HGP5(9 wk.)
Picospritz
C
+
HGP10(6 wk.)
Hamilton
C
+
HG3 (2 mo.)
Tungsten wire
C
+
+
+
HGP9(6 wk.)
Hamilton
C
+
+
+ Indicates
evidence of projections from
labeled
profiles
present in host or graft tissue.
a The tracers were applied using one of five procedures
(see Methods).
b Recipients included in analysis were classified according
to the extent of tracer diffusion as follows: A - Injection
site < 0.5 mm in diameter and confined to graft; B -
Injection larger than 0.5 mm and confined to graft; C -
Injection site within graft, diffused into or slightly over
interface.
c Efferent projections of graft axons. Anterograde axon
label extended into host spinal cord.
d Afferent projections from host neurons. Retrogradely
labeled cells found in host spinal cord.

Figure 4-1. HRP and WGA-HRP tracing revealed intrinsic
interactions and some projections of graft and host axons.
a) Drawing tube tracings of sequential sagittal sections
through a graft with a small (Group A) injection. The center
of the injection site is shown as solid black and the area
containing dense reaction product with no discernable cells
and axons is represented by the hatched region. The area
outlined by a dotted line represents a high density of
labeled cells and axons, and each individual cell within the
graft is indicated by larger dots.
b) Labeled cells and axons were distributed throughout a
transplant (t; HGP11) to the host-graft interface
(arrowheads) following a larger HRP/WGA-HRP injection into
the dorsal region of a graft.
c)Labeled graft axons coursed parallel to this region of the
host-graft interface, but do not penetrate the host spinal
cord (h).
d-g) Axonal projections formed between host and graft
tissues. Graft efferent projections were identified by
retrograde transport into transplant neurons following
injections into the host spinal cord(d) and anterogradely
labeled axons (white arrows) extending into the host spinal
cord after an injection into the transplant (e). f) Example
of short-distance ingrowth of host axons into a transplant
following an injection made 1.9 mm rostral to a graft, g)
Illustration of the potential for greater axonal interactions
following an injection which diffused across the dorsal
region of the host-graft interface (specimen HH5). Note that
retrogradely filled neurons (*) may represent intrinsic
projections labeled by tracer diffusion. However, labeled
fibers can be seen in this ventral section where the
diffusion does not confuse the host-graft border. Axons
extended across the interface region (i.e. arrowhead) between
host (heavily labeled) and graft (lightly labeled) tissues.
Scale in a = 1.0 mm; b,c = 200 nm; d-g = 100 /¿m.

71

72
larger than 0.5 mm in diameter but were still confined to the
transplants. An extensive network of intrinsic graft
projections was again evident. Both labeled cells and axonal
profiles were observed throughout the transplants and up to
the interface in all directions (Fig. 4-1 b,c,e).
The transplants in Group B also suggested the presence
of axonal projections between host and graft tissues.
Similar to findings from preliminary studies (Reier et al.,
'86a), retrogradely filled neurons were occasionally found
within the host spinal cord. Efferent projections from graft
neurons were also observed, as anterogradely-filled axons
could be followed across the interface into the host in two
recipients (Fig. 4-le). Unfortunately, the possibility of
additional labeled axons oriented perpendicular to the plane
of section could not be assessed. In contrast, there were
some regions of each host-graft interface where the two
tissues appeared to be separated by a glial partition. In
these regions axons coursed parallel to the interface, but
they did not extend into the host spinal cord (Fig. 4-1 c).
The recipients in Group C had injection sites which were
not confined to the graft. In each case, however, the outer
zone of TMB reaction product extended beyond either the
rostral or caudal border of the graft, while the opaque
center was confined to the graft. At those regions where the
injection extended over the interface, labeled axons and host
neurons were found within 1.0 mm of the host-graft interface,

73
but few labeled cells were observed farther away. This
labeling pattern differed from the pattern observed following
placement of HRP and WGA-HRP into the normal spinal cord (see
Menetrey et al., '85).
Injections into the host spinal cord
In the reciprocal experiment, HRP and WGA-HRP injections
were made both rostral and caudal to the transplants. The
details of the post-graft intervals, injection site location,
and distances between the injections and the host-graft
interface are summarized in Table 4-3. The injections were
completely confined to the host spinal cord in seven animals.
Evidence for axonal interactions between host and graft
tissues was obtained from specimens which contained large (>
1.5 mm radius) injections that extended to within 2 mm of the
interface region. Of the seven specimens, four contained
retrogradely- filled neurons within the transplants (Fig. 4-
1 d). The number of labeled neurons in these grafts ranged
from a single cell to over 30 cells, with most of these
located within 1.0 mm of the host-graft interface.
In addition to efferent projections from transplant
neurons, the larger HRP/WGA-HRP applications also labeled
axons from host cells that had extended processes into the
transplants. Evidence of such axonal ingrowth was observed
in 3 of 7 of the recipients. In each case, the interface
between host and graft tissues was readily apparent as a
distinct border between the dense axonal labeling in the host

74
TABLE 4-3. HRP/WGA-HRP INJECTIONS INTO HOST SPINAL CORD
Code I.S.a
(Post-Graft)
(Interval)
Dist.b
c.-Int.
(mm)
Dist.c
e.-Int.
(mm)
Eff .d
proj .
Aff ,e
proj .
HH3(14 mo.)
rost.
7.0
2.1
+
+
caud.
10.6
6.8
—
—
HH4(5.5 mo.
)rost.
1.9
1.0
-
+
HH6(2 mo)
rost.
5.0
3.2
—
—
caud.
5.7
4.3
—
-
HH7(2 mo.)
rost.
2.9
1.4
+
—
caud.
3.9
2.8
—
-
HH8(2 mo.)
rost.
2.0
0.6
+
+
caud.
3.7
1.9
—
-
HH9(6.5 mo)
rost.
2.4
1.2
—
—
caud.
2.7
1.7
—
-
HH11(6 mo.)
rost.
3.0
0.0
+
—
caud.
6.2
0.0
“1
—
+ Indicates retrogradely filled cells or anterogradely filled
axons present within the transplant.
a Injection Site. Injection placed either rostral (rost.)
or caudal (caud.) to host-graft interface.
b Measured distance from center of injection site to the
host-graft interface.
c Measured distance from the edge of HRP reaction product
or diffusion to the host-graft interface.
d Efferent projections of transplant neurons. Retrogradely
labeled cells within the graft.
e Afferent projections into the graft. Anterogradely
labeled axons from the host spinal cord.

75
and very sparse axonal profiles in the transplant (Fig. 4-1
f) . These ingrowing host fibers were restricted to a
peripheral border of the graft and most terminated within a
half millimeter of the host-graft interface. In contrast,
labeled axons stopped abruptly at the interface in the
remaining recipients.
The possibility of more extensive axonal integration
within the interface region was illustrated in one case in
which an injection had extended over a dorsal region of the
interface. The labeled axons and cell bodies within this
graft were excluded from consideration of host-graft
projections. However, a region ventral to the area of
diffusion was marked by the presence of labeled axons that
could be followed between host and graft tissues (Fig. 4-1
g) •
Fluoro-Gold Injections: Distribution of Cells
Injections into the host spinal cord
Fluoro-Gold injections were made into the host spinal
cord of 19 recipients at distances ranging from 1.8 - 5.0 mm
from the host-graft interface. Injections in seven of the
rats met three criteria: 1) the injections were confined to
the host spinal cord, 2) they showed no evidence of diffusion
into the cerebral spinal fluid (as seen by a high degree of
non-specific fluorescence throughout the spinal cord) or
central canal region, and 3) they were large enough to label

76
a high percentage of host neurons adjacent to the graft.
Results from these animals are summarized in Table. 4-4.
TABLE 4-4: INJECTIONS OF FLUORO-GOLD INTO
THE HOST SPINAL CORD
Code I.S.
(Post-Graft)(mm)
(Interval)
(host)
# labeled3
cells
in graft
Fusionb
Index
(%+sd)
Glialc
Ratio
G/H
FGC-20(8 mo)
3.6
mm
rost.
0
47%(±13)
2.3 *
FGC-17(9 mo)
4.3
mm
rost.
38
19%(±11)
1.2
FGC-21(8 mo)
2.8
mm
rost.
28
40%(±19)
4.7 *
FGC-23(8 mo)
1.5
mm
rost.
8
8%(±13)
10.0 *
FGC-13(8 mo)
4.4
mm
caud.
697
51%(±20)
3.1 *
FGC-14(5.5m)
3.6
mm
caud.
212
40%(±16)
1.2
FGC-30(6 wk)
1.8
mm
caud.
439
27%(±21)
1.1
3 Total number of retrogradely filled cells within
the transplant. Cell counts corrected according to
the method of Abercrombie (/46).
b Composite Fusion Index: (FI) percentage of the
host-graft interface (closest to injection) which is
devoid of dense glial scar (average of 4 - 10
sections/transplant).
c Ratio of the percent area occupied by glial
elements. * indicates that the glial density within
the graft was significantly higher than that in the
surrounding host tissue (p< 0.01).
The interface between the host gray matter and graft
tissue was characterized by a sharp decrease in the density
of labeled neurons between adjoining host and transplanted
tissue (Fig. 4-2 a) . In all seven grafts, there were regions
of fusion between host and graft tissues as identified with
GFAP staining (see below; Fig. 4-2 b) . Six of the grafts
contained retrogradely labeled cells. These included three

77
transplants (FGC-17,21,23), each with fewer than 50
retrogradely filled neurons within the graft and three others
(FGC-13,14,30) , each with more than 200 labeled cells. In
this sample, the differences between the two groups did not
correspond with the post-grafting interval or the distance
between the injection site and the host-graft interface.
However, more labeled cells were found in the three
recipients with FG placed caudal to the graft than those with
injections placed at rostral levels.
Transplant neurons which projected into the host spinal
cord were distributed throughout the graft tissue (Fig. 4-2
e) . Most of these retrogradely labeled neurons were
multipolar and small, measuring 8 - 20 in diameter (Fig.
4-2 c) , although larger cells were observed occasionally
(Fig. 4-2 d) . Histograms were made for each of these six
grafts to show the distribution of the labeled cells as a
function of distance from the host-graft interface (Fig. 4-
3). In the recipients with few labeled cells (top of Fig. 4-
3), there were more cells within the first millimeter of the
transplant and a decrease in the density of labeled cells
with distance from the host-graft interface. However,
labeled neurons were more evenly distributed in grafts
containing many fluorescent cells, regardless of the absolute
length of the transplants.

Figure 4-2. Identification of graft neurons projecting into
the host spinal cord by retrograde transport of Fluoro-Gold.
a) The interface between host (h) and transplant (t) is
characterized by a marked decrease in density of labeled
neurons.
b) Section adjacent to that shown in a), after staining with
anti-GFAP. A glial scar is present along the dorsal region
of this host-graft interface (bottom half of figure), while
the host and graft tissues are well fused in the ventral
region (between arrowheads).
c) Higher magnification of typical retrogradely labeled cells
within this graft.
d) Example of an occasional large graft neuron that projected
into the host spinal cord.
e) Photo-montage of a sagittal section from specimen FGC-30
to illustrate the distribution of labeled neurons throughout
a transplant following a FG injection into the host spinal
cord (h) . The host-graft interface is marked by a white
dotted line.
Scale in a,b,e = 200 ¿un; c,d = 100 ¿im.

79

Figure 4-3. Individual histograms show the distribution of labeled cells
within six transplants with respect to the host-graft interface closest
to the injection site. The corrected neuron total is in parentheses
above each graft. Each bar represents the number of neurons counted
within successive 1.0 mm segments of the graft. Note that the last bar
in each graph may represent less than a full millimeter of graft tissue.

Numbers of Labeled Cells
DISTRIBUTION OF RETROGRADELY LABELED TRANSPLANT
NEURONS FOLLOWING FLUORO-GOLD INJECTIONS
INTO THE HOST SPINAL CORD
FGC-17
(38)
0 - to 11 - VO *.1-3.0 > XS>
FGC-13
(697)
FGC-21
(28)
FGC-23
o-to u-1.0 *.1 - »u> »ajo
FGC-30
(439)
0-10 11 - to
Distance from Host-Graft Interface (mm)

82
Injections of FG into transplants
In ten of the recipients with FG injections into the
grafts, the extent of tracer diffusion was confined to the
transplant. While seven grafts had some cells labeled in the
host spinal cord, the numbers of labeled cells ranged from
few host neurons to more than 70 host spinal cord cells and
200 dorsal root ganglion (DRG) neurons.
Figure 4-4 summarizes the location and extent of the
graft injections and the distribution of labeled host
neurons. Five of these FG injections were < 0.5 mm in
diameter (FGC- 2a, H6, H7, H8, H10; Fig. 4-5 a). In these
specimens, nearly all of the labeled neurons were found
within the graft itself. The intrinsic neurons were
concentrated in the region nearest the injection site.
However, some retrogradely labeled cells were found in all
regions of these transplants (Fig. 4-5 b) . Labeled neurons
were also found in the adjacent host spinal cord in two of
these recipients. In both cases, the labeled cells were
located within 0.5 mm of the interface zone which was
adjacent to the injection site, and distributed within the
medial or lateral intermediate gray. The greatest number of
labeled host neurons were found in the recipient with an
injection located within 0.2 mm of the host-graft interface
(H10).
In the remaining transplants from this group (FGC-
H9,25,26,27,31) the injection site was larger than 0.5 mm in

Figure 4-4. Distribution of injection sites and labeled neurons
following injections of Fluoro-Gold into transplants. The center panel
contains freehand drawings of a representative section with individual
dots representing cells and their approximate locations within the graft.
Numbers of cells rostral and caudal to the graft are indicated according
to numbers within a range of distances from the interface. DRG= dorsal
root ganglia, totals Left and Right (all grafts were made on the
left).

Animal
(Post-g
Cel Is Rostral
I.) (Distance in
6-4.1 4-2.1 2-1.1
to Transplant
mm)
1.0-0.6 0.5-0
Glial
scar
(FI.X)1
FGC-2a
(2 mo.)
-
-
5
FGC-H6
(2 mo.)
-
-
-
35%
FGC-H7
(2 mo.)
-
-
-
33%
FGC-H8
(6.5 mo.
)
-
-
-
21%
FGC-H9
(6 mo.)
-
-
2
23%
FGC-M10
(5.5 mo.
)
-
-
2 30
42%
FGC-25
(2 mo.)
-
-
-
4
48%
FGC-26
(2 mo.)
-
-
-
-
32%
FGC-27
(14 mo.)
-
-
-
1 3
17X
FGC-31
.
.
.
_
30%
(6 wk.)
Glial
scar
FI.X)1
0-0.5
Cells Caudal
(Distance in
0.6-1.0 1.1-2
to Transplant
mm)
2.1-4 4.1-6
DRG
L R
-
1
-
-
-
35%
-
-
-
-
56X
-
-
-
-
1.0%
-
-
-
-
25%
-
-
-
-
21X
1
-
-
-
49%
58
5
10
13
27
219 2
47%
38
-
-
1
2
6 2
37%
3
-
-
-
-
- -
34%
4
_
_
_
_
7 1
jEZZl
00
Members in each colunn represent the number of cells counted in each region. Scale corresponds to approx. 1.0 mm.
1 Fusion Index: X of linear host-graft interface which is deviod of glial scar (average of 4-10 sections per animal).

85
diameter, but was still confined to the graft. All of these
recipients contained some retrogradely labeled host neurons.
Again, the greatest numbers of host neurons were found when
the injection site was within 0.5 mm of either the rostral or
caudal interface. In addition, the majority of labeled host
neurons were found immediately adjacent to the transplants.
In the best case (FGC-25; Figs. 4-5 c-i) , the FG injection
extended to 0.2 mm from the caudal interface. Retrogradely
labeled neurons were found throughout host spinal cord caudal
to the graft, while few neurons were found rostral to the
graft. Within the sacral spinal cord (Fig. 4-5 h; i.e. 4 -
6 mm away), labeled cells were distributed throughout the
dorsal and intermediate gray regions (laminae I-VII) as well
as regions of the ventral horn. In addition, more cells were
located ipsilateral than contralateral to the graft. In this
animal, a large number of ipsilateral DRG cells were found as
well (Fig. 4-5 i) . However, sections from cervical and
thoracic spinal cord as well as brainstem and brain contained
no labeled cells in any of the recipients. The absence of
retrograde labeling of descending fibers was in contrast with
the pattern observed following a similar injection into a
normal rat.
Comparison of FG and HRP/WGA-HRP injections in normal rats
To compare the patterns of retrograde cell labeling using
the two tracers and the present injection technigues, each of
these tracers was also injected into a normal rat at the T13

Figure 4-5. Retrogradely labeled host neurons following FG
injections into transplants.
a,b) Intrinsic labeled neurons following a small FG injection
(< 0.5 mm diameter) into a transplant (t) . a) The majority of
labeled neurons were located in the immediate vicinity of the
injection, which was adjacent to the rostral host-graft
interface (arrowheads). Cells were also found at the far end
of the graft (b) at a distance of 3 mm from the injection.
c-i) Distribution of retrogradely labeled cells following a
larger injection (dotted line in c) near the caudal border
(arrowheads) of specimen FGC-25. d) Most fluorescent labeled
cells (white arrows) were found immediately adjacent to the
host-graft interface. Inset: Verification of the graft
border was obtained in each case by viewing the interface
region with darkfield optics, using the location of blood
vessels (*) as landmark points, e) GFAP staining of one
section from this specimen illustrates a high degree of
fusion between host and graft (between arrowheads). f) A
patch of retrogradely labeled neurons found approximately 3
mm caudal to the transplant, g) Transverse section of the
sacral spinal cord contains four retrogradely labeled
neurons, h) Composite drawing of 40 sections from the host
sacral spinal cord 4 - 6 mm caudal to the transplant. Left
in the figure is ipsilateral to the graft, right is
contralateral. The photograph in g: was obtained from the
region enclosed in the box. i) Labeled dorsal root ganglion
neurons ipsilateral to the transplant.
Scale in a,c,d,e = 200 /¿m; b,f,g,i = 100 ¿¿m.

87

88
vertebral level. Histological analyses revealed neurons
within the cervical and thoracic spinal cord, brainstem
nuclei, DRG, and cortex of both animals. Similar to findings
from grafted animals, many more labeled cells were found in
each of these regions in the rat which received the pressure
injection of FG than the rat with the HRP/WGA-HRP injection.
PHA-L Injections: Patterns of Axonal Projections
Further definition of the axonal trajectories of host and
graft neurons was made with iontophoretic injections of PHA-
L placed either into transplants or the adjacent host spinal
cord (Table 4-5). The injection region was easily defined by
the presence of darkly filled perikarya. In some specimens,
the injection site contained only a small number of labeled
neurons (Fig. 4-6 a,b). Larger injection sites extended up
to 0.5 mm in diameter.
Projections of transplant neurons
Of the 13 recipients with PHA-L injections into the
transplants, five had acceptable axonal labeling (Table 4-5,
top). All of these injections revealed an extensive network
of axonal projections within the graft. Axons near the
injection site exhibited abundant branching and the nearby
neurons were surrounded by terminal enlargements. Survey
electron micrographs of one such graft showed labeled
terminal boutons throughout the graft neuropil (Jakeman and
Reier, unpublished observations). While the greatest density

89
TABLE 4-5: INJECTIONS OF PHA-L INTO FSC TRANSPLANTS
OR THE HOST SPINAL CORD
Code I.S.a Region6 Axonal Projections
(Post-Graft)
(Interval)
PHAL-8 (2 mo.)Graft Ventral Efferent axons can be traced
across interface. Injury filled
host axons also evident.
PHAL-10(10 wk)Graft dorsal-
medial
Efferent axons in rostral
dorsal horn and intermediate gray
regions of host.
PHAL-23(6 mo) Graft dorsal- Efferent axons present in
medial dorsal horn, dorsal tracts and
intermediate gray, and in rostral
intermediate gray regions.
PHAL-15(6 mo) Graft dorsal- Efferent axons innervate
lateral host lateral motoneurons.
PHAL-32(lOwk) Graft caudal No Efferent axons.
PHAL-11(6
wk)
Host
0.5 mm
caudal
Afferent axons extending
< 0.2 mm into graft.
PHAL-21(6
mo)
Host
1.5 mm
caudal
Afferent axons extending
< 0.3 mm into graft.
PHAL-33(10wk)
Host
1.5 mm
rostral
All axons stop
interface.
a t
PHAL-27(12mo)
Host
0.5 mm
caudal
Many cells and
throughout graft*, most
1.0 mm of interface.
axons
within
PHAL-16(6
mo)
Host
2.0 mm
No afferent axons.
a Injection site. Iontophoretic injection into
graft or host spinal cord.
b Region of graft with injection or distance and
direction between injection in host spinal cord and
the host-graft interface.
* Evidence of both anterograde and retrograde
transport.

90
of fibers was found within the injection region, labeled
axons were found in all areas of the grafts.
Three general axonal projection patterns were seen within
the transplants. Within graft regions containing densely
packed neuronal cell bodies, the labeled axons branched
extensively (Figs. 4-6 b,c). In contrast, where few
perikarya were found, labeled axons remained mostly
unbranched and followed a relatively straight trajectory
(Fig. 4-6 b). Finally, at the interface regions, individual
axons often coursed parallel to the host-graft border, and
occasionally extended into the host neuropil (Fig. 4-6 d).
Labeled axons could be followed into the host spinal cord
in four of these grafts. The pattern of axonal outgrowth was
slightly different for each specimen. In one case (PHAL-8),
the injection was placed ventromedially within the transplant
and resulted in injury to fibers in the ventral white matter
of the host. The appearance of these injury-filled axons was
distinct. These axons were very heavily labeled, and they
exhibited bulbous terminal swellings. Similar profiles were
not found in any of the other animals in this group.
Following injections into two other grafts (PHAL-10, 23),
a high density of labeled axons were seen within the
transplants, especially near the dorsal region of the host-
graft interface. Labeled fibers extended across the
interface and into the adjacent rostral or caudal dorsal horn
(laminae I-III) of the host (Fig. 4-7a).

Figure 4-6. Intrinsic PHA-L labeled profiles following a small injection
into transplant PHAL-8.
a) Sagittal section providing orientation. The injection was placed in
the ventro-medial region of the transplant (t) (double arrowheads).
b) Enlargement from a nearby section of the same transplant. Labeled
axons within acellular regions exhibit little branching (arrows), while
extensive branching is observed around counterstained cell bodies
(arrowheads).
c) Within the transplant, but farther from the injection site, labeled
axons exhibit an extensive pattern of projections.
d) Enlargement of the interface region outlined in the box in (a) .
Labeled axons (arrowheads) within the transplant travel parallel to the
host graft interface. One axon then turns abruptly to enter the adjacent
host spinal cord (h).
Scale in a = 500 /¿m; b,c,d = 100 /im.

92
f' 4 .
t .

Figure 4-7. Examples of efferent projections into the host spinal cord
following PHA-L injections into transplants.
a) Interface between transplant (t) PHAL-10 and the dorsal horn of the
host spinal cord. Labeled axons cross the interface and extend
longitudinally within the rostral dorsal horn (hDH) .
b) Labeled axons approximately 4 mm caudal to a transplant with a PHA-L
injection. Occasional axon collaterals extended into the deeper layers
of the dorsal horn (arrowheads).
c) Example of an axon that extended from a transplant (t) into the host
intermediate gray region (h,G) and branched around the host neurons.
d) Labeled axons in specimen PHAL-15 project out of the transplant and
into the nearby motoneuron pool caudal to the graft. Numerous bouton¬
like profiles are present within the surrounding tissue (arrowheads).
Scale = 100 /zm in all figures.


95
Within the host, the efferent axons were found in both
myelinated and unmyelinated regions. These labeled fibers
continued along a primarily longitudinal trajectory and some
could be followed as far as 4 mm from the interface.
Collaterals from were also observed extending into deeper
regions of the host gray matter (Fig. 4-7 b). In addition,
some axonal outgrowth occurred across the interface where the
graft tissue was apposed to the intermediate gray regions of
the host (Fig. 4-7 c) . Some of these axons could be followed
for several hundred microns before terminating in the host
ventral horn.
The two remaining recipients from this group had longer
(5-7 mm) transplants. In one specimen (PHAL-15) , a large
injection was made near the caudal end of the graft, where
the transplant was well apposed to host motoneurons.
Numerous labeled axons could be followed across the caudal
graft-host border and into the motoneuron pools adjacent to
the graft (Fig. 4-7 d). While labeled fibers were also found
at the rostral pole of this graft, no axons extended into the
host spinal cord across the rostral interface. The other
specimen from this group (PHAL-32) had a small injection in
the ventral region of the graft. Both rostral and caudal
interfaces of this graft were poorly apposed to the host
neuropil, and no labeled axons extended into the recipient
spinal cord.

96
Projections of local host neurons
Successful iontophoretic injections of PHA-L were made
into the host spinal cord at 0.5 - 2.0 mm from the interface
region in five rats. In each case, the host tissues
contained a dense fiber plexus throughout the gray matter
(Figs. 4-8 a,c) and long axonal projections were observed
within the dorsal and ventral myelinated tracts. The
presence of injured axons and resulting injury-fill labeling
patterns were common features among these recipients.
Evidence of host afferent ingrowth was obtained in four
specimens. The pattern of host fiber ingrowth was similar
for 3 of the 4 (PHAL-11,-21,and -33; Figs. 4-8 a,b). A high
density of labeled axons was seen up to the host-graft
interface. From this point, a few labeled fibers extended
into the transplant. The labeled axons within these grafts
terminated shortly after entering the graft, and no fibers
were found at distances greater than 0.3 mm away from the
interface. At the ends of some of these axons, bouton-like
profiles were observed in direct apposition to Cresyl Violet
stained cells.
In the fourth specimen (PHAL-27), a large injection was
made in the host spinal cord 0.5 mm from the host-graft
interface. The presence of several lightly filled neuronal
perikarya far from the injection site suggested that
substantial retrograde transport had occurred (Shu and
Peterson, '88). Both labeled axons and cells were present

Figure 4-8. PHA-L labeled profiles following injections into the host
spinal cord.
a) Short-distance ingrowth of host fibers across the interface (dashed
line) and into FSC transplants in specimen PHAL-11.
b) Higher power micrograph of axonal ingrowth in another rat. Note the
axonal branching and axonal swellings surrounding the transplant neurons.
c) In specimen PHAL-16, no axons crossed the interface following a large
PHA-L injection 2 mm from the host-graft interface.
d) Following a similar injection in specimen PHAL-27, large numbers of
anterogradely filled axons and retrogradely filled cells within the graft
suggested a great deal of interaction between the two tissues. Notice
the axonal profiles crossing between the two tissues in a region of
fusion between host and graft (arrows).
Scale in a,c,d = 100 /im; b = 50 /im.

y*

99
within this graft, and labeled axons were observed coursing
within regions of fusion between the host and graft tissues
(Fig. 4-8 d) . While the greatest concentration of these
profiles was found within 1.0 mm of the host-graft interface,
a small number of cells and axons were found in other regions
of the transplant as well. At the other extreme was specimen
PHAL-16, which did not contain any labeled afferent
projections from local host neurons. Following an injection
placed 2.0 mm rostral to this graft, all labeled fibers
stopped abruptly at the host-graft interface, and some ended
in large retraction terminals (Fig. 4-8 c).
Local Axonal Projections and Glial Reactivity
Fusion of host and graft tissues
The percentage of the host-graft interface which was
devoid of glial scar formation (see Materials and Methods)
was highly variable across sections in each animal (cf. Figs.
4-2 b,5e,9). In the most dramatic example of this
variability, the percentage of fusion at the interface in one
animal ranged from 0.0%, for a section from the medial border
of the transplant, to 57% within a section from where the
graft was apposed to the host dorsal and ventral gray matter.
When the values from sections of each FG recipient were
averaged, a composite Fusion Index (FI) was obtained for the
rostral and caudal interface region of each specimen. These
values ranged from 8% to 56%. (The averages values did not
differ by more than 2% from the values obtained by dividing

100
the total length of host-graft fusion by the total interface
length).
In those animals with acceptable FG injections into the
host spinal cord (n=7) no correlation was found between the
composite FI and the numbers of retrogradely labeled cells
in the transplants (Table 4-4). However, all of the grafts
contained some regions of fusion along the appropriate
interface (e.g., Fig. 4-2 b) . In addition, the recipient
with the fewest number of labeled cells had only 8% fusion of
the rostral interface.
Staining with antisera to GFAP was also completed on 9 of
10 recipients with FG injections into the grafts. The
composite FI for the rostral and caudal interfaces of each
recipient is included in Figure 4-4. Again, each of these
recipients showed some regions of fusion between host and
graft tissues (Fig. 4-5 e) . The 2 recipients with the
greatest number of labeled host neurons had composite FI
measurements of 48-49%. However, not all of the variations
in host neuron ingrowth could be accounted for by the amount
of fusion at the interface.
Both the total numbers of FG labeled cells and the
composite FI reflect characteristics of host-graft
integration across an entire interface. In contrast,
staining of adjacent sections with antibodies to GFAP and
PHA-L allowed a comparison of regional aspects of such
interactions. Using this approach, a correspondence was

101
observed between localized regions of tissue fusion and the
site of axonal projections from transplant neurons into the
surrounding host spinal cord (Fig. 4-9). In contrast, few
axons penetrated a glial interface between host and graft
tissue or within the transplants.
Glial reactivity in adjacent host and graft tissues
In addition to the formation of a glial scar at the
interface, varying amounts of astrocytic hypertrophy were
observed within the transplants and the surrounding host
tissue. The amount of glial reactivity was examined in the
recipients with FG injections into the host spinal cord
(Table 4-4). There was a wide range in the density of glial
staining within each of the grafts, as well as some
variability in the surrounding host gray matter. In order to
control for variations in staining, the ratio of graft/host
glial staining was determined within sections. As described
in methods, the density ratio was determined for each section
as a percentage of field densities in pairs obtained from
within the graft and in the host spinal cord (including 4 -
5 mm either rostral or caudal to the transplant). The
averaged ratio of graft/host glial density for these animals
ranged from 1.1 to 10.0. In four recipients, the glial
reactivity in the graft was significantly higher than that of
the surrounding host tissue. However, there was no
relationship between the values obtained for the graft/host

Figure 4-9. Correspondence between axonal projections and
glial scar formation at the host-graft interface. The left
side of the figure contains darkfield micrographs of PHA-L
stained fibers following an injection into the dorsal
guadrant of transplant PHAL-23 (t) . Adjacent sections stained
with GFAP are on the right.
a,b) A dense scar separates host from graft in the most
lateral regions, and few fibers cross the interface into the
host tissue in this region. Note that very few labeled axons
cross a smaller glial partition within the transplant itself
(*) •
c,d) In this pair, fibers extend from the graft into the host
dorsally, corresponding to a region of fusion between host
and graft tissue (arrowheads). Note that few axons cross
more ventrally, where a dense glial scar is present.
e,f) In the most medial region of the graft, fibers extended
into the host spinal cord across the only part of the
interface where a break is found in the corresponding glial
scar stain.
Scale in a-f = 200 /¿m.

103

104
glial density ratio and the numbers of retrogradely labeled
cells in these animals.
Immunocvtochemical Staining: Patterns of Axonal Interactions
Between Specific Populations of Host Fibers and FSC Grafts
Immunocytochemical staining of adjacent series of
sections with different antisera permitted a comparison of
the patterns of ingrowth of host fibers from several regions
of the neuraxis. A summary of the post-graft intervals and
the observations of fiber ingrowth is provided in Table 4-6.
Elongation of host fibers into FSC transplants
Serotonin (5-HT) immunoreactivitv. Fibers exhibiting
5-HT immunoreactivity were evident throughout the gray matter
of the host rostral to the transplant, where the staining
pattern was identical to that described in normal rats
(Steinbusch, '81; Bullitt and Light, '89). These fibers were
found predominantly in four regions, including parts of the
superficial dorsal horn, the region surrounding the central
canal (lamina X), the intermediolateral cell column of the
thoracic and upper lumbar spinal cord, and around the
motoneurons in the ventral horn. In contrast, very few
stained fibers were found caudal to the transplants.
Graft tissue was apposed to injured host 5-HT fibers
within both dorsal and ventral halves of the host spinal
cord. The vast majority of host 5-HT fibers stopped at the
edge of the grafts (Fig. 4-10 a,c), yet most of these axons
maintained a normal appearance in terms of fiber thickness.

105
TABLE 4-6: IMMUNOCYTOCHEMICALLY STAINED SECTIONS
#
Post-graft
Interval
5HT
TH
OX
CGRP
3
1 m
+
-
-
+
1
6 w
*
-
-
-
5
2 m
*
-
+
*
8
2 m
*
*
-
*
12
2 m
*
*
0
+
S6
2 m
+
-
+
+
S7
2 m
-
-
+
+
S8
2 m
-
-
+
+
S9
2 m
-
-
+
0
S10
2 m
*
-
+
+
Sll
2 m
+
-
0
+
7
3 m
+
*
+
★
S12
3 m
0
-
-
+
S13
3 m
+
-
+
-
S14
3 m
-
-
0
+
S15
3 m
0
-
+
+
S3
4 m
+
-
-
+
S5
4 m
+
-
-
*
16
5.5 m
*
-
+
+
21
8 m
+
*
+
+
25
8 m
+
*
0
+
26
8 m
+
-
0
*
24
12 m
+
*
+
+
% grafts w/axonal ingrowth3
85%
-
71%
94%
% crrafts w/labeled cells
32%
100%
lo%
24%
+ host fibers extended into the transplant;
* labeled cells found within the transplant;
0 no labeled host fibers or cells within the transplant;
- unstained or poorly stained series.
a Specimens with labeled cells in graft were
not included calculating %.
Ingrowth of 5-HT- containing fibers was observed in 11 of
13 grafts. The extent of innervation was similar to that
described in earlier studies (Reier et al., '85, '86a). The

106
majority of these fibers entered from either the dorsal or
ventral regions of the rostral interface (Fig. 4-10 b, 4-12).
In addition, some fibers entered the grafts from the
remaining ventral white matter interface in more medial
sections. While some recipients exhibited more ingrowth
from one region of the interface, there was no obvious
tendency for a greater innervation from ventral or dorsal
interface across animals.
In many regions of the host-graft interface where the two
tissues appeared well fused, 5-HT processes extended just
into the interface in a pattern resembling the ingrowth of
spinal cord axons observed following large injections of WGA-
HRP into nearby segments (Fig. 4-10 a). However, as seen
with FG and WGA-HRP injections, the density of fiber staining
representing the ingrowing fibers was always much less than
that within the host spinal cord rostral to the graft. In
contrast to findings when FSC grafts have been placed into
newborn recipients (Reier et al., '86a; Bregman, '87), the
vast majority of stained fibers within grafts in the adult
were found within 0.5 mm of the host-graft interface. No
fibers were observed greater than 1 mm from the nearest
interface, thus, no fibers extended across the grafts.
At some regions of the host-graft interface, the injured
5-HT fibers were thickened and appeared to ended blindly at
the host-graft interface, forming large terminal bulbs (Fig.
4-12 d). In some of these examples, nearby sections stained

Figure 4-10. Ingrowth of 5-HT fibers from the rostral and ventral host-
graft interface.
a) Small caliber 5-HT axons (arrows) cross the interface (arrowheads)
between the host ventral gray matter (h) and the transplant (t) .
b) A single 5-HT axon (arrow) within a FSC graft.
c) Many of the host 5-HT axons stop within the interface (arrowheads),
while single fibers (arrow) extend into the graft.
d)In some regions of the host-graft interface, 5-HT axons stop abruptly
and form large terminal bulb endings. This region (arrowheads)
corresponds to an area of intense GFAP staining in a nearby section.
Scale in a-d = 50 /¿m.

108

109
with GFAP revealed dense glial scarring in the region where
these bulbous endings were found.
Tyrosine Hydroxylase ÍTH)- immunoreactivitv. All of the
stained grafts contained some cells that exhibited TH- like
immunoreactivity (see below). However, in each case, the
spinal cord rostral to the graft contained a dense plexus of
stained axons, and some fibers appeared to extend into the
grafts across the rostral host-graft interface. The stained
fibers within the transplants were similar in appearance to
those found in the normal host spinal cord.
Oxytocin immunoreactivitv. Immunocytochemical staining
with antisera to oxytocin in both normal spinal cord sections
and rostral to the transplants revealed a small population of
fibers which were concentrated in the dorso-lateral funiculus
and dorsal horn of the spinal cord (Swanson and McKellar,
'79) . Axons were best visualized in longitudinal section,
where they descended in the lateral funiculus and adjacent to
the central canal with little branching evident.
In 12 of 17 transplants, oxytocin immunoreactive fibers
were observed within the parenchyma of the grafts. In each
case, the stained fibers were found near the rostral border
of the transplants (Fig. 4-11 a; 4-12). These fibers were of
very small calibre, and individual axons exhibited
varicosities along their entire length. Little axonal
collateralization was observed. However, the fibers often
traveled in a circuitous manner which was different than that

110
observed in the normal spinal cord. The distance of ingrowth
of these axons was similar to that described for 5-HT and TH-
fibers described above. No oxytocin-containing fibers were
seen found at distances greater than 1.0 mm from the rostral
or lateral interface of the graft.
Where injured Ox- stained fibers were apposed to heavily
scarred regions of the host-graft interface, the axons were
often thickened as compared to their appearance in normal
spinal cord or within the graft tissue. Occasionally, these
thickened fibers assumed a configuration suggesting a
reversal in direction at this point (Fig. 4-11 b).
Calcitonin gene-related peptide (CGRP) immunoreactivitv.
The vast majority of CGRP containing fibers arise from
small diameter primary afferents from DRG cells (Gibson et
al., '84; McNeill et al., '88). Within the host spinal cord,
these fibers were found primarily within laminae II and III,
with axons descending to laminae V and X. In addition to
stained fibers, a population of motoneurons also exhibited
CGRP- immunoreactivity with staining being restricted to the
perikaryon.
When hemisection grafts were cut in the sagittal plane,
host CGRP fibers were commonly seen at three regions of host-
graft apposition: (1) the injured or intact dorsal roots, (2)
the ascending and descending tracts of Lissauer and dorsal
columns, and (3) the central canal or lamina X region. CGRP-
like fibers were observed within the grafts along each of

Figure 4-11. Ingrowth of Ox- and CGRP- containing fibers.
a) Fibers exhibiting Ox-like immunoreactivity enter a FSC transplant (t)
from the rostral host(h) graft interface (arrowheads). The labeled axons
are found mostly in the dorsal portion of the graft (arrows).
b) Higher power micrograph of the interface between a transplant (t) and
the host spinal cord (h) at the rostral host-graft interface (single
arrowheads). Small Ox-like axons are found within the scar (arrows).
In addition, other axons assumed a thickened appearance just rostral to
the interface (double arrowheads).
c) CGRP- containing fibers (arrows) enter a FSC transplant (t) to
innervate the caudal and dorsal quadrant. The host spinal cord (h)
caudal to the graft exhibits normal CGRP- immunoreactivity of fibers in
the dorsal horn and punctate labeling of motoneurons in the ventral horn
(VH). In this section a remnant of a nearby dorsal root (DR) is apposed
to the graft.
d) Higher power micrograph of CGRP- positive fibers at the interface
between a portion of the host dorsal root (h) and a transplant (t) .
Small caliber fibers are seen within the graft (arrows). In addition
some fibers travel parallel to the interface (single arrowheads), while
others within the dorsal root exhibit bulbous endings distal to the graft
(double arrowheads).
Scale in a,c = 200 /m; b,d = 50 /¿m.

112

113
regions. Most of the innervation by CGRP-like fibers was
seen in the dorsal half of the transplants and near the
caudal interface (Fig. 4-11 c, 4-12) , while a few fibers also
entered the grafts from the rostral border. The absolute
distance of ingrowth was difficult to discern, as fibers
entered the grafts from all directions. Most of the axons
were located within 1.0 mm of an interface. However, unlike
the 5-HT- and Ox-containing axons, some CGRP-stained fibers
were also found in deeper regions ventral extent of the
transplants (see Tessler et al., '88; Houle and Reier, '89).
The interface between injured dorsal roots and the dorsal
border of the grafts also revealed CGRP- immunoreactive
fibers that terminated without extending into the grafts. As
seen with other host fiber types, some of these axons also
exhibited large bulbous endings indicative of a failure of
elongation, while others coursed parallel to the interface
(Fig. 4-11 d).
Comparison of different fiber types. The patterns of
axonal ingrowth were compared in three recipients by
superimposing low-magnification tracings of adjacent sections
(Fig. 4-12) . In each case, the fibers entered the grafts
from host regions containing a dense innervation of the
respective fiber type. CGRP-containing axons usually
occupied the dorsal and caudal quadrant of the grafts. These
fibers showed the most robust penetration of the grafts by
aqualitative comparison of fiber density and the graft area

Figure 4-12. Drawing tube tracings of serial sagittal sections
illustrating the distribution of immunocytochemically stained axons in
a FSC graft at 3 months post-grafting. Sections extend from the lateral
(a) to medial (i) extent of the grafts. Caudal is to the left, rostral
is to the right; the dorsal surface is at the top and the ventral surface
is at the bottom of each drawing. Ox- and 5-HT containing fibers enter
the grafts from the rostral host-graft interface, while CGRP- containing
fibers enter from the caudal and dorsal borders. Regions of dense glial
reactivity are shown as lines or stippling within the grafts and at the
borders between host and graft tissues. Many stained axons appear to
enter the grafts across regions containing little scarring (between
arrowheads).
Scale
1.0 mm.

e

116
occupied by labeled axons. In contrast, Ox- and 5-HT-
containing axons were restricted to the rostral borders of
the grafts. Some degree of overlap was apparent in the
distribution these latter fiber types.
Presence of stained cells within FSC transplants
The antisera used in this study were selected because
intrinsic spinal cord cells containing these antigens are
rare or absent, and fibers within the grafts may be presumed
to arise from supraspinal or peripheral origin (Reier et al.,
'86a; Bregman, '87). However, in some of these grafts,
immunoreactive neurons were observed following incubation in
antisera against 5-HT and TH (Table 4-6). In addition, CGRP-
containing cells that stained with a distinctly different
pattern than motoneurons of the normal spinal cord were also
observed within some of the grafts. Similar cells were not
found in the adjacent host spinal cord. These stained
neurons may reflect the differentiation of a small number of
normal cells found in cervical and sacral regions of the
spinal cord (Newton et al., '86; Newton and Hamill, '88;
Mouchet et al., '86). Alternatively, these cells may
represent an abnormal expression of enzymes or peptide due to
the relative deafferented state of the graft tissue (see
Chapter 3, Discussion).
Discussion
Axonal projections formed between transplants of fetal
neural tissue and the host spinal cord may provide an

117
essential anatomical basis for graft-mediated functional
repair after spinal cord injury or disease. In the present
study, an emphasis was placed upon identifying the axonal
projections developed between FSC transplants and the
adjacent segments of the adult rat spinal cord.
Three complementary neuroanatomical tracing techniques
revealed a consistent pattern of axonal interactions between
host and graft tissues. First, a dense network of intrinsic
axonal projections was observed within the transplants.
Second, evidence for axonal outgrowth into the surrounding
host spinal cord was obtained in several graft recipients.
Axons also extended into transplants from neurons within the
adjacent segments of the host spinal cord. In addition,
immunocytochemical staining techniques revealed the
elongation of three distinct populations of host fibers into
FSC transplants.
Together, these axonal interactions suggest an anatomical
substratum that may ultimately serve as a functional neuronal
relay across the site of a spinal cord lesion. In addition,
these results provide some information about the cellular
dynamics that govern axonal outgrowth of developing and
mature spinal cord neurons.
Patterns of Host-Graft Projections
Intrinsic projections
Similar to studies using other transplantation paradigms
(Bolam et al., '87; Walker and McAllister, '87; Fonseca et

118
al., '88), the axonal tracing results demonstrated that cells
within FSC grafts establish complex and widespread networks
of intrinsic fiber projections. The intragraft circuitry
consisted of a predominance of short distance (<1.0 mm)
projections as well as a significant contingent of axons
which extended the length of the transplants.
Graft efferent projections
The transplanted neurons were also able to extend axons
into the adjacent segments of the host spinal cord. These
efferent fibers usually entered the host spinal cord by
penetrating regions of the host-graft interface where an
intervening glial border was not apparent (see below), and
terminated within both dorsal and ventral regions of the
spinal cord. Both the retrograde and anterograde tracing
paradigms provided evidence for axonal outgrowth extending at
least 4 - 5 mm from the host-graft interface, thus confirming
previous results from a more limited neuroanatomical study
(Reier et al., '86a). Similar distances of axonal outgrowth
have been reported for brainstem suspension grafts in rats
with chemical lesions of the spinal cord (Nornes et al.,
'83) .
Host projections into transplants: Local intraspinal neurons
The patterns of afferent ingrowth from local host neurons
were more complex. Injections of FG into either the
periphery or the center of the transplants resulted in
labeling of host neurons. As seen in preliminary WGA-HRP

119
tracing studies following transplantation into either acute
(Reier et al., '86a) or chronic (Houle and Reier, '88) spinal
lesions, most retrogradely labeled cells were located within
0.5 mm of the host-graft interface. This cellular
distribution is similar to that seen with peripheral nerve
(PNS) grafts to the adult spinal cord (David and Aguayo, '81;
Richardson et al., '84), as well as other regions of the CNS
(Benfey and Aguayo, 782). Those experiments suggested that
neurons axotomized close to the cell body may be more likely
to extend a process into a PNS graft than neurons located
farther away. A similar principle may influence axonal
regeneration or sprouting of host axons into FSC transplants
in the adult spinal cord.
Because of proximity of these FG labeled neurons to the
host-graft interface, one cannot exclude the possibility that
they may be grafted neurons that have migrated into the host
neuropil. Recent descriptions of intracerebral transplants
have illustrated the presence of grafted neurons as far as
0.5 mm away from the site of transplantation in both newborn
and adult recipients (McConnell, '85; Finsen and Zimmer,
/86) . Privat et al., ('86) have reported the migration of
transplanted neurons in the adult rat spinal cord. In that
study, serotonergic neurons were identified as far as 15 mm
from brainstem suspension grafts placed below a complete
spinal transection. However, this observation may be related
to the extent of denervation of the host spinal cord, as no

120
migration of serotonin-containing cells was observed
following a similar injection into the lumbar cord of rats
following chemical axotomy of the host serotonin-containing
fibers (Foster et al., '85). In both of these cases the
grafts were made one week after the initial lesion. While
the extent to which a similar mobility of fetal neurons
occurs in the acutely injured adult spinal cord has not yet
been determined, this hypothesis may be testable by combining
prelabeling of the grafted neurons (Lindsay et al., '87) with
a double-labeling strategy in the recipient (Wictorin et al.,
'88) .
Following larger FG injections, retrogradely labeled host
neurons were also found as far as 6 mm from the interface.
In contrast to the ingrowth from neurons bordering the
transplant, these more distant host cells were observed only
when the intragraft tracer injections were positioned next to
the interface (Fig. 4-4). Together with the anterograde
findings, this observation suggested that the distance of
ingrowth from host intraspinal neurons located farther than
1 mm from the host-graft interface is limited.
Host supraspinal projections into transplants
Supraspinal fiber ingrowth from brainstem and
hypothalamic neurons extended into the periphery of the
grafts in a manner resembling the short distance ingrowth of
host spinal cord neurons. It is likely that the failure to
label these supraspinal cells by retrograde transport of FG

121
from injections in the grafts is related to this distribution
of fibers. Specifically, the larger FG injections were not
placed near the rostral interface in these examples and would
not label these short distance projections.
Host primary afferent projections into transplants
In some recipients with FG injections restricted to the
transplants, we also observed labeled dorsal root ganglion
cells. The ingrowth of host primary afferent fibers was
further supported by immunocytochemical staining using
antisera directed against CGRP. Although the dorsal roots
were not intentionally injured in the present study, these
results are similar to those observed when cut dorsal roots
were inserted directly into the fetal tissue at the time of
grafting (Tessler et al., '88). With this latter paradigm,
more recent ultrastructural evidence has shown that such
primary afferent fibers can form synapses with neurons within
the graft (Itoh and Tessler, '88).
Variability in Labeling Patterns
Examination of GFAP- and Nissl- stained sections revealed
regions of intimate fusion between host and graft tissues in
nearly all specimens. Immunocytochemically stained sections
also suggested some degree of host-graft neuritic integration
in the absence of long-distance growth. Except for a few
outstanding examples, however, the quantity of host-graft
interactions revealed with the neuroanatomical tracing
methods was more limited than that expected from preliminary

122
findings (Reier et al., '85, '86a; Houle and Reier, '88).
This difference between the observed labeling patterns and
the anticipated extent of neuronal integration has also been
encountered in other studies (Lund and Harvey, '81; Pritzel
et al., '86; Wictorin et al., '88) and may be inherent to
technical limitations of the labeling procedures.
The use of any axonal tracing methods is dependent upon
the placement of injections relative to the distribution of
terminal fields or neuronal cell bodies of interest. Because
these injections select a small sample of total projections,
it is likely that the absolute fiber and cell numbers
represent a sampling of the total axonal projections formed
between host and transplant tissues (see also Harvey and
Lund, '81; Pritzel et al., '86).
A second concern involves the identification of axonal
projections around the interface region. To prevent the
possibility of false identification of projections, it is
necessary to adopt a conservative interpretation of the
injection site and the region of active tracer uptake (Warr,
'81; Mesulum, '82) . However, similar studies have shown that
retrograde tracer injections that are successfully confined
to a transplant often fail to demonstrate host-graft
projections that have been shown using complementary
anterograde techniques (cf., Lund and Harvey, '81; Harvey and
Lund, '81; also Pritzel et al., '86; Clarke et al., '88a;
McAllister et al., '89). Meanwhile, larger injections, which

123
may label fibers nearer the interface, are often eliminated
due to diffusion into the surrounding tissue.
A final technical consideration is the possibility of
unique axonal transport characteristics of the neurons within
the host and graft tissues. For instance, the transport of
some or all of these axonal tracing molecules may be altered
following axotomy, regeneration, or deafferentation (Feringa
et al., 83, 788, '89; Berkeley and Vierck, '87). Finally,
different tracers may produce different results due to
variations in sensitivity. For example, we observed a
greater degree of retrograde labeling following injections of
FG than we did following injections of HRP/WGA-HRP in normal
and transplanted rats (see also Cabana and Martin, 784) . The
presence of these additional variables emphasizes the need to
compare results of several techniques.
Issues Related to Axonal Growth
The nature and the extent of projections which are formed
between host and graft tissues depend upon both the intrinsic
growth capacity of the neurons and the role played by non¬
neuronal cells and extracellular components in the
surrounding environment. Considering the possibility that
many host-graft interactions may be located at interface
regions, the variability in axonal projection patterns, in
addition to a reflection of technical aspects, may be a
result of the time-course of graft development and the

124
relationship between graft maturation and the sequence of
events consequent to spinal cord injury.
Intrinsic elements related to axonal elongation
The present tracing experiments suggest that the
outgrowth of axons from FSC grafts is greater in terms of
overall distance than the corresponding ingrowth of
supraspinal and more distant intrinsic host spinal cord
neurons. A similar relationship has also been suggested in
other transplantation models in the adult CNS (Oblinger and
Das, '82; McLoon and Lund, '83; Raisman and Ebner, '83;
Freund et al., '85). Differences in the extent of axonal
elongation can be attributed in part to varying growth
capacities that are characteristic of adult and developing
neurons. For example, the production of specific growth-
associated proteins that correlate closely with axonal
elongation is developmentally regulated and decreases with
the maturation of CNS neurons (Jacobson et al., '86) .
Comparisons of afferent projections following transplantation
into hosts of different ages have shown uniformly that axonal
elongation from newborn host brain into transplants is more
extensive than ingrowth from mature brain (Lund and Harvey,
'81; McLoon and Lund, '83) or spinal cord (Bregman et al.,
'89). Similar differences may underlie the greater degree
of ingrowth of CGRP-containing afferents than that of
supraspinal afferent fibers. Furthermore, there is some
evidence that different types of mature CNS neurons exhibit

125
differing capacities for axonal elongation into fetal neural
transplants (Pritzel et al., '86? Nothias et al., '88;
Wictorin et al., '88; Doucet et al., '89).
Astroglial cells and axonal outgrowth
Non-neuronal cells present in adult spinal cord may also
contribute to the patterns of axonal projections. Astroglial
elements within the mature CNS undergo a response to injury
(i.e., gliosis) which includes the hypertrophy of their
processes and an increase in the production of GFAP (rev. in
Nathaniel and Nathaniel, '81; Eng, '88). The formation of
a glial scar between host and graft tissues may prevent the
development of axonal projections between the two tissues
(Raisman and Ebner, '83; Azmitia and Whitaker, '83; Das, '83;
Kruger et al., '86; Reier, '86).
While no quantitative relationship was found between the
amount of glial scarring at the interface and numbers of
retrogradely labeled cells, the influence of the scar on
axonal outgrowth was apparent from qualitative observations.
Although the transplant axons labeled with PHA-L did not
appear to travel through interface regions of dense gliosis,
they rarely formed club-like endings indicative of abortive
axonal elongation (Ramon y Cajal, '28; Sung, '81? Liuzzi and
Lasek, '87). In contrast, such endings were often found in
immunocytochemically stained sections of host fibers at the
host-graft interface or after PHA-L injections into the host
spinal cord.

126
When PHA-L was injected into the grafts, the axonal
trajectories suggested that the presence of reactive
astrocytes may re-direct axonal elongation along the
interface. Interruptions in the scar may then permit some
projections into the host spinal cord. In this regard, it is
possible that these astrocytes may be a favorable substratum
for the elongating axons from the graft, but the distribution
of processes or the orientation of the scar may be important
for directed growth across the host-graft interface (Reier et
al., '89).
Results of other studies have suggested that the degree
of gliosis within a transplant may reflect the extent of
axonal integration between host and graft tissues (Bjorklund,
H. and Dahl, '82; Bjorklund,H. and Olson, '83). Therefore,
we examined the degree of GFAP staining in the graft and host
tissues of seven recipients. Four grafts showed greater
glial reactivity than the surrounding host tissue. There was
no correlation, however, between the ratio of glial fiber
density between graft and host tissue and the numbers of
retrogradely labeled cells. Although this approach provided
a quantitative measure of glial reactivity, the technical
difficulties associated with the quantification of axonal
projections severely limited the interpretation of these
findings.

127
Timing of graft development and axonal interactions
The development of short distance projections, as well
as the presence of host and graft fibers at the interface
region, indicates that the timing of host-graft apposition
may be a critical factor in the development of neuronal
interactions in the adult. This possibility is supported by
the appearance of longer axonal ingrowth from host neurons
situated near the interface than from host cells located
farther away. Since there may be a delay between the time of
injury and when either axotomized or uninjured (i.e.,
sprouting) cells in the host begin to extend fibers,
considerable maturation of the grafts may be underway by the
time that axons have reached the transplants. The
enlargement of the solid transplants within the lesion cavity
may also contribute to variations in the formation of
projections, as host fibers in ventral regions are apposed to
the graft tissue at an earlier stage in its development than
the more dorsal host cells and axons (Kruger et al., '86).
These interactions may be even more complex in cases where
some axonal die-back of host fibers precedes regrowth.
The rapid rate at which rodent tissue matures may also
contribute to the predominance of short distance ingrowth.
While many components within the embryonic CNS tissue should
favor axonal elongation (Aquino et al., '84; Beasley and
Stallcup, '87; Liesi and Silver, '88), it is likely that
these elements are not uniformly available to all host axons

128
in the injured adult CNS at the appropriate time. Likewise,
the progressive expression of non-permissive molecules in the
maturing graft may play a role in the limited ingrowth
observed (e.g. myelin-associated proteins; Caroni and Schwab,
'88). Finally, the timing of axonal elongation relative to
these factors is further complicated by the possibility that
the extent of axonal elongation may be limited by the
formation of synapses within the graft or the denervated host
environment (Bernstein and Bernstein, 'll). The last study
of this series will explore some of these timing issues as
they relate to the interactions between a long myelinated
fiber tract and the local circuitry established at the host-
graft interface.

CHAPTER 5
INTERACTIONS BETWEEN INJURED CORTICOSPINAL TRACT AXONS
AND FETAL SPINAL CORD TRANSPLANTS IN THE ADULT RAT
Introduction
Transplants of fetal spinal cord (FSC) tissue, when
grafted into lesions in the adult spinal cord, may serve to
replace tissue that is destroyed by injury or disease (Reier
et al., '83, '86a; Houle and Reier, '88; Winialski and Reier,
'89; Nothias et al., '89). In addition, areas within these
transplants exhibit some features of normal spinal gray
matter. Thus, such grafts may be used to replace intrinsic
spinal neurons (Jakeman et al., '89; Chapter 3).
Evidence obtained from neuroanatomical tracing and
immunocytochemical studies has shown that axonal projections
develop both within FSC transplants and between the
transplants and the surrounding host spinal cord (Chapter
4). For example, afferent ingrowth from dorsal root fibers
(also Tessler et al., '88), serotonergic fibers from
brainstem nuclei (Reier et al., '86a), oxytocin-containing
fibers from hypothalamus, and local ingrowth from spinal cord
(Reier et al., '86a) neurons has been demonstrated. Finally,
cells distributed throughout the grafts have been shown to
extend into the surrounding spinal cord and terminate around
129

130
host neurons. The axonal projections that are formed between
cells of the host spinal cord and the transplanted tissue may
provide an anatomical basis for the formation of a neuronal
relay across a spinal lesion site (Reier, '85; Reier et al.,
'88; Chapter 4).
The transmission of ascending and descending information
across a spinal cord lesion is likely to require interactions
between long, myelinated fiber tracts in the host spinal cord
and the local circuitry established between host and graft
tissue near the interface. To date, the anatomical basis for
such interactions has not been explored thoroughly.
The rat corticospinal tract (CST) offers several
advantages as a model system for examining the interactions
between long fiber tracts and FSC transplants. In the rodent,
the CST forms a compact fiber system which is easily
identified in histological preparations. Furthermore,
anterograde tracing methods can be readily applied to
determine the projection patterns of this specific population
of descending fibers (Gribnau et al., '86; Joosten et al.,
'87; Casale et al., '88) .
The response of CST axons to different types of lesions
has been well documented. When the spinal cord is injured in
newborn cats, rats, or hamsters, axons of the developing CST
circumvent the lesion and extend through the remaining
immature spinal cord tissue by traversing alternative
pathways. The immature axons continue beyond the lesion site

131
to reinnervate the appropriate target regions (Bregman and
Goldberger, '83; Bernstein and Stelzner, 'S3; Schreyer and
Jones, '83; Kalil, '84; Tolbert and Der, '87). This
developmental plasticity decreases within the first three
weeks postnatal in the rat and is not observed following a
similar lesion in adult animals. Instead, injured adult CST
axons often exhibit a progressive retrograde degeneration
away from the site of the injury (Kalil and Schneider, '75;
Feringa et al., '83; Fishman and Kelly, '84a,b; Tator et al.,
'84; Pallini et al., '88).
To date, there is little evidence to indicate that adult
CST axons are capable of regeneration or collateral sprouting
following axotomy in the spinal cord (Kalil and Reh, '82;
Richardson et al., '84; Kuang and Kalil, '89). If this
limitation is a result of inhibitory influences within the
adult spinal cord environment, it may be possible to promote
the elongation of these axons by transplanting fetal tissue
at the site of a spinal injury. For example, a recent report
has suggested that the presence of FSC transplants at a
lesion site will prolong the period of developmental
plasticity of corticospinal tract fibers in young rats
(Bregman et al., '89). It is unknown whether similar
transplants can prevent retrograde degeneration or support a
sprouting response of CST axons after injury in the adult.
Therefore, the objective of the present study was to
establish whether transplants of FSC tissue would influence

132
the reaction of CST fibers to injury and either allow the
integration of this descending tract with neurons at the
host-graft interface or permit some fibers to extend into
the transplants. Because several studies have shown that the
retraction of injured CST axons can occur as soon as a few
days after injury (Kao et al., '83; Fishman and Kelly, '84b),
an additional paradigm was included to determine if a delay
between the time of injury and placement of graft tissue
would still permit integration of injured CST fibers and FSC
transplants. Portions of this study have been presented in
preliminary form (Jakeman and Reier, '87, '89; Reier et al.,
'88) .
Materials and Methods
Animals and Surgical Procedures
Adult female rats (approximately 8-20 weeks of age)
were used throughout this study. The normal CST was examined
in 3 rats, 22 rats received lesions only, and 53 received
lesions and transplants of fetal rat spinal cord (FSC) tissue
(Fig. 5-1). The experimental results were obtained only from
rats with successful labeling of the CST and complete
bilateral lesions of the dorsal columns (n=56).
The lesion and transplantation procedures were similar
to those described previously (see Chapter 2). After the
rats were anesthetized with ketamine and xylazine, the dorsal
surface of the spinal cord was exposed by performing a
laminectomy of the C6 or T2 vertebrae. Then, a lesion of

133
Bilateral CST Lesions
Close lesion cavity
1 1 r
4 d - 1 wk delay
Eu FSC transplant
0 d-14mo 3xno-17mo
WGA-HRP
2-3 d
transport
WGA-HRP PHA-L
2-3 d 20 d
trans. trans.
1
1
LESION ONLY
(N=18)
DELAYED GRAFTS
(N=6)
Eu FSC transplant
0
d-4xno
WGA-HRP PHA-L
2-3 d 20 d
trans. trans.
1
ACUTE GRAFTS
(N=32)
FIGURE 5-1. Experimental design. Experimental rats received
lesion only, delayed grafts or acute grafts of FSC tissue as
outlined. WGA-HRP or PHA-L were applied bilaterally to the
sensorimotor cortex at various times after the lesion or
transplant. (d=day).

134
2 - 3 mm in length was created by aspiration. The lesion
provided a cavity for transplantation and served to sever the
dorsal columns bilaterally. Some rats received over-
hemisection lesions (Bernstein and Stelzner, '83) , which
extended to the ventral floor of the spinal cord. In others,
more shallow lesions were created by sealing the dorsal vein
with an ophthalmic cautery (Neuromedics, Inc.) at 2 sites
separated by 2 - 3 mm and subsequently removing the
intervening tissue from the dorsal columns and dorsal horn.
FSC tissue was placed into the lesion site and the spinal
cord meninges and muscle incisions were closed in layers as
described in previous studies.
One group of rats (n=6) received transplants according
to a delay paradigm (Fig. 5-1) . After the initial lesion
cavity was made, the wound was closed and the rats were
allowed to recover. Following a delay of 4 days to 1 week,
the rats were reanesthetized and the cavity was surgically
exposed with no further resection of host tissue. A single
piece of Eu FSC was then placed into the cavity and the
meninges closed as described above.
Tracer Application and Tissue Processing
In order to label axons of the CST, the rats were
reanesthetized and placed into a rat head stereotaxic
apparatus (Kopf). A craniotomy was then performed to expose
both hindlimb and forelimb regions of the sensorimotor cortex
(+3 - -4 mm AP; 1-4 mm ML relative to Bregma (Welker '71;

135
Wise and Jones, '77; Gioanni and LaMarche, '85)). On each
side, the overlying dura was removed and superficial bleeding
was controlled with pieces of absorbable gelatin sponge
soaked in bovine thrombin. After the injections were
completed, the bone defects were covered with a small piece
of Durafilm and the overlying skin closed with 4-0 sutures or
surgical wound clips.
HRP and WGA-HRP injections
Rats were labeled at various intervals after either
lesion only or lesion plus transplantation (Table 5-1) .
Multiple (5 - 10) injections of 0.2 - 0.4 nl of 2.0% WGA-HRP
were made into each sensorimotor cortex using a nitrogen-
burst apparatus (picospritzer). In some animals an
alternative method of tracer application was used, whereby
3 tungsten wires containing a bolus (0.5 mm diameter) of
dried HRP and WGA-HRP solution were placed below the surface
of each cortex and removed after the solution had dissolved.
The degree of tract labeling was similar using either method.
After allowing 48 - 72 hours for axonal transport, the
recipients containing HRP/WGA-HRP injections were deeply
anesthetized with sodium pentobarbital and perfused
transcardially with 150 ml heparinized 0.9% NaCl followed by
250 ml fixative (1.0% paraformaldehyde +2.5% glutaraldehyde
in 0.1 M Sorenson's phosphate buffer). Tissue blocks,
including the transplant and 5 mm of host spinal cord rostral
and caudal to the graft, were removed. Vibratome sections of

136
50 /¿m thickness were cut in the sagittal or horizontal plane
and the sections were reacted within 3 hours of sectioning
according to the tetramethylbenzidine (TMB) protocol of de
Olmos et al. ('78). Sections were then mounted onto gelatin-
coated slides and counterstained with 1.0% Neutral Red to
reveal the cytoarchitecture of the host and transplant
tissues.
TABLE 5-1. NUMBERS OF RATS AND TIME POINTS FOR CST LABEL
AFTER LESION OR LESION AND TRANSPLANTATION
WGA-HRP
PHA-L
Lesion only
Transplants
(Acute)
Transplants
(Acute)
C6 T2
C6 T2
C6
3 d
l-2wk
6 wk
3-4 mo
3
4
5 3
3
2
2
12 6
4
6
Transplants
(Delayed)
C6 T2
Transplants
(Delayed)
C6
2 mo 5 -
17 mo - - 1
Time between lesion or transplantation and perfusion.
In all cases, WGA-HRP labeling was done 48-72 hours
before sacrifice, PHA-L labeling was done 20 days prior
to sacrifice.
Distances of retraction of CST at 6 weeks after lesion
or lesion and transplantation were used for quantitative
analysis.

137
The extent of CST retraction or die-back was examined in
HRP/WGA-HRP recipients at 6 weeks after injury (Table 5-1).
Drawing tube tracings were made of each TMB stained
horizontal or sagittal section containing the labeled CST and
the lesion site or transplant. Two lines were superposed
upon the tracings, perpendicular to the axis of the dorsal
CST. One line marked the rostral extent of the space
occupied by a lesion or the rostral border between host and
graft tissue. Where host and graft tissues were well
integrated, the host-graft interface had to be defined by
examining the section (counterstained with Neutral Red) with
brightfield optics (as in Fig. 5-6) . The interface was
identified by a transition between the highly organized
cytoarchitecture of the host spinal cord and the less
organized graft tissue. The second line was then drawn
rostral to the first, at the caudal end of the heavily
labeled CST (referred to as the bulk of the tract) (see Fig.
5-4). For each section, the distance between the two lines
was measured and corrected for the enlargement of the
drawing. An average value was then obtained for each animal
used in the quantitative analysis. Group means were tested
for differences at the p<0.5 level using a one way ANOVA and
individual comparisons were determined by Tukey's hsd (Spence
et al., '83) .

138
Phaseolus vulgaris leucoaaalutinin (PHA-L) injections
Anterogradely-filled CST axons and terminals were
identified by immunocytochemical detection of PHA-L (Vector
Laboratories Inc.)* The tracer was applied to the cortex of
7 rats (4 month grafts, n=6; delayed graft, 17 months, n=l)
using a modification of the methods of Gerfen and Sawchenko
(/84). A 2.5% solution of PHA-L was prepared in 10 mM
phosphate buffer (pH 8.0). Glass micropipettes were cleaned
and broken to a tip diameter of 10-15 /¿m. A craniotomy was
then performed bilaterally (as above) and 3 iontophoretic
injections were made into each cortex.
After allowing 2 0 days for transport of the PHA-L, the
recipients were perfused as above with fixative containing
4.0% paraformaldehyde and 0.25% glutaraldehyde. The cord
blocks including the transplant and 5 - 10 mm of the
surrounding rostral and caudal spinal cord were removed and
postfixed overnight at 4°C. Free-floating Vibratome sections
of 40 /¿m thickness were processed for the identification of
PHA-L containing cells and processes as described in Chapter
4. The sections were first washed in 0.02 M Potassium
Phosphate Buffered Saline (KPBS) and incubated for 2-4 hrs
in a preblocking bath containing 2.0% normal rabbit serum and
0.3% Triton X-100. Triton X-100 was excluded or reduced to
0.1% for sections selected for electron microscopy (below).
All the sections were then incubated in goat anti-PHA-L
(Vector; 1:5000) diluted in KPBS for 36 hours at 4°C and then

139
for 2 additional hours at room temperature. The sections
were rewashed and then processed with biotinylated rabbit
anti-goat IgG (1:225) and Vector Avidin-Biotin-peroxidase
Complex (ABC). The final peroxidase conjugate was reacted
with H202 in the presence of 0.005% diaminobenzidine (DAB).
Nickel-enhanced sections were counterstained with 0.1% Cresyl
Violet or 1.0% Neutral Red prior to coverslipping.
Electron Microscopy
Following incubation in DAB, those sections selected for
electron microscopy were incubated for 1 hr in 1.0% osmium
tetroxide. The sections were then stained en bloc in 0.5%
uranyl acetate in maleate buffer, dehydrated in alcohol and
propylene oxide, infiltrated with EM Bed 812, and mounted
between vinyl slides. The regions of interest were
photographed and then cut and remounted on plastic blocks.
Ultrathin sections were collected on copper grids and
examined with a Zeiss EM10C electron microscope.
Results
WGA-HRP Tract Tracing
Normal CST
Anterogradely labeled corticospinal fibers in normal
adult rats were distributed into three pathways (Fig. 5-2 a)
(Donatelle, '77; Schreyer and Jones, '82; Gribnau et al.,
'86; Casale et al., '88; Bregman et al., '89). The bulk of
the labeled fibers were concentrated in the ventral portion
of the dorsal funiculus (dorsal CST), while a smaller pathway

140
was observed in the dorsolateral funiculus (Schreyer and
Jones, '82) . Within the cervical spinal cord, labeled fibers
were also encountered in the most medial region of the
ventral funiculus (Vahlsing and Feringa, '80). Axonal
labeled terminal fields were concentrated in the medial
dorsal horn and intermediate grey regions (approximate
laminae III - V; Steiner and Turner, '72) , while fewer fibers
were found within the superficial dorsal horn and lateral
intermediate grey (laminae II and VII).
Response of the CST to lesion only
General comments. Some variability in the extent of
labeling was observed with both the pressure injection and
tungsten wire application techniques. While the proximal end
of the injured tract was routinely labeled, in some instances
only light labeling of axons was obtained at distances of 2-
4 mm rostral to the site of injury. This less intense
labeling, however, proved useful in demonstrating the
terminal ends of individual axons rostral to a lesion or
transplant (Fig. 5-2 b).
In animals with complete lesions of the dorsal funiculus,
no labeled fibers were ever observed in the dorsal columns
caudal to the site of the lesion or transplants. Therefore,
references to the caudal extent of the injured dorsal CST
refer exclusively to the portion of the tract rostral to the
lesion site. Following shallow midline lesions, a small
number of CST fibers in the lateral and ventral white matter

Figure 5-2. Darkfield micrographs of the patterns of
HRP/WGA-HRP labeling in normal animals and rostral to the
site of lesion or grafting.
a) Normal labeling pattern in cervical spinal cord following
bilateral cortical injections of HRP and WGA-HRP. The bulk
of the CST is found in the ventral most portion of the dorsal
columns (d). Smaller components are located in the ventral
(v) and lateral columns (1) . Axons innervate the medial
dorsal horn and intermediate gray regions of the spinal cord
(arrowheads) . Transverse 50 ¿im section.
b) Variability in anterograde labeling at 2-3 mm rostral to
a lesion site. The CST on the left of this horizontal
section is labeled much less intensely than that on the
right. This lighter labeling pattern allowed examination of
the individual axons and terminal bulbs (arrowheads) rostral
to a lesion.
Scale in a, b = 100 nm.

142

143
were seen occasionally below the level of the lesion. These
fibers were probably spared by the lesion.
Three days to two weeks post-lesion. Anterograde
transport revealed a histopathology following partial spinal
lesions involving the CST that were similar to that described
in the mouse and rat following complete transection of the
spinal cord (Fishman and Kelly, '84a,b; Pallini et al., '88).
In the first few days after aspirative injury there were
signs of myelin breakdown and retrograde degeneration within
the dorsal CST (Fig. 5-3 a). The caudal extent of this tract
was marked by an accumulation of hematogenous cells, and the
tissue in the region of the tract was necrotic in appearance.
Some retraction bulbs were present at the ends of larger
axons proximal to the lesion. In addition, labeled axons at
the lesion site were thickened (Fig. 5-3b). In comparison,
less hemorrhaging was evident within the dorsolateral and
ventral white matter of the spinal cord rostral to those
lesions which interrupted these tracts. Injured lateral and
ventral labeled fibers remained directly adjacent to the
margin of the lesion while others had retracted a short
distance.
By two weeks post-injury, the lesion site was cleared
most hemorrhagia, and the region just rostral to the lesion
was occupied by necrotic tissue, degenerating fibers and
moderate cystic cavitation (Fig. 5-3 c). The CST within the
dorsal funiculus had undergone retrograde degeneration, and

144
the tract terminated approximately 0.5 - 1.0 mm from the
injury site. At this site, the tract assumed a tapered
configuration under the degenerating dorsal column fibers.
In addition to the observed retraction of the bulk of the
CST, some heterogeneity in axonal die-back of individual
fibers was also noted. Although some of these axons remained
directly at the wound edge or within the necrotic region of
tissue (Fig. 5-3 b), other axons had retracted even farther
from the lesion than the bulk of the tract and exhibited
distinct ovoid retraction bulbs at their terminals. Less
necrosis was evident within the dorsolateral columns, and in
these regions some CST axons had retracted as far as 1 mm
from the lesion while others many remained within 0.2 mm of
the edge of the tissue.
Six weeks post-iniurv. The progressive degeneration of
dorsal CST fibers was still evident in rats sacrificed at six
weeks following injury. Again, the end of the labeled CST
had a tapered appearance as the injured axons had retracted
from the lesion edge (Fig. 5-4 a, c). The region between the
caudal extent of the dorsal CST and the edge of the lesion
was often occupied by cystic cavitation, and contained
necrotic tissue and degenerating fibers. By this six week
time point, the average distance between the end of the
dorsal CST and the edge of the remaining tissue was 1.0 + .26
mm (n=8) . The distance of retraction was greater when the

Figure 5-3. Sagittal sections through a lesion site at 3
days or 2 weeks following lesion only or lesion plus
transplantation.
a) At 3 days after a lesion (L), the injured CST (arrowhead)
is occupied by extensive hemorrhaging. A few thickened axons
and terminal bulbs can be seen rostral to the lesion site
(right hand side of photo). The dorsal columns are seen above
the labeled CST. Within the ventral white matter (bottom of
photo) , hemorrhaging is less extensive (arrow) and
interrupted thickened axons remain closer to the lesion site.
b) Injured CST axons at 3 days after a lesion are thickened
in appearance (arrowheads) and surrounded by hemorrhagic
tissue.
c) By 2 weeks after a lesion, the CST, just above the central
canal (c) , is tapered. The lesion site is occupied by
necrotic tissue and a small amount of hemorrhagia is present
(arrow).
d) Most of the injured CST axons have retracted from the
lesion at 2 weeks post-injury, while a few axons (arrowheads)
remain closer to the injury site.
e) Section through the gray matter of a 2 week transplant
(T) . The tissue was not yet fully apposed to the host spinal
cord (h).
f) By 2 weeks after injury and transplantation, CST axons
have retracted from the injury site. A few axons remain
closer to the edge of the tissue (arrowheads).
Scale in a,c,e = 500 nm; b,d and f= 100 ¿¿m.

146

147
lesions were made at the T2 vertebral level (mean =1.25 + .26
mm; n=3) than when lesions were made at the C6 level (mean
=0.72 + .21 mm; n=5) (p < 0.05) (Fig. 5-5, open bars).
As seen at earlier post-injury times, individual labeled
terminal bulbs were also observed in these specimens. These
retraction bulbs were evident both at the end of the tract
and as far as 2 - 4 mm rostral to the lesion. Many of the
individual fibers were directly apposed to edge or lateral
margins of these cysts.
Three to four months post-injury. The pattern of CST
degeneration and cavitation was similar in lesion recipients
sacrificed at longer survival times (3 - 4 months). The
caudal extent of the CST had retracted approximately 1.0 mm,
while additional enlarged axon terminals were found rostral
to the lesion (Fig. 5-4 e) . Although each specimen was
slightly different, the overall appearance of the tract and
the regions of cystic cavitation were similar to those
observed 6 weeks after injury.
Response of the CST to lesion and transplantation
Three days to two weeks post-grafting. The presence of
a graft in an overhemisection cavity did not have a
noticeable effect upon the initial response of the injured
dorsal CST fibers. In each of the animals sacrificed at
three days or two weeks following transplantation, a small
piece of graft tissue was found along the ventromedial or
lateral wall of the cavity, but it was not adjacent to the

Figure 5-4. Retraction of the CST following lesion only or
lesion plus transplantation. Bilateral injections of WGA-
HRP into the sensorimotor cortex.
a) By 6 weeks after injury, the labeled axons of the CST have
retracted from the edge of the lesion (L). Horizontal section
through the level of the dorsal columns. The distances from
the caudal most extent of the tract to the lesion edge were
measured as shown (white lines). The edge of the lesion is
seen in the very bottom of the figure and verified by
brightfield illumination. Note that individual fibers in the
lateral white matter remain near the edge of the lesion site
(arrowheads).
b) Horizontal sections through the labeled CST at 6 wks
following lesion plus transplantation (T) . A small cyst is
found between the labeled CST and the rostral portion of the
graft. Some labeled axons extend along the margins of the
cyst (arrowhead). The rostral host-graft interface (dotted
line) was determined by examination of the cytoarchitecture
under brightfield illumination.
c) Second example of labeled CST at 6 weeks after lesion
only. Cystic regions separate the injured tract from the edge
of the lesion (L).
d) Example of injured CST directly apposed to a 6 week
transplant (T) . In this example, no cysts are present at the
host-graft interface (dotted line), and labeled fibers are
seen at the edge of the graft.
e) Injured CST fibers retract from the edge of the lesion
(L) at 3 months after lesion only.
f) Three months after lesion plus transplantation, injured
CST axons remain at the host-graft interface.
Scale a - f = 100 ¿un.

149

Figure 5-5. Average distances between the caudal extent of
the dorsal CST and the lesion or host-graft border in T2 and
C6 recipients. Overall average for lesion only (empty bars)
= 1.0 mm + 0.26 (n=8) ; Overall average for lesion and
transplantation (hatched bars) = 0.48 mm + 0.25, (n=18).
These two groups were different by Student's t test (p< .05) .
ANOVA indicated that the means of all four groups were
different (p < .05). Differences were found by Tukey's hsd
between the lesion only and lesion plus transplantation at
both the T2 (and C6 vertebral levels (p< 0.05). Transplants
that were partially apposed to the dorsal CST were included
in the totals. However, three recipients with failed grafts
were not included.

151
Retraction of dCST 6 Weeks
After Lesion or Lesion + Transplant
1 1 Lesion Only
Lesion + Transplant

152
injured fibers of the dorsal funiculus (Fig. 5-3 e) . The
labeled dorsal CST tract was marked by an accumulation of
blood cells and macrophage-like profiles at the earliest time
points. By two weeks post-grafting, most of the labeled
fibers in the dorsal CST had retracted from the edge of the
lesion (Fig. 5-3 c) , and axons ended in retraction bulbs
rostral to the lesion (Fig. 5-3 f).
Six weeks post-grafting. Transplants examined six weeks
after transplantation were well developed, and most had
filled the lesion cavity. The appearance of the cytoarchi-
tecture and degree of myelination were similar to that
described previously (Reier et al., '86a), and suggested that
the grafts were mature and well differentiated.
In the presence of FSC transplants, there was a reduction
in the distance of retrograde degeneration in the dorsal CST
observed at 6 weeks post-injury, as measured using these
methods. In some of these recipients, the qualitative
pattern resembled that seen after a lesion only, as small
cysts were seen between the labeled fibers of the dorsal CST
and the host-graft interface.
However, several of these specimens exhibited regions of
direct apposition between the tapered end of the CST and the
rostral extent of the graft (Fig. 5-4 b, d) . The distance
between the end of the dorsal CST and the host-graft
interface across all the 6 week transplant recipients was
0.48 + .25 mm (n= 18). The distance reflected a decrease in

153
the extent of retrograde degeneration at both the C6 (mean =
0.36 + .18 mm; n=12) and T2 (mean = 0.72 ± .21 mm n=6)
vertebral levels as compared to animals with lesions only
(Fig. 5-5; hatched bars).
Of the 18 recipients used in the quantitative
observations, three contained grafts that were apposed to the
injured CST in the regions near the central canal, but they
were not apposed to the most dorsal portions of the tract.
These recipients did not differ in the average distance of
CST retraction from the remainder of the graft recipients
(distances = 0.91, 0.69, 0.24 mm). In contrast, three
additional graft recipients exhibited good CST labeling, but
had no surviving grafts. Of these failed transplant
recipients, two exhibited CST retraction for distances
similar or greater than those seen in lesion-only recipients
(distances = 2.0, 1.7 mm). The third recipient in this group
had a cyst at the original lesion site which had enlarged to
over 6 mm in length. Injured CST axons were found at the
edge of this cavity.
Three to four months post-grafting. Of five recipients
sacrificed after longer survival times, only one showed
extensive degeneration within the dorsal CST extending as far
as 1.0 mm rostral to the graft. The remaining four specimens
contained regions of integration between the dorsal CST and
the graft similar to that seen in many specimens at six weeks

154
post-grafting. In these animals, labeled CST axons were
directly apposed to the host-graft interface (Fig. 5-4 f).
Delayed grafts When embryonic spinal cord tissue was
placed into lesions that were prepared one week previously
(delayed grafts), the qualitative pattern of axonal labeling
was similar to that observed when grafts were placed into the
acute lesions. Some of the dorsal CST fibers had retracted
from the lesion site, and small cystic cavities were
sometimes present between the caudal end of the tract and the
graft. As seen in the acute (immediate) grafts, labeled
axons were also found along the margins of these cystic
cavities. However, in four of the five recipients, there
were regions of the interface between the host CST and the
graft where the injured tract was apposed to the graft tissue
with no intervening cystic cavities.
Ingrowth of CST axons
In examples that showed the best integration of host and
graft tissue, TMB stained sections contained axonal profiles
resembling individual CST axons within the transplants.
These labeled axons could be followed for up to 200 /im before
they terminated or left the plane of section. The majority
of these ingrowing fibers were found near the end of the
dorsal CST, in the dorsal and rostral edge of the graft (Fig.
5-6 a-d). However, in one case, fibers entered from the
lateral edge of the graft (Fig. 5-6 e). No axonal profiles
were found in the caudal regions of any of these transplants.

Figure 5-6. Injections of WGA-HRP into sensorimotor cortex.
Axonal profiles stained with TMB appear to extend across the
host-graft interface (dotted lines) and into 6 wk FSC
transplants (T) . Horizontal 50 /¿m sections.
a,b) Brightfield photomicrograph of Neutral Red sections
through the rostral host-graft interface of two specimens.
In each case, the interface is evident by the presence of a
glial border (small densely packed cells) along the left
side. A difference in cytoarchitectural organization
indicated the host-graft interface on the right (small black
dotted line). Corresponding blood vessels and background
reaction product landmarks in (a,c) and (b,d) are identified
by (*).
c) Two axons (white arrowheads) enter the FSC transplant
shown in (a).
d) More axons enter another graft from a well fused region
of the ventral host-graft interface as shown in (b).
e) Axons also enter a FSC transplant from the dorsolateral
funiculus.
f) Occasionally, labeled axons travel parallel to the host-
graft interface (white v arrowheads).
Scale in a - f = 100 /¿m.

156
(*â– 

157
Additional axonal profiles within the host gray matter
were directly apposed to graft tissue in many of these
recipients. A few of these labeled axons coursed parallel to
the host-graft interface, while others appeared to terminate
just rostral to the graft or disappear from the plane of
section (Fig. 5-6 f).
Anterograde Transport of PHA-L
Transplants placed into acute lesion cavities
Four months after grafting (earliest time examined), all
of the transplants had filled the lesion cavities and each
was apposed to the host tissue along the rostral and lateral
borders. The PHA-L injection sites resulted in heavy
labeling of cells throughout cortical layers 3-6 (Fig. 5-7
a) . Labeled terminals were found within the dorsolateral
striatum and thalamus in all of these animals, and CST axons
were found both within the tracts and gray matter rostral to
the transplants.
Many of the labeled CST fibers terminated in large
retraction bulbs as far as 4 mm from the rostral border of
the graft (Fig. 5-7 b) . Most of these endings were of a
typical ovoid shape, ranging from (5-10 /xm) in diameter.
Additional axonal endings were more irregular in shape,
similar to those described following injury to the dorsal
columns of rats and other species (Ramon y Cajal, '28; Lance,
'54) .

158
The identified CST axons had extended into three of the
transplants. Based upon the location of these fibers within
the grafts, the axons appeared to have penetrated the
transplants from the rostral and lateral host-graft borders.
The axons extended into the transplants for distances ranging
from 0.1 - 0.3 mm before arborizing (Fig. 5-7 c, d).
Labeled CST axons within the grafts could be followed for
some distance in these 4 0 /¿m sections. Drawing tube tracings
of the labeled fibers across several planes of focus
indicated that only a few CST axons in the grafts had been
labeled with the PHA-L injections. Nevertheless, each of the
ingrowing axons exhibited extensive branching within the
graft (Fig. 5-8). Axon collaterals could be followed along
longitudinal trajectories (700-800 /xm within a section) ,
similar to those reported in the superficial dorsal horn of
the normal cervical spinal cord (Casale et al., '88). In all
examples, enlargements resembling terminal boutons
(Wouterlood and Groenewegen, '85) were found along the
lengths of the axons, as well as at their endings.
In addition to the ingrowth of CST fibers in these
transplants, some labeled axons were also observed adjacent
to the rostral and lateral borders of all six of these
grafts. Axons that did not enter the transplants coursed
parallel to the host-graft interface and exhibited normal
axonal varicosities along their length.

Figure 5-7. Labeled CST fibers following iontophoretic
injections of PHA-L.
a) Example of an injection site within the sensorimotor
cortex.
b) Labeled fibers and terminal bulbs within the dorsal CST,
approx. 2 mm rostral to the site of transplantation.
c) Cresyl Violet-stained section through a 4 month transplant
containing PHA-L labeled axons (arrowheads).
d) Enlargement of labeled CST fibers within a 4 month FSC
transplant.
Scale in a = 100 nm; Scale in b,c,d = 20 /urn.

16o

Figure 5-8. CST axons, labeled by PHA-L injections into the cortex, are seen to enter
and branch within FSC transplants.
a) Horizontal section through a 4 month FSC transplant within the medial region of the
host spinal cord. The dorsal CST is well apposed to the rostral host-graft interface.
b) Drawing tube tracing of axon(s) within the transplant; the vessel along the
interface (arrow) corresponds to the vessel in a.
Scale in a,b = 100/¿m.

162

163
Delayed transplant
An additional recipient received a transplant placed into
a cavity that was created one week prior to the time of
grafting. This rat was retained for 17 months after
transplantation to determine the long-term persistence of
ingrowing fibers. Labeled CST fibers was observed within the
transplanted tissue (Fig. 5-9). A group of labeled fibers
was observed extending across the interface between the
dorsal CST and the graft. These fibers extended a short
distance into the rostral portion of the graft, and showed
much branching near the interface (Fig. 5-9 a). Labeled CST
axons also crossed the host-graft interface from the lateral
border, where a small portion of the dorsolateral funiculus
was adjacent to the transplant (Fig. 5-9 b) . While these
labeled axons were concentrated near the periphery of the
graft, some were also found deeper within the graft. The
branching patterns of labeled axons were similar to those
found in the 4 month transplants. The axons branched and
surrounded the transplant neurons, and varicosities were
found adjacent to and between the counterstained perikarya
(Fig. 5-9 c,d).
Electron microscopy
Regions of the transplants that contained labeled
corticospinal fibers were examined with the electron
microscope (Fig. 5-10). Axonal profiles containing PHA-L
were distributed throughout these areas of the graft neuropil

Figure 5-9. CST axons within a FSC graft placed into a 1 week old lesion and labeled
by PHA-L injections into the cortex at 17 months after grafting.
a) Ingrowth of labeled CST fibers (arrows) across the rostral host-graft interface and
into the transplant (T).
b) Darkfield photomicrograph of axons extending into the graft (T) from the lateral
host (h) border.
c) Labeled axons within Cresyl Violet stained section of the graft.
d) High-power photomicrograph illustrates branching and terminal formation within the
graft neuropil.
Scale in a = 50 nm; b = 100 /¿m; c,d = 20 Jim.


Figure 5-10. Electron microscopy of transplant regions
containing labeled CST axons.
a) Low-power electron micrograph of PHA-L labeled
unmyelinated axons and possible terminals with the transplant
(arrows).
b) Light micrograph of a plastic-embedded section containing
PHA-L labeled fibers within a delayed graft at 17 months
post-transplantation.
c) A PHA-L containing terminal that is near a graft dendrite
(d) .
d) Synaptic endings between an unlabeled axon (u) and PHA-
L labeled CST axon (arrow) and a graft dendrite (d). Post-
synaptic electron-dense regions are evident (arrowheads).
e) Axo-dendritic synapse between a PHA-L labeled terminal
(arrow) and a dendrite (d) within a transplant. Note the
electron-dense post-synaptic thickening (arrowheads).
Scale in a = l.O /¿m; b = 50 /xm; c,d,e = 0.5 ¡im.

167

168
in both the 4 month acute and 17 month delayed grafts. In
these specimens, all of the identified fibers within the
grafts were unmyelinated. However, these labeled axons were
found within both myelinated and myelin-free areas of the
transplants. In addition, labeled boutons containing
mitochondria and pre-synaptic vesicles were found within
these regions. Examples of axo-dendritic synapses were
observed. These were identified by the presence of electron-
dense post-synaptic regions on adjacent dendrites.
Discussion
The return of function following spinal cord injury may
require the establishment of new neural pathways to subserve
the roles of the long sensory and motor tracts that are
disrupted by the injury. In order to determine if such
pathways can be formed by transplantation of fetal tissue at
the lesion site, it is important to identify the capacity of
these fiber tracts for regeneration or sprouting and to
examine the formation and persistence of interactions that
develop between these axons and the transplant tissue. These
results provide anatomical evidence for two ways that the
long descending axons of the corticospinal tract might
integrate with neurons within FSC transplants.
Examination of the CST by anterograde transport of WGA-
HRP at six weeks after injury showed that the presence of a
viable FSC transplant at the site of spinal lesion may
influence the retraction of CST fibers and thus permit

169
apposition of these axons and the graft tissue. These axons
may provide the basis for indirect, polysynaptic interactions
between CST axons and transplant neurons. Alternatively, the
extension of dendrites of the graft neurons across the
rostral interface (Mahalik et al., '86; Clarke et al., '88b)
may permit direct activation of graft neurons by these
persisting fibers.
In addition to these findings, the anterograde transport
of PHA-L demonstrated that adult CST axons exhibit some
capacity for elongation into a graft of FSC tissue. In cases
of good host-graft integration, labeled CST fibers extended
into the neuropil of the grafts and formed synapses within
the transplants. The ingrowth of CST axons may provide a
mechanism for direct cortical activation of neurons within
the grafts. It is important to note, however, that these
axons never extended through the grafts to the spinal cord
levels caudal to the graft, even in the one recipient that
was retained for 17 months after grafting.
CST Axons Persist at the Host-Graft Interface
CST die-back following lesion only
The response of central myelinated axons to injury is
characterized by the dilation of the axonal endings and
subsequent retraction from the site of trauma (Ramon y Cajal,
'28; Lance, '54; Gilson and Stensaas, '74; Kalil and
Schneider, '75). This retraction of injured axons is
associated with repeated extrusion of axoplasm from the

170
dilated terminals (Kao and Chang, 'll; Kao et al., 'll) and
is usually considered to correspond to a failure of
regeneration. In the rodent CST, the process of retrograde
axonal degeneration is further complicated by the vascular
response to spinal trauma. Specifically, the progressive
pathological events following contusive insult to the spinal
cord result in the formation of a conical region of tissue
necrosis. This reaction leads to the formation of cystic
cavities which extend into the base of the dorsal columns in
both the rostral and caudal directions (e.g., Balentine and
Paris, '18a; Nobel and Wrathall, '87). Although the
mechanisms of secondary injury following contusive and
aspirative insult are clearly different, the presence of
hemorrhagic tissue and cysts in the region of the dorsal CST
in this study is similar to the patterns described in those
closed injury models. Thus, the distance between the caudal
end of the cut dorsal CST and the edge of these aspirative
lesions probably involves a combination of the two processes
of axonal retraction and tissue necrosis due to traumatic
injury.
Measurements of CST retraction were based upon the
distance from the bulk of the dorsal CST to the lesion or
graft edge. However, it was clear that individual axons had
retracted at different rates. Evidence from longitudinal
time-course studies of injured adult pyramidal axons support
the hypothesis that the larger CST fibers degenerate most

171
rapidly, while smaller axons persist at the edge of a lesion
for longer times (up to some months after injury) before
degenerating to the nearest axon collateral (Ramon y Cajal,
'28; Tower, MO; Lance, '54; Kalil and Schneider, '75). The
variation in retraction rates within the CST has also been
observed by other authors using anterograde tracing
techniques (e.g. Kao et al., '83; Fishman and Kelly, '84b),
and thus, it may be responsible for the tapered appearance of
the CST seen after injury.
In the present study, the retraction distance of the
injured CST was less when lesions were made in cervical
spinal cord (C6) than when similar lesions were made four
segments lower (T2) . One reason for this finding may be that
the CST axons retracted to the last sustaining collateral,
and therefore the distance reflects a greater density of
axonal collaterals in the cervical enlargement than in upper
thoracic cord (Donatelle, 'll', Gribnau et al., '86).
The distances of CST retraction at six weeks after
partial lesions were less than those reported by Pallini et
al., ('88) at 28 days after complete transection (no
transplant) at the T9 vertebral level. Based upon the
previous argument, this difference may be partly attributed
to the different spinal levels. Alternatively, variations in
axonal retraction may be due to the nature of the lesion
(partial vs. complete transection). However, it is important
to note that the methods used to determine the "die-back"

172
distance were also considerably different. In that study,
TMB stained sections weie examined in brightfield (see also
Fig. 5-3), and individual measurements were made from the
lesion to those axon terminals with distinct retraction
bulbs. This approach allowed the investigators to evaluate
the time course of autolysis for the larger axons. However,
the authors were not likely to count smaller axons that may
have lacked retraction bulbs or that did not retract from the
injury site. Because an important part of this study was
directed at identifying persisting axons, the retraction of
the CST was measured in TMB stained sections using darkfield
illumination (Fig. 5-4).
Influence of FSC transplants on retraction of CST fibers
There are at least three reasons that FSC transplants be
correlated with a decrease in the distance of CST retraction
following injury. One possibility is primarily mechanical in
nature. Specifically, as the grafts enlarged, they may have
expanded into the region of necrosis and filled the area
previously marked by cystic cavitation. This process,
however, was unlikely to have been a major factor in the
present study because the rostral borders of these grafts
were clearly delineated in Nissl sections and the host tissue
did not appear to be compromised by the enlargement of the
grafts. Furthermore, the graft tissue did not expand into
the CST by conforming to the margins of the cystic spaces, as
has been observed when transplants of dissociated FSC tissue

173
were placed into the contused spinal cord (Winialski and
Reier, '89) .
A second factor that may have contributed to the
approximation of the CST axons and transplants is that the
CST axons had retracted from the edge of the tissue after a
lesion and were then induced to regenerate back to the host-
graft interface. The presence of a transplant of FSC tissue
at the lesion site may provide a tropic stimulus for these
small axon sprouts and permit the regeneration of the parent
fibers back to the host-graft interface.
The third potential role of a fetal graft in the
decreased distance of CST retraction is that the transplant
prevented the retraction of a proportion of the injured
axons. It is important to recall that not all axons within
the CST were saved from retrograde degeneration by the
presence of a fetal tissue graft. This is apparent both from
the tapered appearance of the tract and the presence of
terminal bulb structures rostral to the transplants.
In regions where there was no separation between injured
CST axons and the transplants, the graft tissue may have
prevented degeneration according to this third hypothesis by
a combination of several mechanisms. For example, the grafts
may have provided some type of trophic support for the
injured axons, thus preventing them from retracting from the
lesion. Alternatively, the presence of fetal graft tissue,
including both cellular and extracellular components of the

174
developing spinal cord, may provide a beneficial terrain for
the elongation of axon collaterals into the graft tissue
(Bregman et al., '89). Once these fibers have extended into
the graft and formed synapses, they do not undergo any
further degeneration.
An additional possibility is that the effect of FSC
transplants on retrograde degeneration may have been an
indirect result due to a reduction of necrosis and cyst
formation at the lesion site. However, in this study, the
grafts did not appear to influence the process of retrograde
degeneration in the first 2 weeks after injury, suggesting
that the primary effect was not due to a reduction in the
initial traumatic pathology. Nevertheless, this
interpretation must be made with caution, because the grafts
in these few examples were not apposed to the injured tract.
The persistence of injured CST fibers at the edge of FSC
transplants differs from a recent report by Pallini et al.,
('89), in which retrograde degeneration of CST fibers was
examined following transplantation of FSC tissue into
complete transections in adult rats. In that study, there
were no differences in the average retraction of injured
fibers in the presence and absence of grafts. The
discrepancy between the present findings and that report may
reflect either the methods of guantifying the distance of
axonal degeneration (above) or the different levels of the
injury (C6 or T2 vs. T9) . Alternatively, differences in the

175
extent of the initial lesion and pathology (partial lesion
vs. complete transection) and subsequent graft survival and
integration may have resulted in the different outcomes.
CST Axons Regenerate or Sprout into FSC Transplants
The fate of injured CST neurons and their capacity for
regeneration has been controversial. One hypothesis that
received support for many years is that the injured CST
neurons died after spinal cord lesion. This belief was based
upon a decrease in anterograde transport of tritiated proline
(Feringa et al., '84a) and an apparent reduction in the
number of large Betz cells in the cortex (Holmes and May,
'09; Feringa et al., '84b). In addition, fewer CST neurons
were retrogradely labeled with HRP applied proximal to a
spinal transection (Feringa et al., '83; Feringa and
Vahlsing, '85) The contrasting view has been that the
injured neurons survive, but shrink in size (Kalil and
Schneider, '75; Feringa et al., '84b; Barron et al., '88).
The decrease in anterograde and retrograde labeling may
represent an alteration in the axonal transport processes of
these neurons following injury (Goshgarian et al., '83;
Pruitt et al., '88).
There is little evidence to date that adult CST axons are
capable of regeneration after injury (Lance, '54). The
inability of central axons to regenerate has frequently been
attributed to the prohibitive environment of the adult
nervous system. However, some reports have suggested that

176
additional intrinsic limitations may be unique to the long
myelinated fiber tracts of the adult, such as the CST. For
example, Richardson et al. ('82) were able to demonstrate
sprouting of several types of spinal cord and brainstem
neurons into peripheral nerve grafts, but they were unable to
identify regenerating CST axons. One reason for the lack of
corticospinal ingrowth into these peripheral nerves may have
been the distance from the perikarya to the site of
implantation (reviewed in Aguayo, '85) . Alterna- tively, the
retrograde HRP tracing techniques used in these peripheral
nerve studies may have been limited in their sensitivity to
detect small numbers of elongating CST neurons or to identify
axons with abnormal retrograde transport capacities.
Other researchers have claimed that the terminals of
injured CST axons differ from those of injured dorsal column
fibers (Fishman and Kelly, '84a;b). These authors reported
that injured CST axons all formed distinct ovoid terminal
retraction bulbs while injured primary afferents within the
dorsal columns exhibited endings suggestive of growth cones.
From these observations, they have suggested that the
morphological difference may reflect a difference in the
ability to regenerate after injury.
The present results illustrate that adult CST axons are
capable of regeneration or collateral sprouting into
transplants of fetal spinal cord tissue. The distance of
ingrowth is similar to previous observations for other

177
descending and local fiber systems (Reier et al., '86a;
Chapter 4) . The extent of branching and the capacity of
injured CST axons to form synapses upon graft neurons suggest
that the ingrowth of even a few CST fibers within the grafts
may be sufficient to activate a number of neurons within the
transplants.
It is important to note, however, that some abnormal
characteristics of the corticospinal axons were found within
the grafts. For example, the large terminal plexuses (Fig.
5-9 d) and the tortuous pathway of some axons (Fig. 5-7 d) do
not resemble the CST innervation of cells in the dorsal horn.
These differences may be due to the lack of normal
developmental timing cues that are present in the newborn
rat, or different characteristics of the fetal and newborn
target tissues.
Timing of CST Injury and Transplantation
In the brain, a delay of one to two weeks between the
creation of a lesion cavity and transplantation of fetal
tissue can increase the extent of efferent and afferent
projections formed between the transplants and the host
tissue (Gibbs and Cotman, '87; Nieto-Sampedro et al., '88).
In addition, it has been shown in the peripheral nervous
system that a prior conditioning lesion can serve to enhance
the rate of axonal regeneration (McQuarrie, '78). It is
possible that similar factors may influence interactions
between host and graft neurons in the gray matter of the

178
spinal cord. It was unknown whether the die-back of the long
myelinated fiber tracts after injury would exert an opposite
effect with a delay, and thus prevent the ingrowth of these
tracts into FSC transplants. In the present study, a delay
of four days to one week between creating a lesion and
implantation of fetal spinal cord tissue did not prevent the
apposition of CST axons to the graft or the ingrowth of CST
fibers. Yet by this time, some of the axons had already
retracted from the lesion cavity, and necrosis of the dorsal
CST was extensive. Thus, the persistence of fibers at the
graft interface and the ingrowth of injured fibers into the
delayed grafts was probably due to either the regeneration of
axons that had retracted from the lesion or a prevention of
retraction by those axons still at the edge of the lesion at
the time of grafting.
To determine if there is a critical period after which
CST elongation will not occur into transplants, future
studies will involve the placement of FSC transplants into
partial lesions at longer intervals after making the lesions.
Houle and Reier, ('88) have shown that FSC grafts will
survive and integrate with the host when placed into chronic
(two to seven week post-injury) aspiration cavities. Fusion
of host white matter tracts and graft tissue were observed in
some regions, as demonstrated by an absence of gliosis
between the tissues.

179
If experiments with longer delays suggest that a critical
period may prevent CST interactions with FSC transplants,
further studies would be implicated to examine strategies to
improve the outcome in more chronic injury models. For
example, it may be wise to enlarge the initial lesion cavity
in a second lesion paradigm to allow closer integration of
the injured fibers with transplant tissue. This would help
to determine if an ability to elongate after a long term
lesion is due to the lack of apposition between the injured
fibers and the graft or a failure of the host fibers to
initiate axonal elongation.

CHAPTER 6
SUMMARY AND CONCLUSIONS
Spinal cord injury results in severe functional deficits
due to the disruption of neural tissue and the separation of
centers rostral and caudal to the injury. Transplantation
of FSC tissue into a spinal lesion site is one approach that
may promote functional recovery after a spinal cord injury.
The present thesis serves to establish the underlying
anatomical basis for functional reconstruction via a neural
tissue grafting strategy.
Specifically, a battery of complementary neuroanatomical
methods has been used to reveal patterns of axonal
projections established between transplants of rat FSC tissue
and the injured adult rat spinal cord. The interactions
identified in these studies represent the three basic
components - input, intrinsic, and projection neurons - that
define any neural circuit.
Construction of a Relay Across a FSC Graft
The results of these studies support the original
hypothesis underlying the formulation of this thesis; namely
that FSC transplants can establish a neural relay across the
site of a spinal cord injury. The mature graft tissue is
composed primarily of small and intermediate-sized neurons.
180

181
These cells are distributed within both myelinated and
unmyelinated regions of neuropil. Small bundles of
myelinated fibers are also found within the graft tissues.
However, these axons are not organized into long tracts
extending the length of the transplants.
Axonal tracing and immunocytochemical findings
demonstrate the presence of short-distance projections
between host and graft tissues. Afferent input is indicated
by the ingrowth of host axons arising from primary afferent
fibers, local spinal cord neurons, and cells within the host
cortex, hypothalamus, and brainstem. Graft neurons also
extend axons into the surrounding host spinal cord where they
terminate around neurons within the dorsal and ventral gray
matter. Finally, an extensive pattern of intrinsic
projections is observed within the graft tissue.
In addition to interactions established by those axons
that extend across the host-graft interface, there is an
extensive interdigitation of neuritic processes within
regions of fusion between the host and graft tissues.
Together, these anatomical findings serve to define the
elements that may compose a neural relay across FSC grafts.
By combining evidence of afferent, intrinsic, and efferent
projections, it is possible to assemble a variety of
hypothetical relay circuit patterns to subserve the
transmission of ascending, descending and segmental
information across a FSC graft (Fig. 6-1).

182
While several relay circuits are possible, there is no
evidence to date for projections of host CNS axons across
the length of FSC transplants in adults. Thus, these grafts
do not appear to act as a bridge for the regeneration of
injured fibers across the site of a spinal cord injury.
Specificity Issues
The concept of a neural relay implies the transmission
of information from input neurons to the appropriate centers
within the nervous system. In the normal spinal cord, the
precise organization of synaptic relationships that allows
this to occur is determined by the spatial and temporal
constraints present within the developing CNS (reviewed in
Steward, '89a). The anatomical organization, present even
within the interneuronal populations of the spinal cord,
suggests a great deal of specificity in terms of axonal
projection patterns and synaptic connections (Molenaar, '78;
Gobel et al., '80; Bras et al., '89). Thus, the formation of
a functional relay across a FSC graft may require the
replication of some degree of specificity in the patterns of
axonal projections between host and graft tissues.
The adult hippocampus has served as a model system to
examine the specificity of axonal projections between host
and graft tissues (Bjorklund and Stenevi, '84; Raisman and
Ebner '83). These studies have shown that transplanted
neurons will reinnervate only the appropriate target regions
of a denervated hippocampus. The ingrowth of host fibers

Figure 6-1. Diagram of hypothetical relay circuitry
established across a FSC transplant. Every neural circuit is
composed of (a) input neurons, (b) intrinsic neurons, and (c)
output neurons.
Ascending relay: Sensory information may be transmitted to
the higher brain regions through a relay established between
primary afferent fibers, intrinsic graft circuitry, and local
interactions across the rostral host-graft interface.
Descending relay: Descending input to regions below a FSC
graft may be mediated by a similar relay circuit. Evidence
of CST, Ox- and 5-HT fiber ingrowth suggests that graft
neurons may be activated by descending input from three
regions of the neuraxis. Additional descending fibers remain
at the edge of the host-graft interface where they might
activate local spinal cord neurons. Efferent projections of
graft neurons may mediate some form of motor output through
the innervation of motoneurons themselves or other elements
of the caudal segmental circuitry.
Propriospinal and Segmental Relay: Integration of host and
graft neurons near the interface may also provide the basis
for local interactions with propriospinal and reflex
circuitry.

184
Descending Relay
Propriospinal and Segmental Relay

185
is also dependent upon a match between the fiber type and the
normal target region. In this model, a high degree of
synaptic specificity appears to be restored following
transplantation.
Similar specificity has been demonstrated following
transplantation of brainstem suspension grafts into the
transected adult spinal cord (Privat, '86; 88; Lohius,
Hirshfield and Reier, unpublished observations). In this
model, all descending fiber tracts are removed prior to
grafting, yet the transplanted 5-HT immunoreactive neurons
selectively innervate their normal target regions. The
pattern of reinnervation suggests that embryonic neurons
detect some specific post-synaptic cues within the denervated
spinal cord.
The specificity of axonal projections between host and
graft tissues was not directly examined in the present
studies. However, patterns of axonal projections suggested
that axonal outgrowth from FSC grafts may involve a response
to directional cues. In some circumstances, neurons within
the transplants extended axons across the graft and into the
host spinal cord at the most distant host-graft interface.
Elsewhere, efferent axons either changed directions abruptly
after crossing the interface or they extended collateral
branches that continued in divergent directions. While these
observations do not indicate specificity per se. they do

186
suggest that some elements within the host spinal cord
environment may influence the direction of axonal elongation.
Little information has been obtained regarding the
specificity of afferent growth into the transplants. The
differentiation of distinct SG-like regions within the
grafts, however, may provide a suitable model for testing
some aspects of this axonal specificity (Chapter 3) . The
present evidence suggests that these innervation patterns may
be dictated more by the apposition of host fibers than by the
orientation of the graft tissue. The ingrowing fibers do not
appear to seek out appropriate target regions within the
graft. However, it is possible that some cues are presented
by the tissue at the interface region either to promote or
inhibit the elongation of host fibers into the graft.
The evidence described above suggests that there may be
some specificity with regard to interactions between host and
graft fibers. However, the extent of innervation within the
grafts is not as great as that seen in the normal spinal
cord; thus, the synaptic organization within the grafts is
likely to be different as well. Nevertheless, it is possible
that these differences will not prevent the formation of a
neural relay. For example, a similar degree of anatomical
organization is known to exist in the normal rodent striatum
(e.g. Gerfen, '89). Transplants of fetal striatal tissue,
when placed into striatal lesions in adult animals, will
receive host afferent input from appropriate brain regions.

187
Neurons within the grafts also extend efferent projections
into the normal target areas within the brain (Clarke et al.,
'88; Wictorin et al., '88, '89). However, aspects of these
axonal projection patterns are remarkably similar to those
observed in the current study. Specifically, the ingrowth of
most types of afferent fibers is concentrated along the
peripheral regions of the grafts, and the number of axonal
projections that are observed crossing the interface is more
limited than that seen in the normal striatum. In spite of
these differences, it is significant that these transplants
of fetal striatal tissue have been shown to contribute to
functional recovery of specific behavioral tasks that are
impaired in control animals with striatal lesions (Dunnett et
al., '88).
Possible Role of FSC Grafts
in Segmental and Long-Tract Functions
The anatomical characteristics of host and graft
projections can be compared with what is known about the
organization of the normal spinal cord. This information may
then be used to predict the functional capacity that a FSC
graft relay circuit may fill in the injured spinal cord. For
example, the vast majority of connections within the normal
spinal cord are composed of short projections of the spinal
interneurons (Szentagothai, '51,'64a,b; Molenaar, '78;
Yezierski et al., '80; Chung et al., '84). In the context of
spinal cord function, these complex interneuronal circuits

188
play an important role in the integration of segmental
projections with the intersegmental and long-tract systems
(e.g. Szentagothai, '51? Scheibel and Scheibel, '69;
Lundberg, '69; Jankowska and Roberts, '12', Jankowska et al.,
'lb', Bras et al., '89). The predominance of short distance
projections between FSC grafts and the host resembles these
interneuronal circuits in the normal spinal cord. These
characteristics suggest that FSC grafts may be able to
restore local and propriospinal interactions following a
spinal injury.
Through the formation of a relay, the neurons within FSC
grafts may also serve to transmit information across a spinal
cord lesion. There is some evidence that multisynaptic
pathways contribute to the transmission of information over
long distances in the normal spinal cord. Based upon
electrophysiological experiments in the cat, Lloyd (Ml)
concluded that the reticulospinal and propriospinal fibers
form a continuous polysynaptic pathway that originates in the
brainstem and extends the length of the spinal cord. He
proposed that multisynaptic pathways, involving both short
and long propriospinal axons, can play a significant role in
the transmission of descending information. In studies
employing a similar approach to that used by Lloyd, Shik
('83) suggested that multisynaptic pathways may mediate the
transmission of descending locomotor information from the
brainstem to lower spinal cord levels.

189
Long polysynaptic pathways can also contribute to some
types of functional recovery in the absence of long tract
fibers. A number of reports have indicated that residual
sensory and motor functions can be retained after multiple
staggered hemisections are performed to disrupt the long
fiber tracts. Jane et al., ('64) reported recovery of
locomotor behavior in cats following such crossed
hemisections. Similar studies have demonstrated the recovery
of elements of pain sensation after staggered hemisections in
rats and pigs (Breazile and Kitchell, '68; Basbaum, '73).
Thus, the development of a relay circuit across a FSC graft
may improve some degree of function by replacing the long
fiber tracts at the lesion site with newly formed poly¬
synaptic circuits.
Based upon the anatomical evidence, it appears that the
application of FSC tissue grafts to restore function after
injury is most likely to succeed in regions of the spinal
cord where the normal organization is dominated by
interneuronal pools. In these regions, such as the lumbar
and cervical enlargements, interactions within the grafts
and between host and graft tissues may contribute to the
extensive integration of peripheral and central functions. In
contrast, an injury to the mid-thoracic cord might be
repaired more effectively by an approach which would support
the elongation (bridging) of long tract fibers across a
lesion. The use of peripheral nerve (PNS) grafts may be well

190
suited to this latter problem. Initial studies indicated
that CNS neurons, particularly those located near the origin
of a PNS graft, will extend axons for long distances within
the peripheral environment (David and Aguayo, 781). More
recent experiments, involving the placement of PNS grafts
within the optic nerve, have demonstrated that large numbers
of long tract fibers can be directed to an appropriate target
region within the CNS (Aguayo, '85; Bray et al., '87). These
axons then extend for a short distance within the CNS, where
they form functional synapses upon neurons in the target
region (Kierstead et al., '89). Thus, a PNS graft might
provide a simple relay for ascending or descending fibers,
but would not restore segmental interactions.
While many sensory and motor functions can be mediated
by disynaptic or polysynaptic circuits, there are some
aspects of spinal cord function which depend upon the
integrity of the long tract fibers. Researchers conducting
psychophysical studies with primates have found that recovery
of performance on classical discriminative touch and sensory
localization tasks can occur following dorsal column lesions.
However, the ability to discriminate temporal patterns is
lost permanently and cannot be reestablished by training
after similar lesions (Vierck et al., '85). Therefore, if
either FSC or PNS grafts are used to promote functional
recovery after a lesion of the long tract fibers, it is
likely that those sensory functions that are dependent upon

191
precise temporal patterns will not be restored. Similar
limitations may apply to characteristics of motor function
that may be dependent upon monosynaptic projections between
higher centers and motoneurons below a lesion.
Future Directions
These results provide the first step in defining a basis
for functional recovery using FSC transplants. Further
studies must be done to determine if the multisynaptic
pathways which are proposed in this study are actually formed
from these components. Transynaptic labeling methods,
employing traditional axonal tracing molecules or virus
transport, may be used to address this issue (Kristensson et
al., '82; Itaya and van Hoesen, '82; Jankowska and Skoog,
'86) . In addition, methods employing multiple-labeling
procedures may be used to identify the synaptic relationships
between ascending, descending and local circuit neurons at
both the light and ultrastructural levels (Heimer and
Zaborszky, '89).
With the anatomical baseline established by these
studies, future tests of electrophysiological and behavioral
measures are indicated to determine whether the projections
between host and graft tissues represent functional
interactions. Based on these observations, electro-
physiological studies are currently underway to examine
aspects of synaptic function between host and graft tissues.
Results of preliminary single unit extracellular recording

192
experiments suggest that neurons within these grafts are
synaptically activated by stimulation of dorsal root fibers
(Thompson et al., '89). Future studies will examine the
functional correlates of descending and local interactions as
well.
Conclusions
These experiments establish an anatomical setting for the
formation of a neural relay circuit by FSC transplantation at
the site of a spinal cord lesion. If functional pathways are
indeed formed from these elements, the use of FSC grafts may
represent an effective approach to promote the recovery of
some aspects of spinal cord function. Those functions that
involve segmental and propriospinal pathways are most likely
to be restored using this approach. While this strategy may
not ever be effective for the restoration of all types of
spinal cord function, the return of even a small part of a
normal functional repertoire would be of great satisfaction
for a patient with a chronic spinal cord injury.
The biological elements that are expressed during the
formation of host-graft interactions are probably very
different to those that are present in normal development.
Therefore, it is likely that some of the circuits that are
formed will be appropriate for a meaningful functional
outcome, while others may be ineffective or produce
detrimental effects. The ability of these pathways to be
harnessed for effective functional outcome will depend upon

193
the plasticity of these interactions. For example, the
repetitive activation of appropriate pathways through
training or electrical stimulation may lead to the
strengthening of the circuitry through which they are
mediated. In addition, a combination of pharmacological and
molecular strategies may be used sometime in the future to
enhance the responsiveness of neural circuits to such
manipulations. With these possibilities in mind, the
anatomical demonstration of host-graft interactions
represents the first step in evaluating the potential for FSC
transplants to promote the restoration of function after
spinal cord injury.

REFERENCES
Abercrombie, M. (1946) Estimation of nuclear populations from
microtome sections. Anat. Rec. 94:239-247.
Aguayo, A.J. (1985) Axonal regeneration from injured neurons
in the adult mammalian central nervous system. In C.W. Cotman
(ed) : Synaptic Plasticity. New York: Guilford Press, pp.
457-484.
Alvarado-Mallart, R.M., and C. Sotelo (1982) Differentiation
of cerebellar anlage heterotopically transplanted to adult
rat brain: a light and electron microscopic study. J. Comp.
Neurol. 212:247-267.
Anderson,D.K., J.M. Braughler, E.D. Hall, Waters,T.R., J.M.
McCall, and E.D. Means (1988) Effects of treatment with U-
74006F on neurological outcome following experimental spinal
cord injury. J.Neurosura 69:562-567.
Anderson, D.K., P.J. Reier, D.P. Theele, J.B. Munson, L. A.
Ritz, S.A. Brown, and B.Z. O'Steen (1989) Development of
fetal cat neurografts in acute and chronic lesions of adult
cat spinal cord. Soc.Neurosci.Abst. 15:1242.
Aguino, D.A., R.U. Margolis, and R.K. Margolis (1984)
Immunocytochemical localization of a chondroitin sulfate
proteoglycan in nervous tissue. II. Studies in developing
brain. J.Cell Biol. 99: 1130-1139.
Arvidsson, U., S. Cullheim, B. Ulfhake, T. Hokfelt, and L.
Terenius (1989) Altered levels of calcitonin gene-related
peptide (CGRP)-like immunoreactivity of cat lumbar
motoneurons after chronic spinal cord transection. Brain Res.
489:387-391.
Azmitia, E.C., and P.M. Whitaker (1983) Formation of a glial
scar following microinjection of fetal neurons into the
hippocampus or midbrain of the adult rat: An
immunocytochemical study. Neurosci.Lett. 38:145-150.
Balentine, J.D., and D.U. Paris (1978a) Pathology of
experimental spinal cord trauma. I. The necrotic lesion as
a function of vascular injury. Lab.Invest. 3:236-253.
Balentine, J.D., and D.U. Paris (1978b) Pathology of
experimental spinal cord trauma. II. Ultrastructure of axons
and myelin. Lab.Invest. 3:254-266.
194

195
Barron, K.D., M.P. Dentinger, A.J. Popp, and R. Mankes (1988)
Neurons of layer Vb of rat sensorimotor cortex atrophy but do
not die after thoracic cord transection.
J.Neuropath.Exp.Neurol. 47:62-74.
Basbaum, A. I. (1973) Conduction of the effects of noxious
stimulation by short-fiber multisynaptic systems of the
spinal cord in the rat. Exp.Neurol. 40:699-716.
Beasley, L. , and W.B. Stallcup (1987) The nerve growth
factor-inducible large external (NILE) glycoprotein and
neural cell adhesion molecule(N-CAM) have distinct patterns
of expression in the developing rat central nervous system.
J.Neurosci. 7:708-715.
Beattie, M.S., B.T. Stokes, and J.C. Bresnahan (1988)
Experimental spinal cord injury: Strategies for acute and
chronic intervention based on anatomic, physiological, and
behavioral studies. In D.G. Stein, and B.A. Sagel (eds):
Pharmacological Approaches to Treatment of Brain and Spinal
Cord Iniurv. New York: Plenum Publishing Corporation, pp.
43-74.
Benfey, M., and A.J. Aguayo (1982) Extensive elongation of
axons from rat brain into peripheral nerve grafts. Nature
296:150-152.
Berkley, K.J., and C.J. Vierck, Jr. (1987) Transient
reduction in retrograde labeling of diencephalic-projecting
neurons in monkey dorsal column nuclei following removal of
their dorsal column input. In L. Pubols, and B. Sessle (eds) :
Neurology and Neurobioloav Series Vol. 30: Effects of Iniurv
on Trigeminal and Spinal Somatosensory Systems. New York:
Alan R. Liss, Inc., pp 429-436.
Bernstein, D.R., and D.J. Stelzner (1983) Plasticity of the
corticospinal tract following midthoracic spinal injury in
the postnatal rat. J.Comp.Neurol. 221:382-400.
Bernstein, J.J., and M.E. Bernstein (1971) Axonal
regeneration and formation of synapses proximal to the site
of lesion following hemisection of the rat spinal cord.
Exp.Neurol. 30:336-351.
Bernstein, J.J., M.E. Bernstein, and M.R. Wells (1978) Spinal
cord regeneration and axonal sprouting in mammals. In S.G.
Waxman (ed): Physiology and Pathobiology of Axons. New York:
Raven Press, pp. 407-420.
Bernstein, J.J., and W.J. Goldberg (1987) Fetal spinal cord
homografts ameliorate the severity of lesion-induced hind
limb behavioral deficits. Exp.Neurol. 98:633-644.

196
Bjorklund, A., B.J. Hoffer, M.R. Palmer, A. Seiger, and L.
Olson (1983) Survival and growth of neurons with
enkephalin-like immunoreactivity in fetal brain areas grafted
to the anterior chamber of the eye. Neurosci. 10:1387-1398.
Bjorklund, A., 0. Lindvall, O. Isacson, P. Brundin, K.
Wictorin, R.E. Strecker, D.J. Clarke, andS.B. Dunnett (1987)
Mechanisms of action of intracerebral neural implants:
studies on nigral and striatal grafts to the lesioned
striatum. TINS 10:509-515.
Bjorklund, A., and U. Stenevi (1984) Intracerebral neural
implants: Neuronal replacement and reconstruction of damaged
circuitries. Ann Rev. Neurosci. 7:279-308.
Bjorklund, H. , and D. Dahl (1982) Glial disturbances in
isolated neocortex: Evidence from immunohistochemistry of
intraocular grafts. Dev.Neurosci. 5:424-435.
Bjorklund, H. , D. Dahl, K. Haglid, L. Rosengren, and L. Olson
(1983) Astrocytic development in fetal parietal cortex
grafted to cerebral and cerebellar cortex of immature rats.
Dev. Brain Res. 9:171-180.
Bjorklund, H., and L. Olson (1983) Astrocytic development in
intraocular and intracranial cortex cerebri grafts. In
Developing and Regenerating Vertebrate Nervous Systems. New
York: Alan R. Liss, Inc., pp. 239-246.
Blight, A.R., and J.A. Gruner (1987) Augmentation by
4-aminopyridine of vestibulospinal free fall responses in
chronic spinal-injured cats. J.Neurol.Sci. 82:145-159.
Bloch, R.F., and M. Basbaum (1986) Management of Spinal Cord
Injuries. Baltimore,MD: Williams and Wilkins.
Bolam, J.P., T.F. Freund, A. Bjorklund, S.B. Dunnett, and
A.D. Smith (1987) Synaptic input and local output of
dopaminergic neurons in grafts that functionally reinnervate
the host neostriatum. Exp. Brain Res. 68:131-146.
Borgens, R.B., A.R. Blight, D.J. Murphy, and L. Stewart
(1986) Transected dorsal column axons within the guinea pig
spinal cord regenerate in the presence of an applied electric
field. J.Comp.Neurol. 250:168-180.
Borgens, R.B., A.R. Blight, and M.E. McGinnis (1987)
Behavioral recovery induced by applied electric fields after
spinal cord hemisection in guinea pig. Science 238:366-368.
Bras, H. , P. Cavallari, E. Jankowska, and L. Kubin (1989)
Morphology of midlumbar interneurones relaying information

197
from group II muscle afferents in the cat spinal cord.
J.Comp.Neurol. 290:1-15.
Breazile, J.E., and R.L. Kitchell (1968) A study of fiber
systems within the spinal cord of the domestic pig that
subserve pain. J.Comp.Neurol. 133:373-382.
Bregman, B.S. (1987) Spinal cord transplants permit the
growth of serotonergic axons across the site of neonatal
spinal cord transection. Dev.Brain Res. 34:265-279.
Bregman, B.S., and M.E. Goldberger (1983) Infant lesion
effect: III. Anatomical correlates of sparing and recovery of
function after spinal cord damage in newborn and adult cats.
Dev.Brain Res. 9:137-154.
Bregman, B.S., E. Kunkel-Bagden, M. MacAtee, and A. O'Neill
(1989) Extension of the critical period for developmental
plasticity of the corticospinal pathway. J.Comp.Neurol.
282:355-370.
Bregman, B.S., and P.J. Reier (1986) Neural tissue
transplants rescue axotomized rubrospinal cells from
retrograde death. J.Comp.Neurol. 244:86-95.
Buchanan, J.T., and H.O. Nornes (1986) Transplants of
embryonic brainstem containing the locus coeruleus into
spinal cord enhance the hindlimb flexion reflex in adult
rats. Brain Res. 381:225-236.
Bullitt, E., and A.R. Light (1989) Intraspinal course of
descending serotonergic pathways innervating the rodent
dorsal horn and lamina X. J.Comp.Neurol. 286:231-242.
Buzsaki, G. , and F.H. Gage (1988) Mechanisms of action of
neural grafts in the limbic system. Can. J. Neurosci.
15:99-105.
Cabana, T. , and G.F. Martin (1984) Developmental sequence in
the origin of descending spinal pathways. Studies using
retrograde transport techniques in the North American opposum
(Didelphis virginiana). Dev.Brain Res. 15:247-263.
Caroni, P. , and M.E. Schwab (1988) Two membrane protein
fractions from rat central myelin with inhibitory properties
for neurite growth and fibroblast spreading. J.Cell Biol.
106:1281-1288.

198
Casale, E.J., A.R. Light, and A. Rustioni (1988) Direct
projection of the corticospinal tract to the superficial
laminae of the spinal cord in the rat. J. Comp. Neurol.
278:275-286.
Cervero, F., and A. Iggo (1980) The substantia gelatinosa of
the spinal cord. A critical review. Brain 103:717-772.
Chung, K. , G.A. Kevetter, W.D. Willis, and R. Coggeshall
(1984) An estimate of the ratio of propriospinal to long
tract neurons int he sacral spinal cord of the rat.
Neurosci.Lett, 44:173-177.
Clarke, D.J., S.B. Dunnett, O. Isacson, D.J.S.
Sirinathsinghji, and A. Bjorklund (1988a) Striatal grafts in
rats with unilateral neostriatal lesions I. Ultrastructural
evidence of afferent synaptic inputs from the host
nigrostrital pathway. Neurosci. 24:791-801.
Clarke, D.J., P. Brundin, R.E. Strecker, O.G. Nilsson, A.
Bjorklund, and 0. Lindvall (1988b) Human fetal dopamine
neurons grafted in a rat model of parkinson's disease:
ultrastructural evidence for synapse formation using tyrosine
hydroxylase immunocytochemistry. Exp.Brain Res. 73:115-126.
Clemente, C.D. (1964) Regeneration in the vertebrate central
nervous system. Int.Rev.Neurobiol, 6:257-301.
Coimbra, A., B.P. Sodre-Borges, and M.M. Magalhaes (1974) The
substantia gelatinosa Rolandi of the rat. Fine structure,
cytochemistry (acid phosphatase) and changes after dorsal
root section. J.Neurocvt. 3:199-217.
Cole, J.D. (1988) The pathophysiology of the autonomic
nervous system in spinal cord injury. In L.S. lilis (ed) :
Spinal Cord Dysfunction: Assessment. Oxford: Oxford
University Press, pp. 201-236.
Commissiong, J.W. (1984) Fetal locus coeruleus transplanted
into the transected spinal cord of the adult rat: Some
observations and implications. Neurosci. 12:839-853.
Cotman, C.W., and M. Nieto-Sampedro (1985) Progress in
facilitating the recovery of function after central nervous
system trauma. Ann.NY.Acad.Sci. 457:83-104.
Das, G.D. (1983) Neural transplantation in the spinal cord of
adult rats. Conditions, survival, cytology and connectivity
of the transplants. J.Neurol.Sci. 62:191-210.

199
Das, G.D. (1986) Neural transplantation in spinal cord under
different conditions of lesions and their functional
significance. In G.D. Das, and R.B. Wallace (eds) : Neural
Transplantation and Regeneration. New York: Springer-Verlag,
pp. 1-62.
Das, G.D. (1989) Perspectives in anatomy and pathology of
paraplegia in experimental animals. Brain Res. Bull. 22:7-32.
Daube, J.R., B.A. Sandok, T.J. Reagan, and B.F. Westmoreland
(1978) Medical Neurosciences: An Approach to Anatomy.
Pathology, and Physiology by Systems and Levels. Boston,MA:
Little, Brown, and Co.
David, S., and A.J. Aguayo (1981) Axonal elongation into
peripheral nervous system 'bridges' after central nervous
system injury in adult rats. Science. 214:932-933.
de la Torre, J.C. (1980) Chemotherapy of spinal cord trauma.
In W.F. Windle (ed) : The Spinal Cord and Its Reaction to
Traumatic Injury. New York: Marcel Dekker, Inc., pp. 291-310.
de la Torre, J.C. (1984) Spinal cord injury models. Prog.
Neurobiol. 22:289-344.
de Olmos, J., H. Hardy, and L. Heimer (1978) The efferent
connections of the main and accessory olfactory bulb
formations in the adult rat. An experimental HRP study.
J.Comp.Neurol. 181:213-244.
Demopoulos, H.B., E.S. Flamm, D.D. Pietronigro, and M.L.
Seligman (1980) The free radical pathology and the
microcirculation in the major central nervous system
disorders. Acta Physiol. Scand. 492:91-119.
Dohrmann, G.J. (1972) Experimental spinal cord trauma. A
Historical Overview. Arch Neurol 27:468-473.
Donatelle, J.M. (1977) Growth of the corticospinal tract and
the development of placing reactions in the postnatal rat.
J.Comp.Neurol. 175:207-232.
Doucet, G., Y. Murata, P. Brundin, O. Bosler, N. Mons, M.
Geffard, C.C. Ouimet, and A. Bjorklund (1989) Host afferents
into intrastriatal transplants of fetal ventral
mesencephalon. Exp.Neurol. 106:1-19.
Dunnett, S.B., and A. Bjorklund (1987) Mechanisms of function
of neural grafts in the adult mammalian brain. J. exp. Biol.
132:265-289.

200
Dunnett, S.B., O. Isacson, D.J.S. Sirinathsinghji, D.J.
Clarke, and A. Bjorklund (1988) Striatal grafts in rats with
unilateral neostriatal lesions III. Recovery from
dopamine-dependent motor asymmetry and deficits in skilled
paw reaching. Neurosci. 24:813-820.
Dunnett, S.B., G. Toniolo, A. Fine, C.M. Ryan, A. Bjorklund,
and S.D. Iversen (1985) Transplantation of embryonic ventral
forebrain neurons to the neocortex of rats with lesions of
nucleus basalis magnocellularis II. Sensorimotor and learning
impairments. Neurosci. 16:787-797.
Ebner, F.F., R.S. Erzurumlu, and S.M. Lee (1989) Peripheral
nerve damage facilitates functional innervation of brain
grafts in adult sensory cortex. Proc. Natl. Acad. Sci. USA
86:730-734.
Eng, L.F. (1988) Glial fibrillary acidic protein: A review of
structure, function, and clinical application. In Neuronal
and Glial Proteins: Structure. Function and Clinical
Application. New York: Academic Press, Inc., pp. 339-358.
Eriksdotter-Nilsson, M., H. Bjorklund, D. Dahl, and L. Olson
(1986) Growth and development of intraocular fetal cortex
grafts in rats of different ages. Dev. Brain Res. 28:75-84.
Eriksdotter-Nilsson, M., B. Meister, T. Hokfelt, R. Elde, J.
Fahrenkrug, P. Frey, W. Oertel, J.F. Fehfeld, L. Terneius,
and L. Olson (1987) Glutamic acid decarboxylase- and peptide-
immunoreactive neurons in cortex cerebri following
development in isolation: evidence of homotypic and
disturbed patterns in intraocular grafts. Synapse 1:539-551.
Fehlings, M.G., and C.H. Tator (1988) A review of models of
acute experimental spinal cord injury. In L.S. lilis (ed) :
Spinal Cord Dysfunction: Assessment. Oxford: Oxford
University Press, pp. 3-33.
Feringa, E.R., W.J. Gilbertie, and H.L. Vahlsing (1984a)
Histologic evidence for death of cortical neurons after
spinal cord transection. Neurology 34:1002-1006.
Feringa, E.R., R.L. McBride, and J.N. Pruitt,II. (1988) Loss
of neurons in the red nucleus after spinal cord transection.
Exp.Neurol. 100:112-120.
Feringa, E.R., R.L. McBride, S. Varón, and M. Manthorpe
(1989) Continuous nerve growth factor (NGF) treatment
normalizes HRP transport in axotomized rubrospinal neurons.
J.Neuropath.Exp.Neurol. 48:362.

201
Feringa, E.R., H.L. Vahlsing, and B.E. Smith (1983)
Retrograde transport in corticospinal neurons after spinal
cord transection. Neurology 33:478-482.
Feringa, E.R., H.L. Vahlsing, and R.C. Dauser (1984b) The
orthograde flow of tritiated proline in corticospinal neurons
at various ages and after spinal cord injury.
J.Neurol.Neurosura.Psvchiatâ–  47:917-920.
Feringa, E.R., and H.L. Vahlsing (1985) Labeled corticospinal
neurons one year after spinal cord transection.
Neurosci.Lett. 58:283-286.
Finsen, B., and J. Zimmer (1986) Timm staining of hippocampal
nerve cell bodies in the Kyoto rat. A cell marker in allo-
and xenografting of rat and mouse brain tissue, revealing
neuronal migration. Dev.Brain Res. 29:51-59.
Fishman, P.S., and J.P. Kelly (1984a) Identified central
axons differ in their response to spinal cord transection.
Brain Res. 305:152-156.
Fishman, P.S., and J.P. Kelly (1984b) The fate of severed
corticospinal axons. Neurology 34:1161-1167.
Fonseca, M., J. DeFelipe, and A. Fairen (1988) Local
connections in transplanted and normal cerebral cortex of
rats. Exp. Brain Res. 69:387-398.
Forssberg, H., and S. Grillner (1973) The locomotion of the
acute spinal cat injected with clonidine i.v. Brain Res.
50:184-186.
Foster, G.A., M. Schultzberg, F.H. Gage, A. Bjorklund, T.
Hokfelt, H. Nornes, A.C. Cuello, A.A. Verhofstad, and T.J.
Visser (1985) Transmitter expression and morphological
development of embryonic medullary and mesencephalic raphe
neurones after transplantation to the adult rat central
nervous system. I. Grafts to the spinal cord. Exp.Brain Res.
60:427-444.
Freed, W.J., and H.E. Cannon-Spoor (1988) Cortical lesions
increase reinnervation of the dorsal striatum by substantia
nigra grafts. Brain Res. 446:133-143.
Freund, T.F., J.P. Bolam, A. Bjorklund, U. Stenevi, S.B.
Dunnett, J.F. Powell, and A. D. Smith (1985) Efferent synaptic
connections of grafted dopaminergic neurons reinnervating the
host neostriatum: A tyrosine hydroxylase immunocytochemical
study. J.Neurosci. 5:603-616.

202
Gage, F.H. , and G. Buzsaki (1989) CNS grafting: potential
mechanisms of action. In F.J. Seil (ed): Neural Regeneration
and Transplantation. New York: Alan R.Liss,Inc., pp. 99-113.
Gash, D.M., J.R. Sladek,Jr., and C.D. Sladek (1980)
Functional development of grafted vasopressin neurons.
Science 210:1367-1369.
Gerfen, C.R. (1989) The neostriatal mosaic: Striatal patch-
matrix organization is related to cortical lamination.
Science 246:385-388.
Gerfen, C.R., and P.E. Sawchenko (1984) An anterograde
neuroanatomical tracing method that shows the detailed
morphology of neurons, their axons and terminals:
Immunohistochemical localization of an axonally transported
plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L).
Brain Res. 290:219-238.
Gibbs, R.B., and C.W. Cotman (1987) Factors affecting
survival and outgrowth from transplants of entorhinal cortex.
Neuroscience. 21:699-706.
Gibson, S.J., J.M. Polak, S.R. Bloom, and P.D. Wall (1981)
The distribution of nine peptides in rat spinal cord with
special emphasis on the substantia gelatinosa and on the area
around the central canal (lamina X) . J.Comp.Neurol.
201:65-79.
Gibson, S.J., J.M. Polak, S.R. Bloom, I.M. Sabate, P.M.
Mulderry, M.a. Ghatei, G.P. McGregor, J.F.B. Morrison, J.S.
Kelly, R.M. Evans, and M.G. Rosenfeld (1984) Calcitonin
gene-related peptide immunoreactivity in the spinal cord of
man and of eight other species. J.Neurosci. 4:3101-3111.
Giesler, G.J., J.T. Cannon, G. Urea, and J.C. Liebeskind
(1978) Long ascending projections from substantia gelatinosa
Rolandi and the subjacent dorsal horn in the rat. Science
202:984-986.
Gilson, B.C., and L.J. Stensaas (1974) Early axonal changes
following lesions of the dorsal columns in rats. Cell Tissue
Res. 149:1-20.
Gioanni, Y., and M. LaMarche (1985) A reappraisal of rat
motor cortex organization by intracortical microstimulation.
Brain Res. 344:49-61.

203
Gobel, S., W.M. Falls, G.J. Bennett, M. Abdelmoumente, H.
Hayashi, and E. Humphrey (198 0) An EM analysis of the
synaptic connections of horseradish peroxidase-filled stalked
cells and islet cells in the substantia gelatinosa of adult
cat spinal cord. J.Comp.Neurol. 194:781-807.
Goldberger, M.E., and M. Murray (1988) Patterns of sprouting
and implications for recovery of function. In S.G. Waxman
(ed) : Advances in Neurology, Vol.7: Functional Recovery in
Neurological Disease. New York: Raven Press, pp. 361-385.
Goshgarian, H.G., J.M. Koistinen, and E.R. Schmidt (1983)
Cell death and changes in the retrograde transport of
horseradish peroxidase in rubrospinal neurons following
spinal cord hemisection in the adult rat. J.Comp.Neurol.
214:251-257.
Green, B.A., and K.J. Klose (1989) Spinal cord regeneration:
the laboratory/clinical interface. In F.J. Seil (ed): Neural
Regeneration and Transplantation. New York: Alan R.
Liss,Inc., pp. 171-182.
Gribnau, A.A.M., E.J.M. deKort, P.J.W.C. DeDeren, and R.
Nieuwenhuys (1986) On the development of the pyramidal tract
in the rat. II. An anterograde tracer study of the outgrowth
of the corticospinal fibers. Anat Embrvol 175:101-110.
Guth, L. (1975) History of central nervous system
regeneration research. Exp.Neurol. 48:3-15.
Guth, L. , C.P. Barrett, E.J. Donati, F.D. Anderson, M.V.
Smith, and M. Lifson (1985) Essentiality of a specific
cellular terrain for growth of axons into a spinal cord
lesion. Exp.Neurol. 88:1-12.
Guth, L., C.P. Barrett, E.J. Donati, M.V. Smith, M. Lifson,
and E. Roberts (1985b) Enhancement of axonal growth into a
spinal lesion by topical application of triethanolamine and
cytosine arabinoside. Exp.Neurol. 88:44-55.
Harvey, A.R., and R.D. Lund (1981) Transplantation of tectal
tissue in rats. II. Distribution of host neurons which
project to transplants. J.Comp.Neurolâ–  202:505-520.
Harvey, A.R., R.A. Rush, and P.J. Keating (1988) Cultured
fetal tectal tissue grafted to the midbrain of newborn rats:
Morphology of grafts and innervation by host retinal and
cortical axons. Brain Res. 462:89-98.

204
Haun, F. , and T.J. Cunningham (1987) Specific neurotrophic
interactions between cortical and subcortical visual
structures in developing rat: In vitro studies.
J.Comp.Neurol. 256:561-569.
Heimer, L. , and L. Zaborszky (1989) Neuroanatomical
Tract-Tracing Methods 2. Recent Progress. New York: Plenum
Press.
Henschen, A., B. Hoffer, and L. Olson (1985) Spinal cord
grafts in oculo: Survival, growth, histological organization
and electrophysiological characteristics. Exp.Brain Res.
60:38-47.
Henschen, A., T. Hokfelt, R. Elde, J. Fahrenkrug, P. Frey, L.
Terenius, and L. Olson (1988) Expression of eight
neuropeptides in introocular spinal cord grafts:
Organotypical and disturbed patterns as evidences by
immunohistochemistry. Neurosci. 26:193-213.
Holmes, G. , and W.P. May (1909) On the exact origin of the
pyramidal tracts in man and other mammals. Brain 32:1-43.
Houle, J.D., and J.E. Johnson (1989) Nerve growth factor
(NGF)-treated nitrocellulose enhances and directs the
regeneration of adult rat dorsal root axons through
intraspinal neural tissue transplants. Neurosci.Lett.
103:17-23.
Houle, J.D., and P.J. Reier (1988) Transplantation of fetal
spinal cord tissue into the chronically injured adult rat
spinal cord. J.Comp.Neurol. 269:535-547.
Houle, J.D., and P.J. Reier (1989) Regrowth of calcitonin
gene-related peptide immunoreactive axons from the
chronically injured rat spinal cord into fetal spinal cord
tissue transplants. Neurosci.Lett. 103:253-258.
Hunt, S.P. (1983) Cytochemistry of the spinal cord. In P.C.
Emson (ed): Chemical Neuroanatomv. New York: Raven Press, pp.
53-84.
Itaya, S.D., and G.W. vanHoesen (1982) WGA-HRP as a
transneuronal marker in the visual pathways of monkey and
rat. Brain Res. 236:199-204.
Itoh, Y., and A. Tessler (1988) Regenerating CGRP-
immunoreactive dorsal root ganglion axons form terminals in
transplants of fetal spinal cord. Soc.Neurosci.Abst. 14:657.

205
Jacobson, R.D., I. Virag, and J.H.P. Skene (1986) A protein
associated with axon growth, GAP-43, is widely distributed
and developmentally regulated in rat CNS. J.Neurosci.
6:1843-1855.
Jaeger, C.B., and R.D. Lund (1980) Transplantation of
embryonic occipital cortex to the tectal region of newborn
rats: A light microscopic study of the organization and
connectivity of the transplants. J.Comp.Neurol. 194:571-597.
Jakeman, L.B., and P.J. Reier (1987) The response of
corticospinal tract fibers following injury and
transplantation in the adult rat spinal cord.
Soc♦Neurosci.Abst. 13:750.
Jakeman, L.B., and P.J. Reier (1988) Axonal projections
between fetal spinal cord transplants and the adult rat
spinal cord. Soc.Neurosci.Abst. 14:429.
Jakeman, L.B., and P.J. Reier (1989) Regeneration or
sprouting of corticospinal tract axons into fetal spinal cord
transplants in the adult rat. Soc.Neurosci.Abst. 15:1231.
Jakeman, L.B., P.J. Reier, B.S. Bregman, E.B. Wade, M.
Dailey, R.J. Kastner, B.T. Himes, and A. Tessler (1989)
Differentiation of substantia gelatinosa-like regions in
intraspinal and intracerebral transplants of embryonic spinal
cord tissue in the rat. Exp.Neurol. 103:17-33.
Jane, J.A., J.P. Evans, and L.E. Fisher (1964) An
investigation concerning the restitution of motor function
following injury to the spinal cord. J. Neurosurg 21:167-171.
Jankowska, E. , A. Lundberg, W.J. Roberts, and D. Stuart
(1974) A long propriospinal system with direct effect on
motoneurones and on interneurones in the cat lumbosacral
cord. Exp.Brain Res. 21:169-194.
Jankowska, E., and W.J. Roberts (1972) An electro-
physiological demonstration of the axonal projections of
single spinal interneurones in the cat. J.Physiol.
222:597-622.
Jankowska, E., and B. Skoog (1986) Labelling of midlumbar
neurones projecting to cat hindlimb motoneurones by
transneuronal transport of a horseradish peroxidase
conjugate. Neurosci.Lett. 71:163-168.

206
Johnson, M.I., and R.P. Bunge (1983) Plasticity in
neurotransmitter expression and the use of neuronal relays in
spinal cord repair. In C.C. Kao, R.P. Bunge, and P.J. Reier
(eds): Spinal Cord Reconstruction. New York: Raven Press, pp.
329-340.
Joosten, E.A.J., A.A.M. Gribnau, and P.J.W.C. DeDeren (1987)
An anterograde tracer study of the development corticospinal
tract in the rat: Three components. Dev. Brain Res.
36:121-130.
Kalil, K. (1984) Development and regrowth of the rodent
pyramidal tract. TINS 392-396.
Kalil, K., and T. Reh (1982) A light and electron microscopic
study of regrowing pyramidal tract fibers. J.Comp.Neurol.
211:265-275.
Kalil, K., and G.E. Schneider (1975) Retrograde cortical and
axonal changes following lesions of the pyramidal tract.
Brain Res. 89:15-27.
Kao, C.C., and L.W. Chang (1977) The mechanism of spinal cord
cavitation following spinal cord transection. Part 1. A
correlated histochemical study. J. Neurosurg 46:197-209.
Kao, C.C., L.W. Chang, and J.M.B. Bloodworth,Jr. (1977)
Electron microscopic observations of the mechanisms of
terminal club formation in transected spinal cord axons.
J.Neuropath.Exp.Neurol. 36:140-156.
Kao, C.C., J.R. Wrathall, and K. Kyoshima (1983) Axonal
reaction to transection. In C.C. Kao, R.P. Bunge, and P.J.
Reier (eds): Spinal Cord Reconstruction. New York: Raven
Press, pp. 41-57.
Kiernan, J.A. (1979) Hypotheses concerned with axonal
regeneration in the mammalian nervous system. Biol. Rev.
54:155-197.
Kristensson, K. , I.Ninnesmo, L.Persson, and E.Lyke (1982)
Neuron to neuron transfer of herpes simplex virus. Transport
of virus from skin to brainstem nuclei. J. Neurol. Sci.
54:149-156.
Kromer, L.F., A. Bjorklund, and U. Stenevi (1979)
Intracephalic implants: A technique for studying neuronal
interactions. Science 204:1117-1119.

207
Kromer, L.F., A. Bjorklund, and U. Stenevi (1983)
Intracephalic embryonic neural implants in the adult rat
brain. I. Growth and mature organization of brainstem,
cerebellar, and hippocampal implants. J. Comp. Neurol.
218:433-459.
Kruger, S., J. Sievers, C. Hansen, M. Sadler, and M. Berry
(1986) Three morphologically distinct types of interface
develop between adult host and fetal brain transplants:
implications for scar formation in the adult central nervous
system. J.Comp.Neurol. 249:103-116.
Kuang, R.Z., and K. Kalil (1989) Sprouting of corticospinal
fibers into denervated contralateral spinal cord reveals
specificity of new axon arbors. Soc.Neurosci.Abst. 15:92.
Kunkel-Bagden, E., and B.S. Bregman (1989) Transplants alter
the development of sensorimotor function after neonatal
spinal cord damage. Exp. Brain Res.fin press)
LaMotte, C.C. (1986) Organization of dorsal horn
neurotransmitter systems. In T. Yaksh (ed): Spinal Afferent
Processing. New York: Plenum Press, pp. 97-116.
Lance, J.W. (1954) Behaviour of pyramidal axons following
section. Brain 77:314-324.
Liesi,P., and J. Silver (1988) Is astrocyte laminin involved
in axons guidance in the mammalian CNS? Dev.Biol. 130:774-
785.
Light, A.R., and A.M. Kavookjian (1988) Morphology and
ultrastructure of physiologically identified substantia
gelatinosa (Lamina II) neurons with axons that terminate in
deeper dorsal horn laminae (III-V). J.Comp.Neurol. 267:172-
189.
Lindsay, R.M., G. Raisman, and P.J. Seeley (1987)
Intracerebral transplantation of cultured neurons after
reaggregation in a plasma clot. Neurosci. 21:685-698.
Liuzzi, F.J., and R.J. Lasek (1987) Astrocytes block axonal
regeneration in mammals by activating the physiological stop
pathway. Science 237:642-645.
Lloyd, D.P.C. (1941) Activity in neurons of the bulbospinal
correlation system. J.Neurophvsiol. 4:115-134.
Lund, R.D., and A.R. Harvey (1981) Transplantation of tectal
tissue in rats. I. Organization of transplants and pattern
of distribution of host afferents within them. J.Comp.Neurol.
201:191-209.

208
Lundberg, A. (1969) Convergence of excitatory and inhibitory
action on interneurones in the spinal cord. In M.A.B. Brazier
(ed) : The Interneuron. UCLA Forum Med, sci. No. 11. Los
Angeles: Univ. of California Press, pp. 231-365.
Mahalik, T.J., T.E. Finger, I. Stromberg, and L. Olson (1986)
Substantia nigra transplants into denervated striatum of the
rat: Ultrastructure of graft and host interconnections. J.
Comp. Neurol. 249:279-292.
McAllister, J.P., S.R. Cober, E.R. Schaible, and P.D. Walker
(1989) Minimal connectivity between six month neostriatal
transplants and the host substantia nigra. Brain Res.
476:345-350.
McConnell, S.K. (1985) Migration and differentiation of
cerebral cortical neurons after transplantation into the
brains of ferrets. Science 229:1268-1271.
McLoon, S.C., and R.D. Lund (1983) Development of fetal
retina, tectum, and cortex transplanted to the superior
colliculus of adult rats. J. Comp. Neurol. 217:376-389.
McNeill, D.L., R.E. Coggeshall, and S.M. Carlton (1988) A
light and electron microscopic study of calcitonin
gene-related peptide in the spinal cord of the rat.
Exp.Neurol. 99:699-708.
McQuarrie,I.G. (1978) The effect of a conditioning lesion on
the regeneration of motor axons. Brain Res. 152:597-602.
Melzack, R., and P.D. Wall (1965) Pain mechanisms: a new
theory. Science 150:971-979.
Menetrey, D. , J. de Pommery, and F. Roudier (1985)
Propriospinal fibers reaching the lumbar enlargement in the
rat. Neurosci.Lett. 58:257-261.
Mesulum, M.-M. (1982) Tracing Neuronal Connections with
Horseradish Peroxidase. New York: John Wiley and Sons.
Miller, K.E., and V. Seybold (1987) Comparison of
met-enkephalin, dynorphin A- and neurotensin- immunoreactive
neurons in the cat and rat spinal cords: I. Lumbar cord.
J.Comp.Neurol. 255:293-304.
Mize, R.R., R.N. Holdefer, and L.B. Nabors (1988)
Quantitative immunocytochemistry using an image analyzer. I.
Hardware evaluation, image processing and data analysis.
J.Neurosci.Meth. 26:1-24.

209
Molander, C. , Q. Xu, C. Rivero-Melian, and G. Grant (1989)
Cytoarchitectonic organization of the spinal cord in the rat:
II. The cervical and upper thoracic cord. J.Comp.Neurol.
289:375-385.
Molenaar, I. (1978) The distribution of propriospinal neurons
projecting to different motoneuronal cell groups in the cat's
brachial cord. Brain Res. 158:203-206.
Moorman, S.J., J.R. Whalen, and H.O. Nornes (1988) Studies of
hindlimb reflexes affected by implants of fetal brainstem
tissue in the spinal cord of the rat. Soc.Neurosci.Abst.
14:429.
Mouchet, P. , M. Manier, M. Dietl, C. Feuerstein, A. Berod, M.
Arluison, L. Denoroy, and J. Thibault (1986) Immunohisto-
chemical study of catecholaminergic cell bodies in the rat
spinal cord. Brain Res.Bull. 16:341-353.
Mufson, E.J., R. Labbe, and D.G. Stein (1987) Morphologic
features of embryonic neocortex grafts in adult rats
following frontal cortical ablation. Brain Res. 401:162-167.
Naftchi, N.E., S.J. Abrahams, S.M. Crain, E.R. Peterson, J.M.
Hiller, and E.J. Simon (1981) Presence of leucine-enkephalin
in organotypic explants of fetal mouse spinal cord. Peptides
2:57-60.
Nathaniel, E.J.H., and D.R. Nathaniel (1981) The reactive
astrocyte. Adv. Cell. Neurobiol. 2:249-301.
Newton, B.W., and R.W. Hamill (1988) The morphology and
distribution of rat serotoninergic intraspinal neurons: An
immunohistochemical study. Brain Res.Bull. 20:349-360.
Newton, B.W., B.E. Maley, and R.W. Hamill (1986)
Immunohistochemical demonstration of serotonin neurons in
autonomic regions of the rat spinal cord. Brain Res.
376:155-163.
Nieto-Sampedro, M. , J.P. Kesslak, R.B. Gibbs, and C.W. Cotman
(1988) Effects of conditioning lesions on transplant
survival, connectivity, and function. Ann NY Acad.Sci.
495:108-119.
Nobel, L.J., and J.R. Wrathall (1987) The blood-spinal cord
barrier after injury: Pattern of vascular events proximal and
distal to a transection in the rat. Brain Res. 424:177-188.

210
Nornes, H., A. Bjorklund, and U. Stenevi (1974) Temporal
pattern of neurogenesis in spinal cord of rat. I. An
autoradiographic study — Time and sites of origin and
migration and settling patterns of neuroblasts. Brain Res.
73:121-138.
Nornes, H. , A. Bjorklund, and U. Stenevi (1983) Reinnervation
of the denervated adult spinal cord of rats by intraspinal
transplants of embryonic brain stem neurons. Cell Tissue Res.
230:15-35.
Nornes, H., A. Bjorklund, and U. Stenevi (1984)
Transplantation strategies in spinal cord regeneration. In
J.R. Sladek, and D.M. Gash (eds): Neural Transplants:
Development
407-421.
and
Function.
New
York: Plenum Press, pp.
Nothias, F. ,
B.
Onteniente,
M.
Geffard, and M. Peschanski
(1988) Rapid growth of host afferents into fetal thalamic
transplants. Brain Res. 463:341-345.
Nothias, F., J.C. Horvat, M. Pecot-Dechavassine, J.C. Mira,
and M. Peschanski (1989) Neural transplants in an
experimental model of amyotrophic lateral sclerosis.
Soc.Neurosci.Abst. 15:10.
Nygren, L.-G., L. Olson, and A. Seiger (1977) Monoaminergic
reinnervation of the transected spinal cord by homologous
fetal brain grafts. Brain Res. 129:227-235.
Oblinger, M.M., and G.D. Das (1982) Connectivity of neuronal
transplants in adult rats: Analysis of afferents and
efferents of neocortical transplants in the cerebeller
hemisphere. Brain Res. 249:31-49.
Osterholm, J.L. (1974) The pathophysiological response to
spinal cord injury. J. Neurosurg 40:5-33.
Pallini, R., E. Fernandez, and A. Sbriccoli (1988) Retrograde
degeneration of corticospinal axons following transection of
the spinal cord in rats. J. Neurosurg 68:124-128.
Pallini, R. , E. Fernandez, C. Gangitano, A. Del Fa, C.
Olivieri-Sangiacomo, and A. Sbriccoli (1989) Studies on
embryonic transplants to the transected spinal cord of adult
rats. J. Neurosurg 70:454-462.
Patel, U., and J.J. Bernstein (1983) Growth, differentiation,
and viability of fetal rat cortical and spinal cord implants
into adult rat spinal cord. J.Neurosci. 9:303-310.

211
Pritzel, M. , O. Isacson, P. Brundin, L. Wiklund, and A.
Bjorklund (1986) Afferent and efferent connections of
striatal grafts implanted into the ibotenic acid lesioned
neostriatum in adult rats. Exp. Brain Res. 65:112-126.
Privat, A., H. Mansour, A. Pavy, M. Geffard, and F. Sandillon
(1986) Transplantation of dissociated foetal serotonin
neurons into the transected spinal cord of adult rats.
Neurosci.Lett. 66:61-66.
Privat, A., H. Mansour, N. Rajaofetra, and M. Geffard (1989)
Intraspinal transplants of serotonergic neurons in the adult
rat. Brain Res. Bull. 22:123-129.
Pruitt, J.N., E.R. Feringa, and R.L. McBride (1988)
Corticospinal axons persist in cervical and high thoracic
regions 10 weeks after a T-9 spinal cord transection.
Neurology 38:946-950.
Raisman, G., and F.F. Ebner (1983) Mossy fibre projections
into and out of hippocampal transplants. Neurosci. 9:783-801.
Rakic, P.C. (1976) Local Circuit Neurons. Cambridge,MA: MIT
Press.
Ralston, H.J. (1979) The fine structure of Laminae I, II and
III of the Macaque spinal cord. J.Comp.Neurol. 184:619-642.
Ramon y Cajal, S. (1928) Degeneration and Regeneration of the
Nervous System. London: Translated by R.M. May; Hafner
Publishing Company.
Ransohoff, J. (1980) Surgical intervention after traumatic
injury. In W.F. Windle (ed): The Spinal Cord and Its Reaction
to Traumatic Injury. New York: Marcel Dekker, Inc., pp.
311-324.
Reier, P.J. (1985) Neural tissue grafts and repair of the
injured spinal cord. Neuropathol.AppI♦Neurobiol. 11:81-104.
Reier, P.J. (1986) Gliosis Following CNS Injury: The Anatomy
of Astrocytic Scars and Their Influences on Axonal
Regeneration. In S. Federoff (ed): Astrocytes. Volume 3. New
York: Academic Press, Inc., pp. 263-324.
Reier, P.J., and B.S. Bregman (1983) Immunocytochemical
demonstration of substantia gelatinosa-like regions and
serotonergic axons in embryonic spinal cord transplants in
the rat. Soc.Neurosci.Abst. 9:696.

212
Reier, P.J., B.S. Bregman, and J.R. Wujek (1985) Intraspinal
transplants of embryonic spinal cord tissue in adult and
neonatal rats: evidence for topographical differentiation and
axonal interactions with the host CNS. In A. Bjorklund, and
U. Stenevi (eds): Neural Grafting in the Mammalian CNS.
Amsterdam: Elsevier Science Publishers, pp. 257-263.
Reier, P.J., B.S. Bregman, and J.R. Wujek (1986a) Intraspinal
transplantation of embryonic spinal cord tissue in neonatal
and adult rats. J.Comp.Neurol. 247:275-296.
Reier, P.J., B.S. Bregman, and J.R. Wujek (1986b) Intraspinal
transplantation of fetal spinal cord tissue: An approach
toward functional repair of the injured spinal cord. In M.
Goldberger, A. Gorio, and M. Murray (eds): Proceedings of the
International Symposium on Plasticity and Development of the
Mammalian Spinal Cord. Padova: Liviana Press, Fidia Research
Series, pp. 251-269.
Reier, P.J., L.F. Eng , and L.B. Jakeman (1989) Reactive
astrocyte and axonal outgrowth in the injured CNS: Is gliosis
really an impediment to regeneration?. In F.J. Seil (ed) :
Neural Regeneration and Transplantation. New York: Alan R.
Liss, pp. 183-209.
Reier, P.J., and J.D. Houle (1988) The glial scar: Its
bearing on axonal elongation and transplantation approaches
to CNS repair. In S.G. Waxman (ed): Advances in Neurology.
Vol. 47. Functional Recovery in Neurological Disease. New
York: Raven Press, pp. 87-138.
Reier, P.J., J.D. Houle, L.B. Jakeman, D. Winialski, and A.
Tessler (1988) Transplantation of fetal spinal cord tissue
into acute and chronic hemisection and contusion lesions of
the adult rat spinal cord. In D.M. Gash, and J.R. Sladek
(eds): Progress in Brain Research. Vol. 78. Amsterdam:
Elsevier Science Publishers, Biomedical Division. pp.
173-179.
Reier, P.J., M.J. Perlow, and L. Guth (1983a) Development of
embryonic spinal cord transplants in the rat. Dev.Brain Res.
10:201-219.
Reier, P.J., L.J. Stensaas, and L. Guth (1983b) The
Astrocytic Scar as an Impediment to Regeneration in the
Central Nervous System. In C.C. Kao, R.P. Bunge, and P.J.
Reier (eds): Spinal Cord Reconstruction. New York: Raven
Press, pp. 163-195.
Rethelyi, M., and J. Szentagothai (1969) The large synaptic
complexes of the substantia gelatinosa. Exp.Brain Res.
7:258-274.

213
Rexed, B. (1952) The cytoarchitectonic organization of the
spinal cord in the cat. J.Comp.Neurol. 96:415-496.
Richardson, P.M., U.M. McGuiness, and A.J. Aguayo (1982)
Peripheral nerve autografts to the rat spinal cord: Studies
with axonal tracing methods. Brain Res. 237:147-162.
Richardson, P.M., V.M.K. Issa, and A.J. Aguayo (1984)
Regeneration of long spinal axons in the rat. J.Neurocvt.
13:165-182.
Robinson, G.A., and M.E. Goldberger (1986) The development
and recovery of motor function in spinal cats.II.
Pharmacological enhancement of recovery. Exp.Brain Res.
62:387-400.
Sandler, A.N., and C.H. Tator (1976) Review of the effect of
spinal cord trauma on the vessels and blood flow in the
spinal cord. J. Neurosurg 45:638-646.
Saunders, R.D., L.L. Dugan, P. Demediuk, E.D.Means, L. A.
Horrocks, and D.K. Anderson (1987) Effects of
methylprednisolone and the combination of alpha-tacopherol
and selenium on arachidonic acid metabolism and lipid
peroxidation in traumatized spinal cord tissue. J. Neurochem.
49:24-31.
Scheibel, M.E., and A.B. Scheibel (1969) A structural
analysis of spinal interneurons and renshaw cells. In M.A.B.
Brazier (ed): The Interneuron. UCLA Forum Med. Sci. No. 11.
Los Angeles: Univ. of California Press, pp. 159-208.
Schmued, L.C., and J.H. Fallon (1986) Fluoro-Gold: a new
fluorescent retrograde axonal tracer with numerous unique
properties. Brain Res. 377:147-154.
Schreyer, D.J., and E.G. Jones (1982) Growth and target
finding by axons of the corticospinal tract in prenatal and
postnatal rats. Neurosci. 7:1837-1853.
Schreyer, D.J., and E.G. Jones (1983) Growing corticospinal
axons by-pass neonatal rat spinal cord. Neurosci. 9:31-40.
Seybold, V., and R. Elde (1980) Immunohistochemical studies
of peptidergic neurons in the dorsal horn of the spinal cord.
J.Histochem.Cvtochem. 28:367-370.
Seybold, V., and R. Elde (1982) Neurotensin immunoreactivity
in the superficial laminae of the dorsal horn of the rat. I.
Light microscopic studies of cell bodies and proximal
dendrites. J.Comp.Neurol. 205:89-100.

214
Shepard, G.M. (1988) Neurobioloqy. New York: Oxford
University Press.
Shik, M.L. (1983) Action of the brainstem locomotor region on
spinal stepping generators via propriospinal pathways. In
C.C. Kao, R.P. Bunge and P.J. Reier (eds) : Spinal Cord
Reconstruction. New YorkrRaven Press, pp. 421-434.
Shipley, M.T., J. Luna, and J.H. McLean (1989) Processing
and analysis of neuroanatomical images. In L. Heimer, and L.
Zaborszky (eds): Neuroanatomical Tract-Tracing 2. Recent
Progress. New York: Plenum Press, pp. 331-390.
Shu, S.Y., and G.M. Peterson (1988) Anterograde and
retrograde axonal transport of Phaseolus vulgaris
leucoagglutinin (PHA-L) from the globus pallidus to the
striatum of the rat. J.Neurosci.Meth. 25:175-180.
Siegal, J.D., M. Kliot, G.M. Smithe, S. Tyrrell, and J.
Silver (1988) Induced regeneration of cut dorsal root fibers
into adult rat spinal cord. Soc.Neurosci.Abst. 14:1199.
Sladek, J.R. , and D.M. Gash (1984) Neural Transplants:
Development and Function. New York: Plenum Press.
Sobkowicz, H.M., R.W. Guillery, and M.B. Bornstein (1968)
Neuronal organization in long term cultures of the spinal
cord of the fetal mouse. J,Comp.Neurol. 132:365-396.
Sorensen, T. , and J. Zimmer (1988b) Ultrastructural
organization of normal and transplanted rat fascia dentata:
I. A qualitative analysis of intracerebral and intraocular
grafts. J. Comp. Neurol. 267:15-42.
Sorensen, T. , and J. Zimmer (1988a) Ultrastructural
organization of normal and transplanted rat fascia dentata:
II. A quantitative analysis of the synaptic organization of
intracerebral and
267:43-54.
intraocular
grafts. J.
Como.
Neurol.
Spence, J.T., J.W.
Cotton, B.J.
Underwood,
and C.P. Duncan
(1983) Elementary
Prentice-Hall,Inc.
Statistics.
Englewood
Cliffs,
N. J. :
Steinbusch, H.W.M. (1981) Distribution of serotonin-
immunoreactivity in the central nervous system of the rat-
Cell bodies and terminals. Neurosci♦ 6:557-618.
Steiner, T.J., and L.M. Turner (1972) Cytoarchitecture of the
rat spinal cord. J.Physiol. 222:123-125.

215
Stenevi, U., A. Bjorklund, and N.-A. Svengaard (1976)
Transplantation of central and peripheral monoamine neurons
to the adult rat brain: Techniques and conditions for
survival. Brain Res. 114:1-20.
Sternberger, L.A. (1976) Immunocvtochemistrv. New York: John
Wiley and Sons.
Steward, O. (1989a) Principles of Cellular. Molecular, and
Developmental Neuroscience. New York: Springer-Verlag.
Steward, O. (1989b) Nervous system regeneration and repair
following injury. In O. Steward (ed): Principles of Cellular.
Molecular and Developmental Neuroscience. New York: Springer
Verlag, pp. 218-247.
Stokes, B.T., P. Fox, and G. Hollinden (1983) Extracellular
calcium activity in the injured spinal cord. Exp.Neurol.
80:561-572.
Sung, J.H. (1981) Tangled masses of regenerated central nerve
fibers (non-myelinated central neuromas) in the central
nervous system. J.Neuropath.Exp.Neurol. 40:645-657.
Swanson, L.W., and S. McKellar (1979) The distribution of
oxytocin- and neurophysin-stained fibers in the spinal cord
of the rat and monkey. J.Comp.Neurol♦ 188:87-106.
Szentagothai, J. (1951) Short propriospinal neurons and
intrinsic connections of the spinal gray matter. Acta Morph.
Acad. Sci. Hung. 1:81-94.
Szentagothai, J. (1964a) Neuronal and synaptic arrangement in
the substantia gelatinosa Rolandi. J.Comp.Neurol.
122:219-239.
Szentagothai, J. (1964b) Propriospinal pathways and their
synapses. Prog.Brain Res. 11:155-177.
Tator, C.H., A.S. Rivlin, A.J. Lewis, and B. Schmoll (1984)
Effect of acute spinal cord injury on axonal counts in the
pyramidal tract of rats. J. Neurosurg 61:118-123.
Tessler, A., B.T. Himes, J.D. Houle, and P.J. Reier (1988)
Regeneration of adult dorsal root axons into transplants of
embryonic spinal cord. J.Comp.Neurol. 270:537-548.
Thompson, F. , L.B. Jakeman, C.L. Lucas, L. Ray, and P.J.
Reier (1989) Electrophysiological demonstration of host
primary afferent connectivity with neurons in fetal spinal
cord implants. Third Int. Svmp,.Neural Regen. Pacific Grove,
CA:P45.

216
Tolbert, D.L., and T. Der (1987) Redirected growth of
pyramidal tract axons following neonatal pyramidotomy in
cats. J.Comp.Neurol. 260:299-311.
Tower, S.S. (1940) Pyramidal lesion in the monkey. Brain
63:36-89.
Vahlsing, H.L., and E.R. Feringa (1980) A ventral uncrossed
corticospinal tract in the rat. Exp.Neurol. 70:282-287.
Vierck, C.J., R.H. Cohen, and B.Y. Cooper (1985) Effects of
spinal lesions on temporal resolution of cutaneous
sensations. Somatosensory Res. 3:45-56.
Vierck, C.J., J.D. Greenspan, L. A. Ritz, and D.C. Yeomans
(1986) The spinal pathways contributing to the ascending
conduction and the descending modulation of pain sensations
and reactions. In T.L. Yaksh (ed): Spinal Afferent
Processing. New York: Plenum Press, pp. 275-329.
Wagner, F.C., J.C. VanGilder, and G.J. Dohrmann (1978)
Pathological changes from acute to chronic in experimental
spinal cord trauma. J. Neurosurg 48:92-98.
Walker, P.D., and J.P. McAllister (1987) Minimal connectivity
between neostriatal transplants and the host brain. Brain
Res. 425:35-44.
Warr, W.B., J.S. de Olmos, and L. Heimer (1981) Horseradish
Peroxidase. The Basic Procedure. In L. Heimer, and M.J.
RoBards (eds): Neuroanatomical Tract-Tracing Methods. New
York: Plenum Press, pp. 207-262.
Welker, C. (1971) Microelectrode delineation of fine grain
somatotopic organization of SmI cerebral neocortex in albino
rat. Brain Res. 26:259-275.
Wictorin, K., 0. Isacson, W. Fischer, F. Nothias, M.
Peschanski, and A. Bjorklund (1988) Connectivity of striatal
grafts implanted into the ibotenic acid-lesioned striatum I.
Subcortical afferents. Neurosci. 27:547-562.
Wictorin, K., R.B. Simerly, O. Isacson, L.W. Swanson, and A.
Bjorklund (1989) Connectivity of striatal grafts implanted
into the ibotenic acid-lesioned striatum III. Efferent
projecting graft neurons and their relation to host afferents
within the grafts. Neurosci. 30:313-330.
Willis, W.D., and R.E. Coggeshall (1978) Sensory Mechanisms
of the Spinal Cord. New York: John Wiley and Sons.

217
Willis, W.D., R.B. Leonard, and D.R. Kenshalo (1978)
Spinothalamic tract neurons in the substantia gelatinosa.
Science 202:986-988.
Windle, W.F., C.D. Clemente, and W.W. Chambers (1952)
Inhibition of formation of a glial barrier as a means of
permitting a peripheral nerve to grow into the brain.
J.Comp.Neurol. 96:359-369.
Windle, W.F. (1980) The Spinal Cord and Its Reaction to
Traumatic Injury. New York: Marcel Dekker,Inc.
Winialski, D.L., and P.J. Reier (1989) Analysis of rats
receiving fetal spinal cord transplants subsequent to
mid-thoracic contusive spinal injury. Soc.Neurosci.Abst.
15:1242.
Wise, S.P., and E.G. Jones (1977) Cells of origin and
terminal distribution of descending projections of the rat
somatic sensory cortex. J.Comp.Neurol. 175:129-158.
Wouterlood, F.G., and H.J. Groenewegen (1985) Neuroanatomical
tracing by use of Phaseolus vulgaris-leucoagglutinin (PHA-L)
electron microscopy of PHA-L filled neuronal somata,
dendrites, axons and axon terminals. Brain Res. 326:188-191.
Wrathall, J., R. Pettegrew, M. Castro, and L. Verma (1989)
Implantation of immature astrocytes into the contused spinal
cord: Chronic effects on functional deficit and
histopathology. Soc.Neurosci.Abst. 15:1369.(Abstract)
Yezierski, R.P., J.L. Culberson, and P.B. Brown (1980) Cells
of origin of propriospinal connections to cat lumbosacral
gray as determined with horseradish peroxidase. Exp.Neurol.
69:493-512.
Young, W., I. Koreh, V. Yen, and A. Lindsay (1982) Effect of
sympathectomy on extracellular potassium ionic activity and
blood flow in experimental spinal cord contusion. Brain Res.
253:115-124.

BIOGRAPHICAL SKETCH
Lyn Burrell Jakeman was born in Schenectady, New York,
in July of 1961. Her interest in nuroscience began in high
school while studying aspects of neuronal function in both
biology and psychology classes. She attended Hartwick
College in Oneonta, New York, from 1979 until 1983, when she
graduated with a Bachelor of Arts degree in biology.
Attracted by an independent Neuroscience Department and a
diverse research faculty, she began studies at the University
of Florida in August 1983 with a somewhat vaguely defined
interest in neuronal plasticity. Her graduate research
experience began in the laboratory of Drs. Don Walker and
Bruce Hunter. While deciding upon a thesis topic she became
interested in the use of fetal transplants as tools to
examine aspects of axonal plasticity in adult animals. This
interest lead her to the laboratory of Dr. Paul Reier and the
studies contained in this dissertation.
Following completion of her degree, Lyn will begin post¬
doctoral training, under the direction of Dr. C. Anthony
Altar, in the Department of Pharmacological Sciences at
Genentech, Inc., South San Francisco.
218

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
_ < r
Paul J. Reier, Chairman
Matk-df. Overstreet
Professor of Neurological
Surgery and Neuroscience
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate/"i>p scope and quality, as
a dissertation for the degree of poc^or/g^’' ^rtjl^psophy.
B. Munson
5rofessor of Neuroscience
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scops_and
a dissertation for the degree of Doctc
--Char
Profess
Neuroscience
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scop^ and quality, as
a dissertation for the degree of Doctofvcir Philos
Roger M. Reep
Assistant Professoj
of Neuroscience

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Louis A. Ritz
Assistant Professor
of Neuroscience
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in spope and quality, as
a dissertation for the degree of Doctot>of Philosophy.
Donald J. Stehouwer
Associate/Professor
of Psychology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
áJj-aJuí^ xI/SAjL-l —-
Barbara S. Bregman /f
Associate Professor of
Anatomy and Cell Biology
Georgetown University
This dissertation was submitted to the Graduate Faculty
of the College of Medicine and to the Graduate School and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.
May, 1990
Dean, College of Medicine

UNIVERSITY OF FLORIDA
3 1262 08554 3337




PAGE 1

$;21$/ ,17(5$&7,216 %(7:((1 )(7$/ 63,1$/ &25' 75$163/$176 $1' 7+( $'8/7 5$7 63,1$/ &25' %\ /<1 %855(// -$.(0$1 $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( 6&+22/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

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f§ 'UV %DUEDUD %UHJPDQ -RKQ 0XQVRQ 5RJHU 5HHS /RX 5LW] 'RQ 6WHKRXZHU DQG &KXFN 9LHUFN f§ IRU FRQWLQXHG VXSSRUW DQG FRQVWUXFWLYH FULWLFLVPV UHJDUGLQJ P\ ZRUN 7KH GDLO\ SURJUHVV ZDV PDGH PRUH SOHDVDQW ZLWK WKH VXSHUE WHFKQLFDO DQG RUJDQL]DWLRQDO DVVLVWDQFH RI %DUEDUD 2n6WHHQ 0LQQLH 6PLWK DQG 5HJLQD 5HLHU ZKR NHSW WUDFN RI P\ ORRVH SLHFHV RI SDSHU DQG VDYHG PH PRQWKV RI HIIRUW $GGLWLRQDO KHOS ZDV SURYLGHG E\ WKH VHFUHWDULDO DQG VXSSRUW SHUVRQQHO LQ WKH 'HSDUWPHQWV RI 1HXURVFLHQFH DQG 1HXURVXUJHU\ *UDWLWXGH LV DOVR H[WHQGHG WR IHOORZ JUDGXDWH VWXGHQWV HVSHFLDOO\ 'HQLVH DQG *UHJ ZKR WDXJKW PH WR EHOLHYH LQ P\VHOI ZKHQ WKH JDPH VHHPHG ORVW 2YHUVLJKW RI DQLPDO FDUH DQG XVH ZDV NHSW LL

PAGE 3

E\ 'U 'DQ 7KHHOH '90 )LQDOO\ LQVWUXFWLRQ LQ WKH FRQFHSWV RI PRUSKRPHWULF LPDJH DQDO\VLV ZDV PDGH DYDLODEOH E\ 0U 'DQ :LOOLDPV $Q LQFUHGLEOH DPRXQW RI HPRWLRQDO VXSSRUW KDV EHHQ H[WHQGHG RYHU WKH SDVW VL[ \HDUV E\ IDPLO\ DQG IULHQGV HVSHFLDOO\ P\ VLVWHU %DUE P\ SDUHQWV DQG P\ LQODZV +RZHYHU WKH JUHDWHVW DSSUHFLDWLRQ LV H[WHQGHG WR P\ KXVEDQG 'DYLG 7 /HH ZKR SURYLGHG ERWK VXSSRUW DQG HQFRXUDJHPHQW DQG DOVR KHOSHG ZLWK HGLWRULDO VXJJHVWLRQV WHFKQLFDO DVVLVWDQFH DQG VFLHQWLILF FULWLFLVP 7KH ODVW SHUVRQDO DFNQRZOHGJHPHQWV JR WR %RE
PAGE 4

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

PAGE 5

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

PAGE 6

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f YL

PAGE 7

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f WLVVXH ZHUH H[DPLQHG XVLQJ FRQYHQWLRQDO OLJKW DQG HOHFWURQ PLFURVFRSLF WHFKQLTXHV DQG LPPXQRF\WRFKHPLFDO VWDLQLQJ 7KH QRUPDO VXEVWDQWLD JHODWLQRVD ZDV FRPSDUHG ZLWK YLL

PAGE 8

GLVWLQFW P\HOLQIUHH UHJLRQV RI WKH JUDIWV DQG WKH WZR ZHUH IRXQG WR FRQWDLQ VLPLODU F\WRORJLFDO FKDUDFWHULVWLFV DQG VLPLODU SDWWHUQV RI SHSWLGH VWDLQLQJ $[RQDO SURMHFWLRQV EHWZHHQ )6&f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f DV D PRGHO V\VWHP ,QMXUHG &67 D[RQV ZHUH REVHUYHG LQ GLUHFW DSSRVLWLRQ WR )6& WLVVXH DW WKH KRVWJUDIW LQWHUIDFH DQG &67 D[RQV ZHUH DOVR VHHQ H[WHQGLQJ LQWR WKH WUDQVSODQWV 7RJHWKHU WKH UHVXOWV LQGLFDWH WKDW )6& WUDQVSODQWV FDQ EH XVHG WR UHVWRUH DQDWRPLFDO FRQWLQXLW\ WKURXJK f WKH GLIIHUHQWLDWLRQ RI LQWULQVLF VSLQDO FRUG UHJLRQV DW D OHVLRQ VLWH DQG f WKH GHYHORSPHQW RI D[RQDO LQWHUDFWLRQV EHWZHHQ KRVW DQG JUDIW WLVVXHV LQ WKH DGXOW UDW VSLQDO FRUG 7KHVH REVHUYDWLRQV SURYLGH D EDVLV IRU IXWXUH VWXGLHV WR H[DPLQH WKH IXQFWLRQDO LQWHJUDWLRQ RI )6& WUDQVSODQWV DV ZHOO DV D PRGHO IRU LQYHVWLJDWLQJ WKH ELRORJ\ RI D[RQDO HORQJDWLRQ YLLL

PAGE 9

&+$37(5 ,1752'8&7,21 $1' %$&.*5281' 6SLQDO &RUG ,QMXU\ 7UDXPDWLF LQMXU\ WR WKH VSLQDO FRUG EHJLQV D VHTXHQFH RI HYHQWV WKDW UHVXOW LQ WKH GHJHQHUDWLRQ RI VSLQDO FRUG WLVVXH DQG VXEVHTXHQW ORVV RI VHQVRU\ DQG PRWRU IXQFWLRQ ,Q RUGHU WR XQGHUVWDQG WKH FOLQLFDO SLFWXUH WKH XQGHUO\LQJ SDWKRSK\VLRORJ\ KDV EHHQ VWXGLHG H[WHQVLYHO\ LQ H[SHULPHQWDO PRGHOV RI VSLQDO FRUG LQMXU\ 'RKUPDQQ n 6DQGOHU DQG 7DWRU n :DJQHU HW DO n :LQGOH n62 GH OD 7RUUH n %HDWWLH HW DO n )HKOLQJV DQG 7DWRU nf 7KH SURJUHVVLYH GHJHQHUDWLRQ RI VSLQDO FRUG WLVVXH LV LQLWLDWHG E\ D SULPDU\ LQVXOW WR QHXURQV DQG YDVFXODU HOHPHQWV LQ WKH UHJLRQ RI WKH LQMXU\ 6DQGOHU DQG 7DWRU n %DOHQWLQH DQG 3DULV nDEf 7KH DFXWH HYHQWV WKHQ DFWLYDWH D FDVFDGH RI FKHPLFDO UHDFWLRQV GXH WR LVFKHPLD 2VWHUKROP nf LRQLF FRQGXFWDQFH FKDQJHV
PAGE 10

'DXEH HW DO nf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f 5HFHQWO\ VRPH HPSKDVLV KDV DOVR EHHQ SODFHG XSRQ WKH UHFRJQLWLRQ RI ORQJWHUP IXQFWLRQDO HIIHFWV DW GLVWDQW UHJLRQV RI WKH FHQWUDO QHUYRXV V\VWHP DV D UHVXOW RI GHQHUYDWLRQ DQG UHWURJUDGH FKDQJHV DIWHU D[RWRP\ %HDWWLH HW DO nf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

PAGE 11

UHYLHZHG LQ GH OD 7RUUH n %HDWWLH HW DO nf )RU H[DPSOH FRUWLFRVWHURLGV KDYH EHHQ XVHG WR VWDELOL]H PHPEUDQH FKDQJHV DQG UHGXFH HGHPD DIWHU LQMXU\ $QWLR[LGDQWV KDYH EHHQ UHSRUWHG WR SURPRWH IXQFWLRQDO UHFRYHU\ E\ UHGXFLQJ WKH FRQVHTXHQFHV RI OLSLG SHUR[LGDWLRQ 6DXQGHUV HW DO n $QGHUVRQ HW DO nf 2WKHU GUXJV KDYH EHHQ HPSOR\HG WR LPSURYH IXQFWLRQDO FKDUDFWHULVWLFV DWWULEXWHG WR D[RQDO WUDQVPLVVLRQ RI ORQJWUDFW ILEHUV E\ DOWHULQJ LRQLF FKDQQHO FRQGXFWDQFHV %OLJKW DQG *UXQHU nf ,Q DGGLWLRQ WR SKDUPDFRORJLFDO DSSURDFKHV VXUJLFDO SURFHGXUHV LQFOXGLQJ WKH UHPRYDO RI H[WHUQDO FRPSUHVVLRQ VRXUFHV DQG VWDELOL]DWLRQ RI WKH YHUWHEUDO VHJPHQWV VXUURXQGLQJ WKH LQMXU\ VLWH DUH XVHG WR UHGXFH WKH H[WHQW RI IXQFWLRQDO ORVV DIWHU LQMXU\ 5DQVRKRII nf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nf ,Q DGGLWLRQ DSSOLFDWLRQ RI ELFXFXOOLQH D JDPPDDPLQREXW\ULF DFLG *$%$f DQWDJRQLVW KDV EHHQ DVVRFLDWHG ZLWK LPSURYHPHQWV LQ VSLQDO

PAGE 12

VWHSSLQJ DIWHU FKURQLF VSLQDO FRUG LQMXULHV 7KLV ODWWHU HIIHFW DSSHDUV WR EH PHGLDWHG E\ HQKDQFLQJ VHJPHQWDO LQIOXHQFHV RQ WKH UHPDLQLQJ FLUFXLWV 5RELQVRQ DQG *ROGEHUJHU nf &RQYHUVHO\ WKH FOLQLFDO XVH RI WKH *$%$ DJRQLVW EDFORIHQ KDV KDG EHQHILFLDO HIIHFWV LQ UHGXFLQJ H[WUHPH VSDVWLFLW\ LQ VSLQDO FRUG LQMXUHG SDWLHQWV E\ HQKDQFLQJ WKH LQKLELWRU\ LQIOXHQFHV XSRQ VHJPHQWDO UHIOH[HV %ORFK DQG %DVEDXP nf 7KXV PDQ\ SURSRVHG SKDUPDFRORJLFDO WUHDWPHQWV IRU WKH FKURQLF VSLQDO LQMXU\ SDWLHQW UHO\ XSRQ HVWDEOLVKLQJ D EDODQFH RI UHFHSWRUPHGLDWHG QHXURQDO DFWLYLWLHV DW WKH VHJPHQWDO OHYHO 3URPRWLRQ RI $[RQDO 5HJHQHUDWLRQ RU 6SURXWLQJ 2YHU WKH SDVW GHFDGH DGYDQFHV LQ WKHUDSHXWLF VWUDWHJLHV KDYH UHVXOWHG LQ D SURJUHVVLYH UHGXFWLRQ RI PRUWDOLW\ ZLWK D FRQFXUUHQW LPSURYHPHQW LQ WKH TXDOLW\ RI OLIH IROORZLQJ VSLQDO FRUG LQMXU\ HJ %ORFK DQG %DVEDXP n *UHHQ DQG .ORVH nf 1HYHUWKHOHVV D FRQWLQXHG HPSKDVLV PXVW EH SODFHG XSRQ UHVHDUFK HIIRUWV WR SURPRWH WKH UHSDLU RI WKH VSLQDO FRUG DQG WR UHVWRUH IXQFWLRQV QRUPDOO\ PHGLDWHG E\ ERWK VHJPHQWDO DQG ORQJWUDFW V\VWHPV 6LQFH QHXURJHQHVLV LV HVVHQWLDOO\ ODFNLQJ ZLWKLQ WKH DGXOW PDPPDOLDQ FHQWUDO QHUYRXV V\VWHP &16f WKHUH LV QR PHFKDQLVP IRU VSRQWDQHRXV UHSODFHPHQW RI QHXURQV DIWHU LQMXU\ 5HSDLU RI WKH VSLQDO FRUG LV WKHUHIRUH GHSHQGHQW XSRQ WKH UHJHQHUDWLRQ RU VSURXWLQJ RI D[RQV IURP H[LVWLQJ QHXURQV $Q HPSKDVLV LQ PDPPDOLDQ UHJHQHUDWLRQ UHVHDUFK KDV

PAGE 13

EHHQ SODFHG XSRQ WKH HYDOXDWLRQ RI GLIIHUHQFHV EHWZHHQ WKH SHULSKHUDO QHUYRXV V\VWHP 316f ZKHUH LQMXUHG D[RQV UHJHQHUDWH DQG DUH DEOH WR VXFFHVVIXOO\ UHLQQHUYDWH WKHLU WDUJHW WLVVXHV DQG WKH &16 ZKHUH D[RQV GR QRW UHJHQHUDWH VXFK WKDW WKH\ UHWXUQ WR WKHLU RULJLQDO SRVWV\QDSWLF VLWHV UHYLHZHG LQ &OHPHQWH n *XWK n %HUQVWHLQ HW DO n .LHUQDQ nf 7KH DVSHFWV RI WKHVH V\VWHPV ZKLFK KDYH EHHQ FRQWUDVWHG PRVW RIWHQ DUH WKH UHJHQHUDWLYH RU VSURXWLQJ FDSDFLW\ RI WKH D[RQV DQG WKH SHUPLVVLYH RU LQKLELWRU\ QDWXUH RI WKHLU HQYLURQPHQW 7KLV LQWHUDFWLRQ EHWZHHQ D[RQV DQG VXUURXQGLQJ FHOOV UHFHLYHG HDUO\ DWWHQWLRQ E\ 5DPRQ \ &DMDO nf ZKR REVHUYHG VPDOO UHJHQHUDWLYH VSURXWV IROORZLQJ H[SHULPHQWDO LQMXU\ WR VSLQDO FRUGV RI \RXQJ NLWWHQV 7KH VSURXWV IDLOHG WR SHUVLVW DIWHU WZR ZHHNV DQG QR IXQFWLRQDO UHFRYHU\ ZDV REVHUYHG 0RUH UHFHQWO\ $JXD\R DQG KLV FROOHDJXHV FRQILUPHG 5DPRQ \ &DMDOnV QRWLRQ WKDW WKH HQYLURQPHQW FDQ SURIRXQGO\ LQIOXHQFH UHJHQHUDWLRQ %\ LPSODQWLQJ SLHFHV RI SHULSKHUDO QHUYH LQWR WKH EUDLQ DQG VSLQDO FRUG WKH\ GHPRQVWUDWHG WKDW &16 QHXURQV FDQ H[WHQG DQG PDLQWDLQ ORQJ D[RQDO VSURXWV ZLWKLQ WKH SHULSKHUDO HQYLURQPHQW 'DYLG DQG $JXD\R 5LFKDUGVRQ HW DO nf 2WKHU UHVHDUFKHUV KDYH SURYLGHG HYLGHQFH RI YDU\LQJ GHJUHHV RI V\QDSWLF UHRUJDQL]DWLRQ IROORZLQJ LQMXU\ ZLWKLQ WKH DGXOW EUDLQ DQG VSLQDO FRUG A HJ &RWPDQ DQG 1LHWR6DPSHGUR n *ROGEHUJHU DQG 0XUUD\ n 6WHZDUG nEf 7RJHWKHU WKLV ZRUN KDV LQVSLUHG

PAGE 14

UHQHZHG HQFRXUDJHPHQW LQ WKH ILHOG RI &16 DQG VSLQDO FRUG UHSDLU 6WUDWHJLHV DLPHG DW SURPRWLQJ WKH UHJHQHUDWLRQ RI VSLQDO FRUG D[RQV LQFOXGH FKDQJHV WR WKH &16 PLFURHQYLURQPHQW DV ZHOO DV PHWKRGV WKDW PLJKW VWLPXODWH D[RQDO VSURXWLQJ DQG HORQJDWLRQ *XWK HW DO nDf GHPRQVWUDWHG WKDW D[RQV ZLWKLQ WKH VSLQDO FRUG ZLOO QRW H[WHQG LQWR D YDFDQW OHVLRQ VLWH LQVWHDG WKH\ PXVW HQFRXQWHU D FHOOXODU WHUUDLQ IRU VXFFHVVIXO HORQJDWLRQ ,Q DGGLWLRQ WKH HVWDEOLVKPHQW RI D GHQVH PHVKZRUN RI JOLDO DQG FRQQHFWLYH WLVVXH HOHPHQWV DW WKH OHVLRQ VLWH PD\ DOVR SUHVHQW D SUREOHP IRU JURZLQJ D[RQV 7KH FRQFHSW RI ILEURJOLDO VFDUULQJ DV DQ LPSHQHWUDEOH EDUULHU WR HORQJDWLQJ D[RQV ZDV FKDPSLRQHG E\ 5DPRQ \ &DMDO f DQG KDV EHHQ D WRSLF RI GHEDWH IRU WKH VHYHUDO GHFDGHV UHYLHZHG LQ 5HLHU HW DO nE 5HLHU DQG +RXOH nf :LWK WKLV LQ PLQG VHYHUDO DSSURDFKHV KDYH EHHQ WDNHQ WR SUHYHQW RU UHGXFH WKH H[WHQW RI JOLDOILEUREODVWLF VFDU IRUPDWLRQ DIWHU LQMXU\ DQG WKXV SURPRWH D[RQDO HORQJDWLRQ 7KH LQYDVLRQ RI ILEUREODVWV FDQ EH PLQLPL]HG E\ XVLQJ FORVHG VSLQDO FRUG LQMXU\ PRGHOV VXFK DV WKH ZHLJKWGURS FRQWXVLRQ RU FOLS FRPSUHVVLRQ DSSURDFKHV ,Q DGGLWLRQ SKDUPDFRORJLFDO DJHQWV KDYH EHHQ DSSOLHG WR SUHYHQW WKH IRUPDWLRQ RI VFDU WLVVXH DW D OHVLRQ VLWH HJ :LQGOH HW DO n *XWK HW DO nEf 7KHVH UHVXOWV KDYH VXJJHVWHG WKDW D[RQV PD\ H[WHQG D VKRUW GLVWDQFH LQWR D VSLQDO OHVLRQ ZKHUH VXFK D VFDU LV UHGXFHG

PAGE 15

6RPH UHFHQW LQYHVWLJDWLRQV KDYH EHHQ GLUHFWHG DW SURPRWLQJ WKH HORQJDWLRQ RI LQMXUHG VSLQDO FRUG D[RQV XVLQJ GLIIHUHQW PHWKRGV 2QH VXFK DSSURDFK LQYROYHV WKH LPSODQWDWLRQ RI FXOWXUHG FHOOV LQWR D OHVLRQ 6LHJDO HW DO n :UDWKDOO HW DO nf 7KHVH VWXGLHV UHIOHFW D GHVLUH WR DSSO\ VXEVWDQFHV NQRZQ WR LQGXFH D[RQDO HORQJDWLRQ LQ YLWUR WR WKH LQMXUHG VSLQDO FRUG 7KH UHVXOWV VXJJHVW WKDW WKH HIIHFWV PD\ EH PRUH FRPSOH[ LQ YLYR EHFDXVH RI PDQ\ XQFRQWUROOHG YDULDEOHV )LQDOO\ WKH DSSOLFDWLRQ RI HOHFWULFDO ILHOGV KDV DOVR EHHQ LQYHVWLJDWHG DV D PHDQV WR LQFUHDVH WKH GLVWDQFH RI D[RQDO HORQJDWLRQ %RUJHQV HW DO n nf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

PAGE 16

D[RQV RU SDWWHUQV RI UHLQQHUYDWLRQ FDQQRW EH YHULILHG XVLQJ WKHVH WHFKQLTXHV )HWDO 1HXUDO 7UDQVSODQWV DQG 6SLQDO &RUG 5HSDLU 5HSRUWV RI VRPH GHJUHH RI IXQFWLRQDO UHFRYHU\ IROORZLQJ WUDQVSODQWDWLRQ RI IHWDO &16 WLVVXH LQWR WKH EUDLQ HJ *DVK HW DO n %MRUNOXQG DQG 6WHQHYL n 'XQQHWW HW DO n %X]VDNL DQG *DJH nf VXJJHVWHG WKDW HPEU\RQLF QHXUDO WLVVXH PLJKW SURPRWH QHXURQDO UHSDLU IROORZLQJ LQMXU\ RU GLVHDVH 7KHVH ILQGLQJV KDYH OHG WR WKH DSSOLFDWLRQ RI VLPLODU WUDQVSODQWDWLRQ VWUDWHJLHV LQ WKH VSLQDO FRUG 7KH ILUVW LQWUDVSLQDO IHWDO JUDIWLQJ VWXGLHV UHVXOWHG LQ ORZ WUDQVSODQW VXUYLYDO UDWHV DV FRPSDUHG WR VLPLODU H[SHULPHQWV LQ WKH EUDLQ 1\JUHQ HW DO n 3DWHO DQG %HUQVWHLQ n 'DV n &RPPLVVLRQJ nf 6XFK GLIILFXOWLHV VHUYHG WR XQGHUVFRUH WKH H[WUHPH SDWKRORJLFDO FRQVHTXHQFHV RI VSLQDO FRUG LQMXU\ ,W KDV EHHQ SURSRVHG WKDW PDQ\ RI WKH JUDIWV IDLOHG WR VXUYLYH EHFDXVH WKH\ GLG QRW LQWHJUDWH ZLWK WKH SDUHQFK\PD RI WKH LQMXUHG VSLQDO FRUG 'DV nf )ROORZLQJ LPSURYHPHQWV LQ VXUJLFDO SURFHGXUHV DQG FDUHIXO VHOHFWLRQ RI GRQRU WLVVXH DJHV 1RUQHV HW DO n 5HLHU HW DO nD 5HLHU nf PRUH UHFHQW VWXGLHV RI LQWUDVSLQDO WUDQVSODQWDWLRQ KDYH PHW ZLWK JUHDWHU VXFFHVV 2QH DSSURDFK LQYROYHV LQMHFWLQJ VXVSHQVLRQV RI GLVVRFLDWHG HPEU\RQLF EUDLQVWHP FHOOV FDXGDO WR WKH VLWH RI LQMXU\ 1\JUHQ HW DO n 1RUQHV HW DO n 3ULYDW HW DO nf 7KH IRFXV RI WKLV VWUDWHJ\ LV WR UHVWRUH VXSUDVSLQDO PRGXODWRU\ LQIOXHQFHV

PAGE 17

WR GHQHUYDWHG UHJLRQV EHORZ WKH OHYHO RI D VSLQDO OHVLRQ $Q HPSKDVLV KDV EHHQ SODFHG XSRQ WKH GHVFHQGLQJ PRQRDPLQHUJLF V\VWHPV ZKLFK KDYH EHHQ DVVRFLDWHG ZLWK WKH PRGXODWLRQ RI VHJPHQWDO UHIOH[ DQG ORFRPRWRU FLUFXLWULHV 7KHVH VWXGLHV KDYH LQGLFDWHG WKDW WKH LQMXUHG VSLQDO FRUG FDQ EH UHLQQHUYDWHG E\ JUDIWHG HPEU\RQLF EUDLQVWHP QHXURQV )XUWKHUPRUH VXFK JUDIWV FDQ PHGLDWH VRPH W\SHV RI UHIOH[ FKDQJH DIWHU VSLQDO LQMXU\ RU FKHPLFDO GHQHUYDWLRQ %XFKDQDQ DQG 1RUQHV n 0RRUPDQ HW DO n 3ULYDW HW DO n n f :KLOH WUDQVSODQWV SODFHG EHORZ D OHVLRQ VLWH PD\ FRQWULEXWH WR WKH UHSODFHPHQW RI PRGXODWRU\ LQIOXHQFHV UHFRYHU\ RI VHQVDWLRQ DQG YROXQWDU\ PRWRU FDSDFLWLHV ZLOO UHTXLUH DSSOLFDWLRQV WKDW UHVWRUH FRQWLQXLW\ DW WKH OHVLRQ VLWH 7KHUHIRUH DQ DOWHUQDWLYH DSSURDFK WRZDUG VSLQDO FRUG UHSDLU LQYROYHV WKH WUDQVSODQWDWLRQ RI IHWDO WLVVXH GLUHFWO\ LQWR D OHVLRQ FDYLW\ HJ IHWDO VSLQDO FRUG )6&f JUDIWVf 5HLHU HW DO nD nnD +RXOH DQG 5HLHU nf 7KLV DSSURDFK GLIIHUV IURP WKH WUDQVSODQWDWLRQ RI WLVVXH FDXGDO WR DQ LQMXU\ DQG LW GLUHFWO\ DGGUHVVHV WKUHH PDMRU FRQVHTXHQFHV RI VSLQDO FRUG LQMXU\ )LUVW WKH SUHVHQFH RI HPEU\RQLF WLVVXH DW WKH VLWH RI D OHVLRQ PD\ SURYLGH WURSKLF LQIOXHQFHV WR SUHYHQW GHJHQHUDWLYH FKDQJHV DIWHU LQMXU\ )RU H[DPSOH IHWDO JUDIWV SODFHG LQWR WKH LQMXUHG VSLQDO FRUG RU FRUWH[ RI QHRQDWDO UDWV KDYH EHHQ DVVRFLDWHG ZLWK D VLJQLILFDQW UHGXFWLRQ LQ WKH H[WHQW RI FHOO GHDWK WKDW LV

PAGE 18

FKDUDFWHULVWLF RI VXFK OHVLRQV LQ WKH LQIDQW &16 %UHJPDQ DQG 5HLHU n +DXQ DQG &XQQLQJKDP nf ,Q DGGLWLRQ WKHUH KDV EHHQ DW OHDVW RQH VXJJHVWLRQ WKDW WKH SUHVHQFH RI IHWDO WLVVXH LQ D VSLQDO OHVLRQ FDYLW\ PD\ SUHYHQW GHJHQHUDWLRQ RI ZKLWH PDWWHU ILEHU WUDFWV LQ DGXOW UHFLSLHQWV 'DV f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n 5HLHU nf :KLOH UHVXOWV IURP UHFHQW VWXGLHV VXJJHVW WKDW GHVFHQGLQJ D[RQV FDQ H[WHQG DFURVV D )6& JUDIW LQ QHZERUQ UDWV WKHUH LV QR HYLGHQFH WR GDWH WR LQGLFDWH WKDW LQMXUHG &16 D[RQV LQ WKH DGXOW ZLOO EULGJH D IHWDO JUDIW WR UHLQQHUYDWH WKHLU RULJLQDO VSLQDO FRUG WDUJHW UHJLRQV +RZHYHU DQ DOWHUQDWLYH SRVVLELOLW\ LV WKDW WUDQVSODQWV PD\ HVWDEOLVK D QHXURQDO

PAGE 19

UHOD\ SDWKZD\ DFURVV D VSLQDO FRUG OHVLRQ -RKQVRQ DQG %XQJH n 1RUQHV HW DO nr 5HLHU n 5HLHU HW DOn -DNHPDQ DQG 5HLHU f 'HYHORSPHQW RI D 1HXUDO 5HOD\ $FURVV D 6SLQDO ,QMXU\ 6LWH 7KH FRQFHSW RI D QHXUDO UHOD\ KDV EHHQ GLVFXVVHG DW LWV PRVW EDVLF OHYHO E\ 6KHSDUG nf 7KH WKUHH FRPSRQHQWV RI WKH V\QDSWLF WULDG WKDW IRUP DQ\ QHXURQDO FLUFXLW LQFOXGH LQSXW QHXURQV LQWULQVLF QHXURQV DQG SURMHFWLRQ QHXURQV &RPSOH[ YDULDWLRQV RI WKHVH FRPSRQHQWV IRUP WKH EDVLV IRU ORFDO FLUFXLWV WKURXJKRXW WKH &16 5DNLF nf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

PAGE 20

QHXURQV 'DV n 0DKDOLN HW DO n &ODUNH HW DO nEf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n 'XQQHWW DQG %MRUNOXQG n *DJH DQG %X]VDNL nf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

PAGE 21

VSLQDO FRUG LV WR GHILQH WKH DQDWRPLFDO EDVLV IRU LQWHJUDWLRQ RI KRVW DQG JUDIW WLVVXHV 3UHOLPLQDU\ VWXGLHV RI LQWHUDFWLRQV EHWZHHQ VXFK WUDQVSODQWV DQG WKH LQMXUHG DGXOW UDW VSLQDO FRUG KDYH LQGLFDWHG WKDW VRPH D[RQDO SURMHFWLRQV FDQ IRUP EHWZHHQ WKH WLVVXHV 5HLHU HW DO n nDf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

PAGE 22

&+$37(5 *(1(5$/ 0(7+2'6 $ QXPEHU RI VSLQDO FRUG LQMXU\ PRGHOV KDYH EHHQ XVHG WR HYDOXDWH SRWHQWLDO VWUDWHJLHV IRU LQWHUYHQWLRQ DQG UHSDLU 7KHVH LQFOXGH GLVFUHWH OHVLRQV RI VSHFLILF ILEHU WUDFWV FKHPLFDO D[RWRP\ RI ILEHU W\SHV EOXQW FRQWXVLRQ RU FRPSUHVVLRQ LQMXULHV DQG FRPSOHWH RU SDUWLDO WUDQVHFWLRQ PRGHOV UHYLHZHG LQ GH OD 7RUUH n %HDWWLH HW DO n 'DV nf 7KH SUHVHQW LQYHVWLJDWLRQV KDYH HPSOR\HG D PRGHO RI WUDQVSODQWDWLRQ LQWR SDUWLDO WUDQVHFWLRQ FDYLWLHV SUHSDUHG E\ DVSLUDWLRQ LPPHGLDWHO\ EHIRUH JUDIWLQJ DFXWH OHVLRQVf 7KH WUDQVSODQWDWLRQ PHWKRGV XVHG WKURXJKRXW WKHVH VWXGLHV DUH VLPLODU WR WKRVH GHWDLOHG LQ SUHYLRXV UHSRUWV 5HLHU HW DOnDnDf (DFK H[SHULPHQWDO GHVLJQ KDV HPSOR\HG RQO\ PLQRU PRGLILFDWLRQV RI WKH SURFHGXUHV GHVFULEHG EHORZ ([SHULPHQWDO $QLPDOV $GXOW IHPDOH LQEUHG 6SUDJXH'DZOH\ UDWV ZHUH XVHG WKURXJKRXW WKHVH VWXGLHV $OO RI WKH UDWV ZHUH REWDLQHG IURP =LYLF0LOOHU /DERUDWRULHV $OOLVRQ 3DUN 3$f DQG ZHLJKHG JUDPV DW WKH VWDUW RI WKH H[SHULPHQWV 7KH UDWV ZHUH KRXVHG WZR SHU FDJH LQ WKH 8QLYHUVLW\ RI )ORULGD DQLPDO UHVRXUFHV IDFLOLW\ DFFUHGLWHG E\ WKH $PHULFDQ $VVRFLDWLRQ RI /DERUDWRU\ $QLPDO &DUHWDNHUVf DFFRUGLQJ WR WKH JXLGHOLQHV

PAGE 23

HVWDEOLVKHG E\ WKH 1DWLRQDO ,QVWLWXWHV RI +HDOWK 3XEOLFDWLRQ QXPEHU f 7KH\ ZHUH H[DPLQHG GDLO\ E\ D YHWHULQDULDQ DQGRU YHWHULQDU\ WHFKQLFLDQ IRU JHQHUDO KHDOWK FRQGLWLRQV DQG IRU DQ\ SRVWRSHUDWLYH FRPSOLFDWLRQV GXH WR WKH VSLQDO FRUG LQMXU\ $OO VXUJLFDO SURFHGXUHV ZHUH SHUIRUPHG ZLWK LQVWUXPHQW SUHSDUDWLRQ LQ DQWLPLFURELDO EHQ]DONRQLXP FKORULGH VROXWLRQ =HSKDULQ +& :LQWKURS %UHRQ /DERUDWRULHVf DQG b (WKDQRO 3UHSDUDWLRQ RI /HVLRQ &DYLWLHV 7KH WUDQVSODQW UHFLSLHQWV ZHUH DQHVWKHWL]HG ZLWK LQWUDPXVFXODU LQMHFWLRQV RI D PL[WXUH RI NHWDPLQH .HWDVHW PJNJ $YHFR &Rf DQG [\OD]LQH 5RPSXQ PJNJ 0RED\ &RUSf 7KH VSLQRXV SURFHVVHV ZHUH WKHQ H[SRVHG E\ ORQJLWXGLQDO LQFLVLRQV RI WKH RYHUO\LQJ PXVFXODWXUH %OHHGLQJ IURP WKH VXSHUILFLDO PXVFOHV ZDV FRQWUROOHG ZLWK HSLQHSKULQHLPSUHJQDWHG FRWWRQ SHOOHWV *LQJLSDN %HOSRUW &R,QFf $ ODPLQHFWRP\ ZDV WKHQ SHUIRUPHG DW WKH DSSURSULDWH YHUWHEUDO OHYHO XVLQJ ILQHWLSSHG URQJHXUV DQG WKH KRVW VSLQDO FRUG ZDV H[SRVHG E\ D ORQJLWXGLQDO LQFLVLRQ RI WKH GRUVDO PHQLQJHV MXVW ODWHUDO WR PLGOLQH 8VLQJ LULGHFWRP\ VFLVVRUV DQG D JODVV PLFURSLSHWWH ZLWK OLJKW DVSLUDWLYH SUHVVXUH D FDYLW\ RI PP LQ OHQJWK ZDV FUHDWHG LQ WKH SDUHQFK\PD RI WKH VSLQDO FRUG DQG EOHHGLQJ ZDV FRQWUROOHG ZLWK VPDOO SHOOHWV RI JHODWLQ VSRQJH *HOIRDP 8SMRKQ &Rf VRDNHG LQ D VDOLQH VROXWLRQ FRQWDLQLQJ ERYLQH WKURPELQ 7KURPERVWDW 3DUNH 'DYLVf

PAGE 24

3UHSDUDWLRQ RI 'RQRU 7LVVXH )RU HDFK WUDQVSODQWDWLRQ VHVVLRQ D SUHJQDQW UDW ( HPEU\RQLF GD\ ( GD\ RI LQVHPLQDWLRQf ZDV DQHVWKHWL]HG ZLWK b FKORUDO K\GUDWH PJNJ LSf $ ODSDURWRP\ ZDV SHUIRUPHG DQG LQGLYLGXDO GRQRU HPEU\RV DSSUR[LPDWH FURZQUXPS OHQJWK RI PPf ZHUH UHPRYHG DV QHHGHG DQG SODFHG LQWR D VWDQGDUG WLVVXH FXOWXUH PHGLXP +DQNnV %DODQFHG 6DOW 6ROXWLRQf 7KH HPEU\RQLF VSLQDO FRUG WLVVXH ZDV WKHQ GLVVHFWHG E\ UHPRYDO RI RYHUO\LQJ VNLQ OD\HUV DQG WKH GHWDFKPHQW RI WKH VSLQDO JDQJOLD 7KH VSLQDO FRUG ZDV VWULSSHG RI HPEU\RQLF PHQLQJHDO OD\HUV DQG XVHG IRU WUDQVSODQWDWLRQ ZLWKLQ RQH KRXU RI UHPRYDO IURP WKH PRWKHU $W WKH HQG RI WKH WUDQVSODQWDWLRQ VHVVLRQ WKH SUHJQDQW UDW ZDV HXWKDQL]HG ZLWK DQ LQWUDFDUGLDF LQMHFWLRQ RI VRGLXP SHQWREDUELWDO %XWOHU &Rf 7UDQVSODQWDWLRQ 3URFHGXUH 2QFH KHPRVWDVLV ZDV DFKLHYHG LQ WKH KRVW HLWKHU RQH RU WZR SLHFHV RI GRQRU VSLQDO FRUG WLVVXH ZHUH FXW WR DSSUR[LPDWH WKH OHQJWK RI WKH SUHSDUHG FDYLW\ 7KH GRQRU WLVVXH DQG D VPDOO DPRXQW RI WLVVXH FXOWXUH PHGLXP ZHUH SODFHG LQWR WKH FDYLW\ ZLWK D IODPHWLSSHG PLFURSLSHWWH $IWHU SODFHPHQW RI WKH JUDIWV WKH H[FHVV WLVVXH FXOWXUH PHGLXP ZDV UHPRYHG ZLWK OLJKW PDQXDO VXFWLRQ $ VLQJOH SLHFH RI K\GURFHSKDOXV VKXQW ILOP 'XUDILOP &RGPDQ 6KXUWOHII ,QFf ZDV XVXDOO\ FXW WR D VL]H MXVW ODUJHU WKDQ WKH OHVLRQ FDYLW\ DQG SRVLWLRQHG GLUHFWO\ DERYH WKH JUDIW

PAGE 25

7KH GXUDO LQFLVLRQ ZDV FORVHG ZLWK LQWHUUXSWHG VXWXUHV DQG WKH VSLQDO FRUG ZDV WKHQ FRYHUHG ZLWK D VHFRQG SLHFH RI V\QWKHWLF GXUDO FRYHULQJ 7KH RYHUO\LQJ PXVFOHV ZHUH WKHQ VXWXUHG LQ OD\HUV XVLQJ VLON DQG WKH VNLQ LQFLVLRQ FORVHG ZLWK ZRXQG FOLSV )LVKHUf 3RVW2SHUDWLYH &DUH )ROORZLQJ VXUJHU\ DOO UDWV UHFHLYHG D VXEFXWDQHRXV LQMHFWLRQ RI ORQJDFWLQJ SHQLFLOOLQ 'XDO3HQ 7HFK $PHULFD 8f 7KH\ ZHUH FDUHIXOO\ PRQLWRUHG DQG NHSW RQ D KHDWLQJ SDG RU XQGHU D PLOG KHDW ODPS XQWLO UHFRYHU\ IURP DQHVWKHVLD :LWKLQ KRXUV WKH UDWV ZHUH UHWXUQHG WR WKH DQLPDO FDUH IDFLOLW\ ZKHUH WKH\ ZHUH IHG UDW FKRZ DQG ZDWHU DG OLELWXP DQG PDLQWDLQHG XQGHU D KRXU OLJKW DQG KRXU GDUN VFKHGXOH ,Q WKH VXEVHTXHQW ZHHNV DIWHU VXUJHU\ DSSUR[LPDWHO\ b RI WKH DQLPDOV LQLWLDWHG PLOG DXWRWRP\ RI WKH LSVLODWHUDO IRUHOLPE RU KLQGOLPE FRUUHVSRQGLQJ WR OHVLRQ OHYHOf DQG ZHUH WUHDWHG ZLWK GDLO\ DSSOLFDWLRQ RI YHWHULQDU\ DXWRSKDJLF UHSHOOHQW &KHZJXDUG 6XPPLW +LOO /DEVf 7KRVH IHZ UDWV WKDW IDLOHG WR UHVSRQG WR VXFK WUHDWPHQW ZLWKLQ D IHZ GD\V ZHUH VDFULILFHG VKRUWO\ WKHUHDIWHU DQG LQFOXGHG LQ WKH GDWD DQDO\VLV 7KLV FRQVLGHUDWLRQ FRQWULEXWHG WR WKH UDQJH RI SRVWJUDIW VXUYLYDO WLPHV SRVWJUDIW LQWHUYDOVf UHIHUUHG WR WKURXJKRXW WKH ZRUN

PAGE 26

&+$37(5 ',))(5(17,$7,21 2) 68%67$17,$ *(/$7,126$/,.( 5(*,216 ,1 ,175$63,1$/ 75$163/$176 2) (0%5<21,& 63,1$/ &25' 7,668( ,QWURGXFWLRQ ,QWUDFHUHEUDO JUDIWV RI IHWDO &16 WLVVXH KDYH EHHQ VKRZQ WR FRPSHQVDWH IRU D YDULHW\ RI IXQFWLRQDO GHILFLWV LQ H[SHULPHQWDO DQLPDO PRGHOV 7KLV PD\ RFFXU WKURXJK WKH UHSODFHPHQW RI QHXURQDO FLUFXLWULHV RU QHXURWUDQVPLWWHUV RU E\ WKH SURGXFWLRQ RI QHXURQRWURSKLF VXEVWDQFHV ZLWKLQ WKH KRVW EUDLQ UHYLHZHG LQ %MRUNOXQG DQG 6WHQHYL n 6ODGHN DQG *DVK n %MRUNOXQG HW DO n 'XQQHWW DQG %MRUNOXQG nf 7KH GHJUHH WR ZKLFK VXFK JUDIWV FDQ VXEVHUYH WKH IXQFWLRQV RI WKH GDPDJHG EUDLQ UHJLRQ PD\ GHSHQG XSRQ WKH DELOLW\ RI WKH JUDIWHG WLVVXH WR GLIIHUHQWLDWH DQG LQWHJUDWH ZLWK WKH KRVW QHXUDO FLUFXLWU\ 7KXV VHYHUDO LQYHVWLJDWRUV KDYH H[DPLQHG WKH H[WHQW WR ZKLFK IHWDO QHXUDO WUDQVSODQWV ZLOO GLIIHUHQWLDWH DQG H[KLELW RUJDQRW\SLF FKDUDFWHULVWLFV ZKHQ SODFHG LQWR KRPRWRSLF DQG KHWHURWRSLF VLWHV ZLWKLQ WKH &16 HJ -DHJHU DQG /XQG n $OYDUDGR0DOODUW DQG 6RWHOR n .URPHU HW DO n (ULNVGRWWHU1LOVVRQ HW DO n +DUYH\ HW DO n 6RUHQVHQ DQG =LPPHU nDEf )URP WKHVH VWXGLHV LW LV FOHDU WKDW JUDIWHG UHJLRQV RI WKH &16 H[KLELW

PAGE 27

GLIIHUHQW GHJUHHV RI GLIIHUHQWLDWLRQ GHSHQGLQJ XSRQ WKH DJH RI WKH HPEU\RQLF GRQRU WLVVXH DQG WKH UHJLRQ IURP ZKLFK LW LV REWDLQHG ,Q UHFHQW \HDUV WKHUH KDV EHHQ VRPH HQWKXVLDVP IRU WKH DSSOLFDWLRQ RI IHWDO &16 WLVVXH WUDQVSODQWDWLRQ WHFKQLTXHV WR WKH SUREOHP RI VSLQDO FRUG LQMXU\ HJ 'DV n &RPPLVVLRQJ n 3ULYDW HW DO n 5HLHU HW DOn nD +RXOH DQG 5HLHU nf 7RJHWKHU WKHVH UHSRUWV LQGLFDWH WKDW LQWUDVSLQDO WUDQVSODQWDWLRQ UHVXOWV LQ WKH VXUYLYDO DQG LQWHJUDWLRQ RI HPEU\RQLF GRQRU WLVVXH LQ ERWK QHRQDWDO DQG DGXOW UHFLSLHQWV 2QH DSSURDFK LQYROYHV WKH LQWURGXFWLRQ RI IHWDO QHXURQV LQWR WKH OHVLRQ VLWH 5HLHU n 5HLHU HW DO nf ,Q SDUWLFXODU KRPRWRSLF JUDIWV SODFHG LQWR WKH VLWH RI D VSLQDO OHVLRQ PD\ VHUYH DV D VRXUFH RI VSHFLILF LQWUDVSLQDO QHXURQDO SRSXODWLRQV ZLWK DQ LQKHUHQW SRWHQWLDO IRU LQWHJUDWLQJ ZLWK V\QDSWLF FLUFXLWV DERYH DQG EHORZ WKH LQMXU\ 6RPH GHJUHH RI KRPRW\SLF GLIIHUHQWLDWLRQ KDV EHHQ LQGLFDWHG E\ VWXGLHV LQ ZKLFK IHWDO VSLQDO FRUG WUDQVSODQWV ZHUH SODFHG LQWR LQWUDFHUHEUDO RU LQWUDVSLQDO FDYLWLHV 5HLHU DQG %UHJPDQ n 5HLHU HW DO n nDEf ,Q WKHVH LQLWLDO LQYHVWLJDWLRQV GLVWLQFW P\HOLQIUHH UHJLRQV ZHUH REVHUYHG LQ PDWXUHG JUDIWV OHDGLQJ WR WKH K\SRWKHVLV WKDW WKHVH XQP\HOLQDWHG DUHDV FRUUHVSRQGHG WR WKH VXSHUILFLDO GRUVDO KRUQ f§ HVSHFLDOO\ WKH VXEVWDQWLD JHODWLQRVD 6*f f§ RI WKH QRUPDO VSLQDO FRUG 7KLV UHJLRQ RI WKH VSLQDO FRUG LV HDVLO\ LGHQWLILHG LQ QRUPDO WLVVXHV EDVHG XSRQ WKH SDXFLW\ RI

PAGE 28

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n -DNHPDQ HW DO f 0DWHULDOV DQG 0HWKRGV $QLPDOV DQG 6XUJLFDO 3URFHGXUHV 7HQ DGXOW UDWV UHFHLYHG LQWUDVSLQDO LPSODQWV RI (X UDW VSLQDO FRUG WLVVXH 7KH VXUJLFDO SURFHGXUHV ZHUH VLPLODU WR WKRVH GHVFULEHG LQ SUHYLRXV UHSRUWV 5HLHU HW DO nD nD &KDSWHU WKLV YROXPHf (DFK UDW ZDV DQHVWKHWL]HG ZLWK NHWDPLQH DQG [\OD]LQH DQG D ODPLQHFWRP\ ZDV SHUIRUPHG DW WKH 7 YHUWHEUDO DSSUR[LPDWHO\ ,A /M VSLQDOf OHYHO $Q DVSLUDWLYH OHVLRQ FDYLW\ PP LQ OHQJWK ZDV FUHDWHG DW WKH H[SRVHG VLWH 7KH OHVLRQ ZDV WKHQ H[WHQGHG WR LQFOXGH HLWKHU DQ H[WHQVLYH GRUVDO IXQLFXORWRP\ RU ODWHUDO

PAGE 29

KHPLVHFWLRQ :KROH VHJPHQWV RI IHWDO VSLQDO FRUG PP LQ OHQJWK ZHUH GLVVHFWHG IURP GRQRU HPEU\RV DQG LQWURGXFHG LQWR WKH OHVLRQ FDYLWLHV 5HLHU HW DO nDf /LJKW DQG (OHFWURQ 0LFURVFRS\ $W WR PRQWKV DIWHU WUDQVSODQWDWLRQ UHFLSLHQWV ZHUH DQHVWKHWL]HG ZLWK D OHWKDO GRVH RI VRGLXP SHQWREDUELWDO DQG SHUIXVHG WKURXJK WKH KHDUW ZLWK b 1D&O IROORZHG E\ b JOXWDUDOGHK\GH DQG b SDUDIRUPDOGHK\GH LQ 0 SKRVSKDWH EXIIHU )ROORZLQJ WKH SHUIXVLRQ VHJPHQWV RI WLVVXH LQFOXGLQJ WKH WUDQVSODQW DQG VXUURXQGLQJ VSLQDO FRUG ZHUH WKHQ H[FLVHG DQG GLYLGHG LQWR VHYHUDO WUDQVYHUVH RU ORQJLWXGLQDO VOLFHV 7KH VSHFLPHQV ZHUH VXEVHTXHQWO\ RVPLFDWHG GHK\GUDWHG DQG HPEHGGHG LQ (0 %HG (OHFWURQ 0LFURVFRS\ 6FLHQFHVf IRU SODVWLF WKLFN VHFWLRQLQJ 5HJLRQV RI WKH JUDIWV FODVVLILHG DV 6*OLNH DW WKH OLJKW PLFURVFRSLF OHYHO ZHUH WULPPHG DQG WKLQ VHFWLRQV ZHUH VXUYH\HG DW WKH XOWUDVWUXFWXUDO OHYHO XVLQJ D =HLVV (0& HOHFWURQ PLFURVFRSH ,PPXQRFYWRFKHPLVWUY $W VLPLODU LQWHUYDOV WKH UHPDLQLQJ LQWUDVSLQDO JUDIW UHFLSLHQWV Q f ZHUH SHUIXVHG ZLWK b SDUDIRUPDOGHK\GH LQ 0 6RUHQVRQnV SKRVSKDWH EXIIHU S+ f 7LVVXH EORFNV LQFOXGLQJ WKH WUDQVSODQWV ZHUH H[FLVHG DQG SUHSDUHG IRU 9LEUDWRPH MXPf VHFWLRQLQJ $GMDFHQW cLP VHFWLRQV ZHUH SURFHVVHG IRU WKH SUHVHQFH RI P\HOLQ EDVLF SURWHLQ 0%3f RU QHXURWHQVLQ 17f OLNH LPPXQRUHDFWLYLW\ ZLWK WKH LQGLUHFW

PAGE 30

SHUR[LGDVH DQWLSHUR[LGDVH 3$3f PHWKRG 6WHUQEHUJHU nf XVLQJ SULPDU\ DQWLVHUD UDLVHG LQ UDEELWV 7KH DQWLVHUD WR 17 ZDV REWDLQHG IURP ,PPXQRQXFOHDU &RUS 6WLOOZDWHU 01f DQG WKH DQWLVHUXP WR 0%3 ZDV SURYLGHG E\ 'U / ) (QJ 3DOR $OWR &$ 9$ 0HG &HQWHUf )RU 3$3 VWDLQLQJ WLVVXH VHFWLRQV ZHUH LQFXEDWHG RYHUQLJKW DW URRP WHPSHUDWXUH LQ SULPDU\ DQWLVHUXP GLOXWHG WR ZLWK D VROXWLRQ RI b 7ULWRQ ; LQ SKRVSKDWH EXIIHUHG VDOLQH 3%6f $OO LQFXEDWLRQV ZHUH SHUIRUPHG LQ WKH SUHVHQFH RI b QRUPDO JRDW VHUXP 7KH VHFWLRQV ZHUH ZDVKHG VHYHUDO WLPHV LQ 3%6 DQG WKHQ LQFXEDWHG LQ JRDW DQWLUDEELW ,J* &RRSHU %LRPHGLFDO RU 6WHUQEHUJHU 0H\HUf GLOXWHG ZLWK WKH DQWLVHUXP GLOXHQW IRU PLQXWHV DW URRP WHPSHUDWXUH $IWHU D ULQVH ZLWK 3%6 WKH VHFWLRQV ZHUH LQFXEDWHG IRU PLQXWHV LQ UDEELW 3$3 &RRSHU %LRPHGLFDO RU 6WHUQEHUJHU0H\HUf GLOXWHG )ROORZLQJ VHYHUDO ZDVKHV LQ 3%6 DQG 0 7ULV EXIIHU S+ f WKH LPPXQRF\WRFKHPLFDO UHDFWLRQ SURGXFW ZDV GHYHORSHG LQ D 0 7ULV EXIIHU VROXWLRQ FRQWDLQLQJ b nGLDPLQREHQ]LGLQH K\GURFKORULGH '$% 6LJPDf DQG b + $QWLERG\ VSHFLILFLW\ ZDV YHULILHG E\ WKH DQDWRPLFDO GLVWULEXWLRQ RI LPPXQRUHDFWLYH HOHPHQWV LQ QRUPDO VSLQDO FRUG WLVVXH DQG E\ WKH DEVHQFH RI LPPXQRUHDFWLYH HOHPHQWV ZKHQ SULPDU\ DQWLERG\ ZDV UHSODFHG ZLWK QRUPDO VHUXP DORQH &RUUHVSRQGHQFH %HWZHHQ 3ODVWLF DQG ,PPXQRFYWRFKHPLVWUY ,Q WKUHH VSHFLPHQV XVHG IRU LPPXQRF\WRFKHPLVWU\ LP YLEUDWRPH VHFWLRQV ZHUH REWDLQHG WR FRUUHVSRQG WR WKH

PAGE 31

VHFWLRQV VWDLQHG ZLWK DQWLVHUD WR 0%3 DQG 17 7KHVH VHFWLRQV ZHUH RVPLFDWHG DQG GHK\GUDWHG DV GHVFULEHG DERYH DQG HPEHGGHG EHWZHHQ YLQ\O VOLGHV ZLWK (0 %HG )URP WKHVH VHFWLRQV cLUD VHFWLRQV ZHUH FXW RQ DQ /.% XOWUDPLFURWRPH DQG VWDLQHG ZLWK b 7ROXLGLQH %OXH 6LPLODU P DQG XUQ VHFWLRQV ZHUH DOVR REWDLQHG IURP WKH WKRUDFLF VSLQDO FRUG RI QRUPDO UDWV WR LOOXVWUDWH QRUPDO FKDUDFWHULVWLFV DQG VWDLQLQJ SDWWHUQV ZLWKLQ WKH VXEVWDQWLD JHODWLQRVD 5HVXOWV /LJKW 0LFURVFRS\ DQG &YWRORDLFDO &KDUDFWHULVWLFV 6HFWLRQV RI QRUPDO UDW VSLQDO FRUG ZKHQ VWDLQHG ZLWK DQWLVHUXP GLUHFWHG DJDLQVW P\HOLQ EDVLF SURWHLQ 0%3f H[KLELW D FKDUDFWHULVWLF SDWWHUQ RI P\HOLQ GLVWULEXWLRQ 6SHFLILFDOO\ WKH ORQJ ILEHU WUDFWV DSSHDU GHQVHO\ VWDLQHG ZKLOH PRGHUDWH VWDLQLQJ UHIOHFWV WKH SUHVHQFH RI P\HOLQDWHG ILEHUV FRXUVLQJ WKURXJKRXW PRVW RI WKH FHQWUDO JUD\ PDWWHU 7KH PRVW VWULNLQJ IHDWXUH RI WKLV SUHSDUDWLRQ DV DOVR VHHQ ZLWK FRQYHQWLRQDO P\HOLQ VWDLQV LV WKDW UHJLRQ ZLWKLQ WKH GRUVDO KRUQ ZKLFK FRUUHVSRQGV WR WKH F\WRDUFKLWHFWXUDOO\ GHILQHG VXEVWDQWLD JHODWLQRVD 6*f 7KLV UHJLRQ VWDQGV RXW DJDLQVW WKH EDFNJURXQG RI LQWHQVH P\HOLQ LPPXQRUHDFWLYLW\ GXH WR WKH SDXFLW\ RI P\HOLQDWHG D[RQV )LJ DEf )HWDO VSLQDO FRUG WUDQVSODQWV ZHUH VWDLQHG ZLWK DQWL0%3 WR H[DPLQH WKH GLIIHUHQWLDWLRQ RI WKH JUDIWV $OO RI WKH WUDQVSODQWV H[DPLQHG ZLWK WKLV WHFKQLTXH ZHUH KHDYLO\

PAGE 32

)LJXUH ,GHQWLILFDWLRQ RI P\HOLQIUHH DUHDV Df 3DWWHUQ RI 3$3 VWDLQLQJ ZLWK DQWLVHUXP DJDLQVW 0%3 LQ D WUDQVYHUVH VHFWLRQ RI D QRUPDO UDW WKRUDFLF VSLQDO FRUG VKRZLQJ WKH P\HOLQIUHH UHJLRQV LQ WKH VXSHUILFLDO GRUVDO KRUQ DUURZVf Ef +LJKHU PDJQLILFDWLRQ RI $QWL0%3 VWDLQLQJ LQ D QRUPDO GRUVDO KRUQ LOOXVWUDWLQJ WKH ODUJH P\HOLQDWHG ILEHUV ZKLFK RIWHQ WUDYHUVH WKH VXSHUILFLDO ODPLQDH Ff $QWL0%3 VWDLQLQJ RI D PRQWK LQWUDVSLQDO WUDQVSODQW Wf DQG VXUURXQGLQJ KRVW LQWHUPHGLDWH JUD\ K@*f DQG ODWHUDO ZKLWH PDWWHU KJf +RUL]RQWDO VHFWLRQf 7KUHH P\HOLQ IUHH SDWFKHV DUH VHHQ DW WKH HGJH RI WKH WUDQVSODQW DUURZVf Gf (QODUJHPHQW RI ER[HG UHJLRQ LQ Ff 0\HOLQDWHG D[RQV DUURZf FURVV WKURXJK WKLV RWKHUZLVH XQP\HOLQDWHG DUHD 'RWWHG OLQH KLJKOLJKWV WKH KRVWJUDIW LQWHUIDFH 6FDOH LQ DF LP EG LP

PAGE 34

P\HOLQDWHG DV LQGLFDWHG E\ DUHDV RI YHU\ GHQVH VWDLQLQJ ,Q DGGLWLRQ ODUJH UHJLRQV H[KLELWLQJ PRGHUDWH LPPXQR UHDFWLYLW\ ZHUH HYLGHQW )LQDOO\ WKH WUDQVSODQWV XVXDOO\ FRQWDLQHG RQH RU PRUH DUHDV ZKLFK ZHUH FRQVSLFXRXV GXH WR WKH PDUNHG DEVHQFH RI 0%3OLNH VWDLQLQJ )LJ FGf 7KHVH P\HOLQIUHH DUHDV W\SLFDOO\ DVVXPHG D FRQYROXWHG FRQILJXn UDWLRQ ZLWKLQ WKH JUDIWV DQG DSSHDUHG DV HLWKHU VLQJOH RU PXOWLSOH SDWFKHV RU ORQJ VWULSV RI QHXURSLO GHSHQGLQJ XSRQ WKH SODQH RI VHFWLRQ ,Q PDQ\ FDVHV WKHVH UHJLRQV ZHUH ORFDWHG QHDU WKH SHULSKHU\ RI WKH JUDIWV )LJ Ff KRZHYHU VRPH P\HOLQIUHH DUHDV ZHUH ORFDWHG PRUH FHQWUDOO\ 0\HOLQDWHG D[RQV IUHTXHQWO\ FXUYHG DORQJ WKH VXUIDFHV RI WKH XQVWDLQHG UHJLRQV DQG RIWHQ VPDOO EXQGOHV RI DQWL0%3 VWDLQHG SURFHVVHV WUDYHUVHG WKH P\HOLQIUHH ]RQHV LQ D UDGLDO IDVKLRQ UHPLQLVFHQW RI WKH SDWWHUQ RI P\HOLQDWHG SULPDU\ DIIHUHQWV SURMHFWLQJ WR GHHSHU OD\HUV RI WKH JUD\ PDWWHU LQ WKH QRUPDO VSLQDO FRUG )LJ EGf :LWK WKH SHUVSHFWLYH GHULYHG IURP 0%3VWDLQHG JUDIWV H[DPLQDWLRQ RI WROXLGLQH EOXHVWDLQHG VHFWLRQV RI )6& WUDQVSODQWV UHYHDOHG DUHDV WKDW FRUUHVSRQGHG WR SDWWHUQV RI 0%3 LPPXQRUHDFWLYLW\ )LJ DF )LJ Ef 7KH [P VHFWLRQV FRQWDLQHG UHJLRQV RI H[WHQVLYH P\HOLQDWLRQ DV ZHOO DV QXPHURXV P\HOLQGHILFLHQW DUHDV ZLWKLQ WKH JUDIW WLVVXH 7R GHWHUPLQH ZKHWKHU WKH P\HOLQIUHH DUHDV LGHQWLILHG ZLWKLQ WKH JUDIWV E\ LPPXQRKLVWRFKHPLVWU\ RU WROXLGLQH EOXH VWDLQLQJ ZHUH LQGHHG HTXLYDOHQW DGMDFHQW VHFWLRQV ZHUH

PAGE 35

)LJXUH &RPSDULVRQ RI 0%3 DQG WROXLGLQH EOXH VWDLQHG VHFWLRQV Df 7ROXLGLQH EOXH VWDLQHG VHPLWKLFN VHFWLRQ ZLWKLQ DQ LQWUDVSLQDO )6& WUDQVSODQW VKRZLQJ P\HOLQDWHG UHJLRQV Pf FRQWDLQLQJ ODUJHU QHXURQV DQG XQP\HOLQDWHG SDWFKHV RXWOLQHG LQ DUURZKHDGVf ZLWK VPDOOHU QHXURQV DQG SURFHVVHV Ef 0%3 VWDLQHG VHFWLRQ UHYHDOV D P\HOLQIUHH UHJLRQ DUURZKHDGVf QHDU WKH KRVWJUDIW LQWHUIDFH LQ DQRWKHU UHFLSLHQW 6HYHUDO UHJLRQV RI WKH JUDIW ODFN P\HOLQ \HW WKLV UHJLRQ FRUUHVSRQGV WR DQ DUHD FRQWDLQLQJ VPDOO WLJKWO\ SDFNHG QHXURQV DQG SURFHVVHV LQ Ff 7UDQVSODQW Wf DQG KRVW JUD\ PDWWHU K,*f DUH ODEHOHG Ff $GMDFHQW WROXLGLQH EOXH VWDLQHG VHFWLRQ IURP WKH VDPH DUHD ZLWKLQ DUURZKHDGVf ZKLFK LV RFFXSLHG E\ VPDOO FHOOV DQG SURFHVVHV 6FDOH LQ DF LP E [P

PAGE 37

SURFHVVHG VR WKDW IRU HDFK 0%3VWDLQHG VHFWLRQ WKHUH ZDV D FRUUHVSRQGLQJ [P VHFWLRQ HPEHGGHG LQ SODVWLF ,Q WKHVH H[DPSOHV ]RQHV WKDW IDLOHG WR VKRZ 0%3 LPPXQRUHDFWLYLW\ ZHUH FORVHO\ LQ UHJLVWHU ZLWK KRPRJHQRXV XQP\HOLQDWHG DUHDV LQ WKH DGMDFHQW WROXLGLQH EOXH VWDLQHG VHFWLRQ )LJ EFf )XUWKHU H[DPLQDWLRQ RI WKHVH UHJLRQV LQ SODVWLF VHFWLRQV UHYHDOHG RWKHU VLPLODULWLHV EHWZHHQ WKH P\HOLQIUHH DUHDV RI )6& JUDIWV DQG WKH QRUPDO 6* ODPLQDH $V LQ WKH QRUPDO VXEVWDQWLD JHODWLQRVD )LJ DFf WKH XQP\HOLQDWHG ]RQHV RI PDWXUHG WUDQVSODQWV FRQVLVWHG RI QXPHURXV VPDOO FHOOV /WPf FKDUDFWHUL]HG E\ D WKLQ ULP RI F\WRSODVP VXUURXQGLQJ D SURPLQHQW QXFOHXV 7KH QXFOHL RI WKHVH FHOOV DV RI WKRVH LQ WKH QRUPDO VXEVWDQWLD JHODWLQRVD ZHUH URXQG RU RYDO DQG RIWHQ H[KLELWHG ODUJH FOHIWV FI )LJ FGf 7KHVH FHOOV ZHUH TXDOLWDWLYHO\ GLVWLQFW IURP WKH ODUJHU QHXURQV [P GLDPHWHUf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

PAGE 38

)LJXUH &\WRORJ\ RI WKH QRUPDO 6* DQG 6*OLNH UHJLRQV LQ )6& JUDIWV Df $ S WROXLGLQH EOXH VWDLQHG WUDQVYHUVH VHFWLRQ IURP WKH QRUPDO DGXOW UDW GRUVDO KRUQ /DPLQDH DUH GHVLJQDWHG LQ URPDQ QXPHUDOV DV ODPLQDH ,9 DQG WKH 6* DV ODPLQD ,, Ef $Q XQP\HOLQDWHG UHJLRQ IURP DQ (X LQWUDVSLQDO JUDIW Wf DGMDFHQW WR WKH KRVW ZKLWH PDWWHU Kf 6HYHUDO DVSHFWV RI WKHVH UHJLRQV UHVHPEOH WKRVH RI WKH QRUPDO GRUVDO KRUQ DOWKRXJK WKH GLYLVLRQV EHWZHHQ XQP\HOLQDWHG SDWFKHV VHH DUURZKHDGVf VXJJHVW WKDW WKH JUDIW UHJLRQ KDV DVVXPHG D VHUSHQWLQH RULHQWDWLRQ 1RWH ODUJHU QHXURQV Qf LQ FORVH DSSUR[LPDWLRQ WR WKHVH UHJLRQV LQ WKLV ILJXUH 7KH LQWHUIDFH LV LQGLFDWHG E\ WKH GRWWHG OLQH Ff 6* QHXURQV LQ WKH QRUPDO VSLQDO FRUG DW D KLJKHU PDJQLILFDWLRQ 1HXURQV ZLWKLQ WKH 6* DUH WLJKWO\ SDFNHG DQG FRQWDLQ ODUJH QXFOHL ZLWK SURPLQHQW LQGHQWDWLRQV DUURZKHDGVf 1RWH WKH SDUDOOHO RULHQWDWLRQ RI D[RQV LQ WUDQVYHUVH VHFWLRQ Gf ,Q P\HOLQIUHH UHJLRQV RI WKH WUDQVSODQWV WKH QHXURQV DUH VLPLODU DOWKRXJK WKH SURFHVVHV RIWHQ ODFN WKH ORQJLWXGLQDO RULHQWDWLRQ FKDUDFWHULVWLF RI WKH QRUPDO GRUVDO KRUQ 6FDOH LQ DE LP FG /WP

PAGE 39

r

PAGE 40

EHWZHHQ WKH WZR UHJLRQV 7KLV ZDV SDUWLFXODUO\ HYLGHQW ZLWK UHJDUG WR WKH GLVWULEXWLRQ RI FHOOV DQG QHXULWLF SURFHVVHV ,Q WKH LQWDFW VSLQDO FRUG 6* QHXURQV FDQ EH GLIIHUHQWLDWHG LQWR WZR OD\HUV LH +L DQG ,,R 5DOVWRQ n 6]HQWDJRWKDL nDf +RZHYHU QR REYLRXV F\WRDUFKLWHFWXUDO ODPLQDWLRQ ZDV VHHQ LQ WKHVH UHJLRQV RI WKH WUDQVSODQWV ,Q DGGLWLRQ PDQ\ VPDOO FLUFXODU QHXULWLF SURILOHV ZHUH VHHQ LQ WUDQVYHUVH VHFWLRQV RI WKH QRUPDO 6* WKXV UHIOHFWLQJ WKHLU RULHQWDWLRQ SDUDOOHO WR WKH ORQJLWXGLQDO D[LV RI WKH VSLQDO FRUG )LJ Ff ,Q FRQWUDVW WKH QHXULWLF SURFHVVHV LQ WKH P\HOLQIUHH DUHDV RI WKH JUDIWV VHHPHG PRUH UDQGRPO\ RUJDQL]HG DQG VHFWLRQHG SURILOHV DVVXPHG PDQ\ RULHQWDWLRQV (OHFWURQ 0LFURVFRS\ 1HXURQV ZLWKLQ WKH QRUPDO VXEVWDQWLD JHODWLQRVD ZHUH JHQHUDOO\ VSKHURLG )LJ Df RU IXVLIRUP )LJ Ef LQ VKDSH 7KH SHULNDU\D UDQJHG IURP 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

PAGE 41

)LJXUH 8OWUDVWUXFWXUH RI WKH QRUPDO VXEVWDQWLD JHODWLQRVD Df 7UDQVYHUVH VHFWLRQ FRQWDLQV D QHXURQDO FHOO ERG\ Qf DQG WKH FRPSDFW QHXURSLO FRQWDLQLQJ DEXQGDQW D[RGHQGULWLF V\QDSVHV DGf LQWHUVSHUVHG ZLWK ORQJLWXGLQDO EXQGOHV RI XQP\HOLQDWHG SURFHVVHV DUURZVf /DUJH JORPHUXODU D[RQDO SURFHVVHV ZHUH RIWHQ REVHUYHG VWDUf Ef 2EOLTXH VHFWLRQ WKURXJK WKH 6* UHJLRQ RI DQRWKHU QRUPDO VSHFLPHQ 7KH IDVFLFOHV DUH PRUH GLIILFXOW WR GLVFHUQ WKDQ LQ Df 6FDOH LQ DE [P

PAGE 43

DGGLWLRQ ODUJH JORPHUXODU FRPSOH[HV ZHUH HYLGHQW LQ WKH VXEVWDQWLD JHODWLQRVD RI WKHVH QRUPDO VHFWLRQV DV ZHOO $ VXUYH\ RI WKH XQP\HOLQDWHG UHJLRQV ZLWKLQ )6& WUDQVSODQWV LQ ORZ SRZHU HOHFWURQ PLFURJUDSKV UHYHDOHG PDQ\ FKDUDFWHULVWLFV VLPLODU WR WKH QRUPDO 6* )LJ f 7KH QHXURQV LQ WKHVH DUHDV ZHUH DOVR VPDOO DQG FRQWDLQHG ODUJH QXFOHL ZLWK SURPLQHQW LQGHQWDWLRQV 7KH FHOOV ZHUH FORVHO\ VSDFHG DQG ZHUH VXUURXQGHG E\ FRPSDFW QHXURSLO FRQVLVWLQJ RI VPDOO D[RQV [Pf DQG LQWHUPHGLDWHVL]HG GHQGULWHV QPf 2FFDVLRQDOO\ VPDOO EXQGOHV RI XQP\HOLQDWHG SURFHVVHV ZHUH VHHQ ZKLFK UHVHPEOHG WKH IDVFLFOHV LQ WKH QRUPDO 6* +RZHYHU PDQ\ RI WKH D[RQV DQG GHQGULWHV ZHUH PRUH UDQGRPO\ RULHQWHG )LJ f ([FHSW IRU DQ RFFDVLRQDO VZROOHQ QHXULWLF SURILOH FRQWDLQLQJ O\VRVRPHV DQG GHJHQHUDWLQJ PLWRFKRQGULD D[RQDO DQG GHQGULWLF SURFHVVHV GLG QRW GLVSOD\ LUUHJXODU F\WRORJLFDO FKDUDFWHULVWLFV ,Q VRPH WUDQVSODQWV K\SHUWURSKLF DVWURF\WLF SURFHVVHV ZHUH REVHUYHG SDUWLFXODUO\ QHDU WKH SHULSKHU\ RI WKH JUDIWV )LJ f 0DQ\ RI V\QDSWLF FRQWDFWV REVHUYHG ZLWKLQ WKH XQP\HOLQDWHG JUDIW UHJLRQV ZHUH D[RGHQGULWLF ZLWK D[Rn D[RQLF V\QDSVHV RFFDVLRQDOO\ EHLQJ SUHVHQW DV ZHOO ,Q DGGLWLRQ D UDUH D[RVRPDWLF V\QDSVH FRXOG EH IRXQG LQ WKH QRUPDO DQG JUDIW P\HOLQIUHH DUHDV 7KH ERXWRQV ZLWKLQ WKH P\HOLQIUHH UHJLRQV XVXDOO\ FRQWDLQHG DJJUHJDWHV RI VPDOO DJUDQXODU YHVLFOHV )LJ f 8VXDOO\ WKH YHVLFOHV ZHUH

PAGE 44

)LJXUH /RZ SRZHU HOHFWURQ PLFURJUDSK RI DQ 6*OLNH UHJLRQ LQ DQ ( LQWUDFHUHEUDO WUDQVSODQW 7KH VPDOO QHXURQV LQ WKHVH UHJLRQV ZHUH VLPLODU WR WKH QRUPDO 6* UHJLRQV LQ VL]H VKDSH DQG WKH SUHVHQFH RI QXFOHDU FOHIWV $Q D[RGHQGULWLF DGf DQG D[RVRPDWLF DVf V\QDSVH DUH VKRZQ :KLOH VRPH XQP\HOLQDWHG D[RQV WUDYHOHG LQ IDVFLFOHV DUURZVf PRVW D[RQDO DQG GHQGULWLF SURFHVVHV DVVXPHG D YDULHW\ RI RULHQWDWLRQV DQG DSSHDUHG WR ODFN VRPH RI WKH RUJDQL]DWLRQ RI WKH QRUPDO VSLQDO FRUG /DUJH ILODPHQWRXV JOLDO SURFHVVHV rf ZHUH VRPHWLPHV VHHQ LQ WKHVH JUDIWV 6FDOH MP

PAGE 46

)LJXUH +LJKHU PDJQLILFDWLRQ RI 6*OLNH UHJLRQV LQ DQ LQWUDVSLQDO WUDQVSODQW 7KH YDVW PDMRULW\ RI V\QDSVHV ZHUH D[RGHQGULWLF DGf DQG YHVLFOHV ZHUH XVXDOO\ FOHDU DJUDQXODUf DQG URXQG DUURZVf DOWKRXJK IODWWHQHG YHVLFOHV DQG GHQVHFRUHG YHVLFOHV DUURZKHDGVf ZHUH DOVR REVHUYHG $VWURF\WLF SURFHVVHV ZHUH VRPHWLPHV SUHVHQW rf 6FDOH P

PAGE 48

URXQG UDWKHU WKDQ IODWWHQHG DQG VRPH WHUPLQDOV FRQWDLQHG VPDOO GHQVHFRUHG YHVLFOHV 6LPLODU SUHV\QDSWLF VWUXFWXUHV ZHUH DOVR VHHQ LQ WKH QRUPDO 6* +RZHYHU WKH VFDOORSHG WHUPLQDOV DQG UHODWHG JORPHUXOL FKDUDFWHULVWLF RI QRUPDO SULPDU\ DIIHUHQW LQQHUYDWLRQ RI WKH 6* ZHUH QRW IRXQG ZLWKLQ WKH JUDIWV H[DPLQHG LQ WKLV VWXG\ 1HXURWHQVLQOLNH ,PPXQRUHDFWLYLWY :KHQ VHFWLRQV RI QRUPDO VSLQDO FRUG ZHUH UHDFWHG ZLWK DQWLVHUD WR QHXURWHQVLQ 17f VWDLQLQJ ZDV UHVWULFWHG WR WKH ODPLQD ,, UHJLRQ RI WKH VXSHUILFLDO GRUVDO KRUQ ZKHUH LW ZDV VHHQ LQ WZR GLVWLQFW OD\HUV RI YHU\ ILQH SURFHVVHV )LJ D 6H\EROG DQG (OGH nf /DEHOHG D[RQDO SURILOHV ZHUH QRW IRXQG LQ DQ\ RWKHU UHJLRQ RI WKH QRUPDO VSLQDO FRUG DQG QR LPPXQRUHDFWLYH FHOO ERGLHV ZHUH IRXQG LQ WKHVH VHFWLRQV 6WDLQLQJ RI )6& WUDQVSODQWV ZLWK DQWLVHUD UDLVHG DJDLQVW 17 UHYHDOHG UHJLRQV WKURXJKRXW WKH JUDIWV ZKLFK FRQWDLQHG VPDOO LPPXQRUHDFWLYH SURFHVVHV 0DQ\ RI WKHVH SDWFKHV FRUUHVSRQGHG ZLWK P\HOLQIUHH UHJLRQV LQ QHLJKERULQJ VHFWLRQV VWDLQHG ZLWK DQWL0%3 )LJ EGf +RZHYHU LQ FRQWUDVW WR WKH QRUPDO VSLQDO FRUG 17 VWDLQLQJ ZLWKLQ WKH WUDQVSODQWV GLG QRW IRUP WZR GLVWLQFW EDQGV 2WKHU GLIIHUHQFHV ZHUH DOVR REVHUYHG EHWZHHQ WKH SDWWHUQV RI 17 VWDLQLQJ DQG WKH QRUPDO 6* ,Q DGGLWLRQ WR WKH SDWFKHV RI ILEHUV WKDW FRUUHVSRQGHG WR 0%3IUHH UHJLRQV RI WKH JUDIWV VRPH 17 ILEHUV ZHUH GLVWULEXWHG WKURXJKRXW WKH P\HOLQDWHG DUHDV RI WKH WUDQVSODQWV )XUWKHUPRUH 17OLNH FHOOV ZHUH DOVR IRXQG

PAGE 49

)LJXUH 1HXURWHQVLQ LPPXQRUHDFWLYLW\ LQ QRUPDO VSLQDO FRUG DQG LQWUDVSLQDO WUDQVSODQWV Df 7UDQVYHUVH VHFWLRQ WKURXJK WKH VXEVWDQWLD JHODWLQRVD RI D QRUPDO VSLQDO FRUG 17 OLNH LPPXQRUHDFWLYLW\ LV UHVWULFWHG WR VPDOO ILEHUV LQ WKLV UHJLRQ RI WKH VSLQDO FRUG /DEHOHG D[RQV DUH VHSDUDWHG LQWR WZR GLVWLQFW EDQGV DUURZVf Ef 6DJLWWDO VHFWLRQ WKURXJK D )6& WUDQVSODQW Wf DQG WKH DGMDFHQW KRVW VSLQDO FRUG Kf DIWHU VWDLQLQJ ZLWK DQWL0%3 $ UHJLRQ RI WKH JUDIW QHDU WKH LQWHUIDFH EHWZHHQ K DQG Wf H[KLELWV D ODFN RI P\HOLQ VWDLQLQJ Ff &HOOV FRQWDLQLQJ 17OLNH LPPXQRUHDFWLYLW\ ZHUH IRXQG WKURXJKRXW WUDQVSODQWV EXW WKH\ ZHUH QRW REVHUYHG LQ WKH QRUPDO DGXOW VSLQDO FRUG Gf 6DJLWWDO VHFWLRQ DGMDFHQW WR WKDW VKRZQ LQ Ef DIWHU VWDLQLQJ ZLWK DQWL17 7KH XQP\HOLQDWHG UHJLRQ FRQWDLQV D OLJKWO\ VWDLQHG SDWFK RI 17OLNH LPPXQRUHDFWLYLW\ FRQWDLQLQJ YHU\ ILQH 17OLNH ILEHUV 6LPLODU D[RQDO SURILOHV ZHUH QHYHU VHHQ ZLWKLQ WKH GHHSHU ODPLQDH RI QRUPDO VSLQDO FRUG VHFWLRQV VXJJHVWLQJ WKDW WKH D[RQV LQ WKLV VHFWLRQ KDYH H[WHQGHG D VKRUW GLVWDQFH LQWR WKH KRVW VSLQDO FRUG IURP ZLWKLQ WKH WUDQVSODQW 6FDOH LQ DF QP EG [P

PAGE 51

ZLWKLQ WKH JUDIWV )LJ Ef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n nEf 7KH LQGLFDWLRQ WKDW WKHVH UHJLRQV PLJKW UHIOHFW VRPH RUJDQRW\SLF GLIIHUHQWLDWLRQ ZDV H[DPLQHG IXUWKHU LQ DQ H[SHULPHQW LQ ZKLFK SUHJQDQW UDWV ZHUH LQMHFWHG ZLWK WULWLDWHG WK\PLGLQH RQ HLWKHU GD\ ( RU (X /DEHOHG GRQRU WLVVXH ZDV UHPRYHG IURP IHWXVHV LQ RQH XWHULQH KRUQ DQG WUDQVSODQWHG IHWXVHV LQ WKH FRQWUDODWHUDO KRUQ ZHUH OHIW WR FRPSOHWH JHVWDWLRQ 5HLHU HW DO nDf $W RQH PRQWK SRVWWUDQVSODQWDWLRQ DXWRUDGLRJUDSK\ LQGLFDWHG WKDW WKH QXFOHL RI QHXURQV LQ WKH P\HOLQIUHH UHJLRQV RI WKHVH WUDQVSODQWV H[KLELWHG WKH VDPH UHODWLYH GHJUHH RI ODEHOLQJ DV GLG WKH QXFOHL RI WKRVH FHOOV SUHVHQW LQ WKH VXSHUILFLDO ODPLQDH RI WKH LQWDFW VSLQDO FRUGV RI WKH OLWWHUPDWHV RI WKH GRQRU IHWXVHV 5HLHU HW DO nDnEf

PAGE 52

,W WKXV DSSHDUHG WKDW WKH P\HOLQGHILFLHQW UHJLRQV LQ WKH IHWDO VSLQDO FRUG JUDIWV ZHUH WKH FRXQWHUSDUWV RI WKH QRUPDO VXEVWDQWLD JHODWLQRVD 7KHVH JHQHUDO FULWHULD WKRXJK LQWULJXLQJ GLG QRW SURYLGH VXIILFLHQW SURRI RI WKH H[DFW QDWXUH RI WKH P\HOLQIUHH DUHDV LQ WKH JUDIWV ,Q WKLV FRQWH[W LW LV ZHOO UHFRJQL]HG WKDW RWKHU FKDUDFWHULVWLFV PDNH WKLV UHJLRQ GLVWLQFW IURP WKH UHVW RI WKH JUD\ PDWWHU LQ WKH LQWDFW VSLQDO FRUG ,Q SDUWLFXODU WKH DEXQGDQFH RI VPDOO FHOOV OHG 5H[HG nf WR PDNH WKH GLVWLQFWLRQ RI ODPLQD ,, RI WKH FDW VSLQDO FRUG DQG VLPLODU VWXGLHV KDYH VKRZQ WKLV UHJLRQ ZLWKLQ WKH UDW VSLQDO FRUG DV ZHOO 0RODQGHU HW DO nf ([DPLQDWLRQ RI WKH XOWUDVWUXFWXUH RI WKLV UHJLRQ KDV DOVR VHUYHG WR GHILQH WKH W\SHV RI SURFHVVHV DQG V\QDSWLF SURILOHV WKDW GLVWLQJXLVK WKH VXEVWDQWLD JHODWLQRVD ODPLQD ,,f IURP WKH VXUURXQGLQJ PDUJLQDO OD\HU ODPLQD ,f DQG ODPLQD ,,, 5DOVWRQ n" 6]HQWDJRWKDL nDf $GGLWLRQDO LGHQWLI\LQJ IHDWXUHV KDYH EHHQ QRWHG WKURXJK WKH XVH RI LPPXQRF\WRFKHPLFDO WHFKQLTXHV ZKLFK KDYH GHPRQVWUDWHG D YDULHW\ RI SHSWLGHFRQWDLQLQJ FHOOV DQG ILEHUV UHYLHZHG LQ 6H\EROG DQG (OGH n *LEVRQ HW DO n +XQW n /D0RWWH nf :LWK WKHVH FKDUDFWHULVWLF F\WRORJLFDO DQG LPPXQRF\WRFKHPLFDO IHDWXUHV DV D EDVLV IRU FRPSDULVRQ WKH SUHVHQW VWXG\ KDV SURYLGHG DGGLWLRQDO HYLGHQFH LQ VXSSRUW RI WKH RUJDQRW\SLF GLIIHUHQWLDWLRQ RI VXEVWDQWLD JHODWLQRVDOLNH UHJLRQV LQ WUDQVSODQWV RI IHWDO VSLQDO FRUG WLVVXH

PAGE 53

0YHOLQIUHH $UHDV DQG 3HSWLGHUJLF (OHPHQWV 3UHYLRXV VWXGLHV KDYH GHPRQVWUDWHG UHJLRQV RI GHQVH LPPXQRUHDFWLYLW\ REWDLQHG ZLWK DQWLERGLHV WR VHYHUDO SHSWLGHV ZKLFK DUH QRUPDOO\ DVVRFLDWHG ZLWK WKH VXEVWDQWLD JHODWLQRVD 5HLHU DQG %UHJPDQ n 5HLHU HW DO nf 3DWFKHV RI WLVVXH ZLWKLQ WKH WUDQVSODQWV VWDLQHG KHDYLO\ ZLWK DQWLERGLHV WR PHW DQG OHXHQNHSKDOLQ VRPDWRVWDWLQ DQG VXEVWDQFH 3 7KHVH SDWFKHV KDYH EHHQ LGHQWLILHG ZLWKLQ )6& WUDQVSODQWV SODFHG LQWR WKH DGXOW EUDLQ RU QHRQDWDO DQG DGXOW VSLQDO FRUG ,Q DGGLWLRQ VLPLODU ILQGLQJV KDYH EHHQ UHSRUWHG UHJDUGLQJ WKH GLIIHUHQWLDWLRQ RI UHJLRQV RI GHQVH SHSWLGH VWDLQLQJ LQ )6& WUDQVSODQWV WKDW GHYHORS LQ RFXOR +HQVFKHQ HW DO nf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

PAGE 54

KRZHYHU 17FRQWDLQLQJ D[RQV ZHUH IRXQG RQO\ ZLWKLQ WKH VXEVWDQWLD JHODWLQRVD UHJLRQ RI WKH VSLQDO FRUG 'HYHORSPHQWDO ,PSOLFDWLRQV 7KH REVHUYDWLRQ WKDW IHWDO VSLQDO FRUG WLVVXH FDQ H[KLELW VRPH GHJUHH RI RUJDQRW\SLF GHYHORSPHQW LV FRQVLVWHQW ZLWK UHSRUWV WKDW WUDQVSODQWV RI WLVVXH IURP YDULRXV HPEU\RQLF EUDLQ UHJLRQV FDQ DFKLHYH F\WRDUFKLWHFWXUDO DQG XOWUDVWUXFWXUDO FKDUDFWHULVWLFV FRUUHVSRQGLQJ WR WKRVH RI WKH KRPRORJRXV DUHDV LQ WKH LQWDFW &16 HJ .URPHU HW DO n n /XQG DQG +DUYH\ nf )HWDO VSLQDO FRUG WLVVXH KDV DOVR EHHQ VKRZQ WR H[KLELW VRPH F\WRDUFKLWHFWXUDO RU LPPXQRF\WRFKHPLFDO FKDUDFWHULVWLFV UHVHPEOLQJ WKH QRUPDO GRUVDO KRUQ ZKHQ JURZQ LQ WLVVXH FXOWXUH 1DIWFKL HW DO n 6RENRZLF] HW DO nf RU LQ RFXOR +HQVFKHQ HW DO nf 7KH SUHVHQW ILQGLQJV VKRZ WKDW WKH GLIIHUHQWLDWLRQ RI WKH P\HOLQIUHH DUHDV DOVR RFFXUV ZKHQ )6& WLVVXH JUDIWV GHYHORS ZLWKLQ WKH DGXOW VSLQDO FRUG ,Q UHODWHG VWXGLHV FXUUHQWO\ LQ SURJUHVV VLPLODU XQP\HOLQDWHG UHJLRQV KDYH EHHQ REVHUYHG LQ FHOO VXVSHQVLRQ JUDIWV RI UDW )6& ZLWKLQ WKH FRQWXVHG UDW VSLQDO FRUG :LQLDOVNL HW DO nf DQG LQ VROLG SLHFH JUDIWV RI FDW IHWDO VSLQDO FRUG WLVVXH LQ WKH DGXOW FDW VSLQDO FRUG $QGHUVRQ HW DO n 5HLHU HW DO LQ SUHSDUDWLRQf 7KHVH UHJLRQV KDYH WKXV IDU EHHQ LGHQWLILHG RQ WKH EDVLV RI LLP WKLFN SODVWLF VHFWLRQV DQG DQWL0%3 VWDLQHG VHFWLRQV RI WKH JUDIWV $GGLWLRQDO REVHUYDWLRQV VXJJHVW WKDW WKH P\HOLQIUHH

PAGE 55

DUHDV PD\ DOVR EH DQDORJRXV WR VPDOO ILEHUIUHH DUHDV UHYHDOHG ZLWK LPPXQRF\WRFKHPLFDO VWDLQLQJ XVLQJ SRO\FORQDO DQWLERGLHV UDLVHG DJDLQVW WKH SKRVSKRU\ODWHG IRUP RI WKH KHDY\ QHXURILODPHQW SURWHLQ VHH VLOYHU VWDLQLQJ SDWWHUQV LQ /XQG DQG +DUYH\ f +RZHYHU RQH UHFHQW UHSRUW KDV LQGLFDWHG WKDW 6*OLNH UHJLRQV DUH QRW VHHQ IROORZLQJ LQMHFWLRQV RI FHOO VXVSHQVLRQV IURP ( ( IHWDO UDW VSLQDO FRUG LQWR WKH LERWHQLF DFLG OHVLRQV RI WKH OXPEDU VSLQDO FRUG 1RWKLDV HW DO nf 7KLV GLIIHUHQFH PD\ UHIOHFW WKH GLIIHUHQW GRQRU DJHV XVHG .URPHU HW DO nf KRZHYHU 6* OLNH DUHDV KDYH EHHQ LGHQWLILHG LQ JUDIWV RI ( UDW VSLQDO FRUG WLVVXH SODFHG LQWR WKH UDW EUDLQ 5HLHU HW DO nDf 7KXV ZKLOH WKH SURFHGXUHV XVHG LQ WKLV VWXG\ ZHUH QRW DSSOLHG E\ 1RWKLDV HW DO LW PD\ EH SRVVLEOH WR REVHUYH 6* OLNH UHJLRQV ZLWKLQ WKHLU JUDIWV SODFHG LQWR LERWHQLF DFLG OHVLRQV E\ XWLOL]LQJ VSHFLILF PDUNHUV VXFK DV WKH DQWLVHUD UDLVHG DJDLQVW GRUVDO KRUQ SHSWLGHV RU 0%3 7KH SUHVHQFH RI D GRUVDO KRUQ FRPSRQHQW LQ )6& JUDIWV LV OLNHO\ WR EH UHODWHG WR WKH GHYHORSPHQWDO WLPLQJ RI WKLV UHJLRQ RI WKH VSLQDO FRUG (YDOXDWLRQV RI VSLQDO FRUG KLVWRJHQHVLV LQ WKH UDW $OYDUGR0DOODUW DQG 6RWHOR n 1RUQHV HW DO nf KDYH LQGLFDWHG WKDW WKHUH LV D SHDN DW DSSUR[LPDWHO\ (( LQ WKH JHQHUDWLRQ RI QHXURQV ZKLFK XOWLPDWHO\ FRPSULVH WKH GRUVDO KRUQ 7KHVH FHOOV WKHQ PLJUDWH ZLWK WKH PDMRULW\ RI QHXUREODVWV UHDFKLQJ WKH SUHVXPSWLYH GRUVDO KRUQ UHJLRQ WZR GD\V ODWHU 7KH

PAGE 56

PDWXUDWLRQ RI WKH 6*OLNH DUHDV LQ WKHVH IHWDO VSLQDO FRUG JUDIWV PXVW WKHUHIRUH RFFXU DIWHU WUDQVSODQWDWLRQ VLQFH GRQRU WLVVXH LQ WKHVH H[SHULPHQWV ZDV REWDLQHG DW (X( 7KHVH FRQVLGHUDWLRQV SHUWDLQLQJ WR FHOO ELUWKGDWHV DQG RQVHW RI PLJUDWLRQ DOVR VXJJHVW WKDW WKH FOXVWHULQJ RI VPDOO QHXURQV LQWR WKH 6*OLNH UHJLRQV PD\ EH GXH WR WKH SHUVLVWHQFH RI LQWULQVLF UHFRJQLWLRQ FXHV ZKLFK LQIOXHQFH WKH DJJUHJDWLRQ RI WKHVH FHOOV GXULQJ QRUPDO GHYHORSPHQW VHH DOVR .URPHU HW DO nf 5HODWHG VWXGLHV KDYH DOVR VKRZQ WKDW WKHVH LQWULQVLF FXHV DUH DOVR UHWDLQHG LI WKH JUDIW LV SODFHG LQWR KHWHURWRSLF VLWHV RU ZKHQ LW LV HQWLUHO\ LVRODWHG IURP KRVW DIIHUHQW LQSXWV E\ WUDQVSODQWLQJ WKH IHWDO VSLQDO FRUG ZLWK WKH VXUURXQGLQJ PHQLQJHV DWWDFKHG -DNHPDQ HW DO nf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P SODVWLF VHFWLRQV RU ZLWK E\ LPPXQRF\WRFKHPLFDO VWDLQLQJ ZLWK DQWLVHUXP WR 17 )LQDOO\ ZKLOH WKHVH JUDIW UHJLRQV

PAGE 57

FRQWDLQHG VRPH EXQGOHV RI XQP\HOLQDWHG D[RQV WKH SDUDOOHO ORQJLWXGLQDO DUUDQJHPHQW RI QHXURQDO SURFHVVHV FKDUDFWHULVWLF RI WKH QRUPDO GRUVDO KRUQ ZDV DEVHQW IURP WKH P\HOLQIUHH UHJLRQV RI WKH JUDIWV ,W LV OLNHO\ WKDW VRPH RI WKHVH GLIIHUHQFHV DUH UHODWHG WR VSHFLILF DVSHFWV RI WKH JUDIWLQJ SURFHGXUH VXFK DV WKH LQLWLDO RULHQWDWLRQ RI WKH JUDIW WLVVXH GRQRU DJH DQG FKDQJHV LQ WKH SUHFLVH WLPLQJ RI GHYHORSPHQWDO FXHV .URPHU HW DO n 6WHQHYL HW DO nf ,Q DGGLWLRQ WKH WRSRJUDSK\ RI VRPH RI WKHVH WUDQVSODQWV PD\ EH GLVWRUWHG E\ VSDWLDO UHVWUDLQWV ZKLFK FDQ FRQWULEXWH WR WKH RUJDQL]DWLRQDO GLIIHUHQFHV REVHUYHG 7KH GLIIHUHQWLDWLRQ RI RUJDQRW\SLF UHJLRQV ZLWKLQ DQ DEQRUPDO F\WRDUFKLWHFWXUDO IUDPHZRUN KDV DOVR EHHQ REVHUYHG LQ RWKHU W\SHV RI IHWDO &16 WUDQVSODQWV HJ .URPHU HW DO n 0XIVRQ HW DO n 6RUHQVHQ DQG =LPPHU nEf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n &RLPEUD HW DO nf :KLOH VXFK

PAGE 58

FRPSOH[HV KDYH EHHQ IRXQG LQ GRUVDO UHJLRQV RI )6& JUDIWV LQWHQWLRQDOO\ LQQHUYDWHG E\ SULPDU\ DIIHUHQW ILEHUV ,WRK DQG 7HVVOHU nf 6*OLNH DUHDV GHHSHU ZLWKLQ WKH WUDQVSODQWV UHFHLYH IHZ VXFK DIIHUHQWV ,QQHUYDWLRQ RI )6& WUDQVSODQWV IURP DGXOW KRVW ILEHUV LV ODUJHO\ UHVWULFWHG WR WKH SHULSKHU\ RI WKH JUDIWV &KDSWHU f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n

PAGE 59

6H\EROG DQG (OGH n 0LOOHU DQG 6H\EROG nf 7KXV WKH SUHVHQFH RI WKHVH FHOOV WKURXJKRXW )6& WUDQVSODQWV LQ WKH DEVHQFH RI VXFK WUHDWPHQWV PD\ UHIOHFW DOWHUDWLRQV LQ SHSWLGH H[SUHVVLRQ RU D[RQDO WUDQVSRUW PHFKDQLVPV 6LPLODU GLVFUHSDQFLHV ZLWK UHJDUG WR SHSWLGH H[SUHVVLRQ KDYH EHHQ IRXQG UHFHQWO\ LQ FRUWLFDO DQG VSLQDO JUDIWV WKDW GHYHORSHG LQ RFXOR (ULNVGRWWHU1LOVVRQ HW DO n +HQVFKHQ HW DO nf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f LPPXQRUHDFWLYLW\ RI KLQGOLPE PRWRQHXURQV DIWHU FKURQLF VSLQDO FRUG WUDQVHFWLRQ $UYLGVVRQ HW DO nf ,PSOLFDWLRQV IRU 5HSDLU 7KH IXQFWLRQDO UROH RI FHOOV LQ WKH PDWXUH 6* RI WKH VSLQDO FRUG LV D WRSLF VWLOO XQGHU LQWHQVH LQYHVWLJDWLRQ UHYLHZHG LQ :LOOLV DQG &RJJHVKDOO n &HUYHUR DQG ,JJR nf 7KH WHUPLQDWLRQ RI XQP\HOLQDWHG SULPDU\ DIIHUHQW

PAGE 60

ILEHUV LQ WKLV UHJLRQ SURYLGHV WKH EDVLV IRU WKHRULHV FRQFHUQLQJ LWV UROH LQ WKH PRGXODWLRQ DQG JDWLQJ RI SDLQ DQG UHIOH[ IXQFWLRQV 0HO]DFN DQG :DOO n :LOOLV DQG &RJJHVKDOO nf 6]HQWDJRWKDL nDf XVHG *ROJL VWDLQV DQG GHJHQHUDWLRQ WHFKQLJXHV WR UHYHDO WKH PRUSKRORJ\ DQG SURMHFWLRQ SDWWHUQV RI 6* QHXURQV DQG SURSRVHG WKDW WKH 6* LV SULPDULO\ D FORVHG V\VWHP GRPLQDWHG E\ ORFDO SURMHFWLRQ QHXURQV WKDW H[WHQG QR PRUH WKDQ VHJPHQWV 0RUH UHFHQW HYLGHQFH REWDLQHG ZLWK D[RQDO WUDFLQJ WHFKQLTXHV KDV VKRZQ WKDW DW OHDVW VRPH RI WKHVH 6* QHXURQV FDQ SURMHFW DV IDU DV WKH PHGXOOD *LHVOHU HW DO nf DQG WKDODPXV :LOOLV HW DO nf ,W LV QRZ NQRZQ WKDW D[RQDO SURMHFWLRQV IURP WKH 6* DOVR H[WHQG LQWR GHHSHU ODPLQDH RI WKH VSLQDO FRUG DV ZHOO /LJKW DQG .DYRRNMLDQ nf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

PAGE 61

LQWHUPHGLDWH DQG YHQWUDO UHJLRQV RI WKH VSLQDO FRUG PD\ EH XVHG WR DGGUHVV WKLV LVVXH +RZHYHU RQH IHDWXUH RI WKH QRUPDO VSLQDO JUD\ PDWWHU WKDW LV UDUHO\ VHHQ LQ WKHVH WUDQVSODQWV REWDLQHG IURP (X UDW HPEU\RV LV WKH GHYHORSPHQW RI JURXSV RI ODUJH PRWRQHXURQV ZLWKLQ WKH JUDIWV *UHDWHU QXPEHUV RI PRWRQHXURQV KDYH EHHQ LGHQWLILHG LQ JUDIWV GHULYHG IURP \RXQJHU (f HPEU\RV 5HLHU HW DO nDf 7KXV VHOHFWLRQ RI GLIIHUHQW DJHG GRQRU WLVVXH PD\ EH XVHIXO IRU HQULFKLQJ WUDQVSODQWV LQ HLWKHU YHQWUDO RU GRUVDO QHXURSLO HJ 1RWKLDV HW DO nf ,W LV QRW NQRZQ ZKLFK UHJLRQ RI WKH HPEU\RQLF QHXUD[LV LV EHVW VXLWHG WR UHVWRUH IXQFWLRQ LQ WKH LQMXUHG VSLQDO FRUG +RZHYHU VXFFHVVIXO UHSDLU RI GDPDJHG QHXUDO QHWZRUNV PD\ UHJXLUH WKH UHFRQVWUXFWLRQ RI FHUWDLQ VXSUDVHJPHQWDO DQG LQWUDVSLQDO FLUFXLWV 5HFHQW VWXGLHV KDYH VKRZQ WKDW LQ VRPH LQVWDQFHV KRPRWRSLF JUDIWV DUH LQQHUYDWHG LQ YDU\LQJ GHJUHHV E\ KRVW VHURWRQHUJLF DQG SULPDU\ DIIHUHQW ILEHUV %UHJPDQ n 5HLHU HW DO n nD 7HVVOHU HW DO nf %RWK RI WKHVH D[RQDO V\VWHPV DV ZHOO DV PDQ\ RWKHU LGHQWLILDEOH ILEHU SRSXODWLRQV QRUPDOO\ SURMHFW WR WKH 6* %HFDXVH WKHVH 6*OLNH DUHDV DUH HDVLO\ LGHQWLILHG ZLWKLQ )6& WUDQVSODQWV DQG EHFDXVH WKH DIIHUHQW LQQHUYDWLRQ RI WKH QRUPDO 6* LV ZHOO FKDUDFWHUL]HG WKLV WUDQVSODQWDWLRQ PRGHO VKRXOG SURYLGH D YDOXDEOH RSSRUWXQLW\ IRU WHVWLQJ WKH DELOLW\ RI KRVW D[RQV WR UHFRJQL]H UHJLRQV RI WKHVH JUDIWV ZLWK VLPLODU F\WRORJLFDO DQG SHSWLGHUJLF FKDUDFWHULVWLFV WR WKH 6* RI WKH QRUPDO

PAGE 62

VSLQDO FRUG 6XFK LQIRUPDWLRQ FDQ EH XVHIXO LQ IXUWKHU XQGHUVWDQGLQJ WKH SRWHQWLDO RI WKH JUDIWV WR UHFRQVWUXFW VSHFLILF FLUFXLWULHV LQ WKH LQMXUHG VSLQDO FRUG 7KH GLIIHUHQWLDWLRQ RI DW OHDVW RQH UHJLRQ RI WKH QRUPDO VSLQDO FRUG ZLWKLQ )6& JUDIWV VXJJHVWV WKDW WKHVH JUDIWV PD\ UHSODFH SRSXODWLRQV RI LQWULQVLF VSLQDO FRUG QHXURQV 7KH IROORZLQJ VWXGLHV DUH GHVLJQHG WR GHWHUPLQH ZKHWKHU WKHVH QHXURQV IRUP SURMHFWLRQV ERWK ZLWKLQ WKH JUDIWV DQG EHWZHHQ WKH WUDQVSODQW DQG KRVW VSLQDO FRUG

PAGE 63

&+$37(5 $;21$/ 352-(&7,216 %(7:((1 )(7$/ 63,1$/ &25' 75$163/$176 $1' 7+( $'8/7 5$7 63,1$/ &25' 1(852$1$720,&$/ 75$&,1* $1' ,00812&<72&+(0,&$/ 678'< 2) +267*5$)7 ,17(5$&7,216 ,QWURGXFWLRQ ,Q QHRQDWDO UDWV WUDQVSODQWV RI IHWDO VSLQDO FRUG )6&f WLVVXH KDYH EHHQ VKRZQ WR SURYLGH DQ HQYLURQPHQW FRQGXFLYH WR WKH HORQJDWLRQ RI VRPH GHVFHQGLQJ D[RQV WKURXJK D VSLQDO LQMXU\ VLWH %UHJPDQ nf ,Q DGGLWLRQ ZKHQ SODFHG LQWR KHPLVHFWLRQ OHVLRQV LQ WKHVH QHZERUQ UDWV )6& WUDQVSODQWV KDYH EHHQ VKRZQ WR LPSURYH WKH GHYHORSPHQW RI VSHFLILF DVSHFWV RI KLQGOLPE IXQFWLRQ DV FRPSDUHG ZLWK UDWV ZLWK KHPLVHFWLRQV RQO\ .XQNHO%DJGHQ DQG %UHJPDQ nf ,Q DGXOW UDWV KRZHYHU WKHUH LV QR HYLGHQFH WR VXSSRUW WKH FRQFHSW RI D[RQDO UHJHQHUDWLRQ RI GHVFHQGLQJ D[RQV DFURVV IHWDO VSLQDO FRUG WUDQVSODQWV 1HYHUWKHOHVV WKH SURSDJDWLRQ RI VRPH DVSHFWV RI DVFHQGLQJ DQG GHVFHQGLQJ LQIRUPDWLRQ PLJKW EH DFKLHYHG E\ IHWDO JUDIWV WKURXJK WKH HVWDEOLVKPHQW RI D QHXURQDO UHOD\ EHWZHHQ WKH URVWUDO DQG FDXGDO UHJLRQV RI WKH UHFLSLHQW VSLQDO FRUG 5HLHU HW DO n nf 7R WHVW WKLV K\SRWKHVLV WKH SUHVHQW VWXG\ ZDV GHVLJQHG WR LGHQWLI\ DQG FKDUDFWHUL]H SDWWHUQV RI D[RQDO LQWHUDFWLRQ HVWDEOLVKHG EHWZHHQ )6& JUDIWV DQG DGMDFHQW UHJLRQV RI WKH

PAGE 64

KRVW VSLQDO FRUG 7KHUHIRUH WKH SXUSRVH RI WKH ILUVW H[SHULPHQW ZDV WR H[WHQG SUHOLPLQDU\ :*$+53 WUDFLQJ VWXGLHV ZKLFK KDG VXJJHVWHG VRPH D[RQDO LQWHJUDWLRQ EHWZHHQ KRVW DQG JUDIW 5HLHU HW DO nDf $ IOXRUHVFHQW UHWURJUDGH WUDFHU )OXRUR*ROGf ZDV WKHQ XVHG WR GHWHUPLQH WKH GLVWULEXWLRQ RI FHOOV FRQWULEXWLQJ WR D[RQDO LQWHUDFWLRQV 7R FRPSOHWH WKH D[RQDO WUDFLQJ VWXGLHV WKH DQWHURJUDGH WUDQVSRUW RI WKH SODQW OHFWLQ 3KDVHROXV YXOJDULV OHXFRDJJOXWLQLQ 3+$/f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f R[\WRFLQ 2[f W\URVLQH K\GUR[\ODVH 7+f DQG FDOFLWRQLQ JHQHUHODWHG SHSWLGH &*53f )LQDOO\ DGGLWLRQDO VHFWLRQV IURP ERWK D[RQDO WUDFLQJ DQG LPPXQRF\WRFKHPLFDO VSHFLPHQV ZHUH VWDLQHG ZLWK DQWLVHUXP DJDLQVW JOLDO ILEULOODU\ DFLGLF SURWHLQ *)$3f WR H[DPLQH WKH UHODWLRQVKLS EHWZHHQ KRVWJUDIW SURMHFWLRQV DFURVV WKH LQWHUIDFH DQG WKH SDWWHUQV RI JOLDO UHDFWLYLW\ 3RUWLRQV RI

PAGE 65

WKLV VWXG\ KDYH EHHQ VXPPDUL]HG SUHYLRXVO\ -DNHPDQ DQG 5HLHU nf 0DWHULDOV DQG 0HWKRGV $QLPDOV DQG 7UDQVSODQWDWLRQ 6XUFUHUY $ WRWDO RI IHPDOH DGXOW UDWV UHFHLYHG WUDQVSODQWV RI )6& WLVVXH DFFRUGLQJ WR D PRGLILFDWLRQ RI SUHYLRXVO\ GHVFULEHG PHWKRGV 5HLHU HW DO nD nD &KDSWHU f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n +RXOH DQG 5HLHU nf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

PAGE 66

WKH RULJLQDO ODPLQHFWRP\ URVWURFDXGDOO\ $IWHU WKH WUDFHU ZDV LQMHFWHG WKH VXUIDFH RI WKH FRUG ZDV ZDVKHG ZLWK SK\VLRORJLFDO VDOLQH DQG D GURS RI PLQHUDO RLO ZDV SODFHG RYHU WKH VSLQDO FRUG WR PLQLPL]H GLIIXVLRQ RI WKH WUDFHU LQWR WKH VXUURXQGLQJ WLVVXHV 7KH VSLQDO FRUG ZDV WKHQ FRYHUHG ZLWK D SLHFH RI 'XUDILOP DQG WKH ZRXQG ZDV FORVHG DV GHVFULEHG DERYH +RUVHUDGLVK 3HUR[LGDVH +53f DQG :KHDW *HUP $JJOXWLQLQ +53 FRQMXJDWH :*$+53f $QWHURJUDGH DQG UHWURJUDGH ODEHOLQJ $ FRPELQDWLRQ RI +53 7\SH 9,f DQG :*$+53 6LJPD &KHPLFDOf ZHUH XVHG IRU ERWK UHWURJUDGH ODEHOLQJ RI FHOOV DQG DQWHURJUDGH ILOOLQJ RI D[RQV 0HVXOXP nf DV GHVFULEHG LQ SUHYLRXV VWXGLHV ,Q WKH ILUVW SDUW RI WKH H[SHULPHQW PL[WXUHV RI WKH WZR WUDFHUV ZHUH DSSOLHG WR WUDQVSODQWV Q f XVLQJ D YDULHW\ RI WHFKQLTXHV Df 6HYHQ UDWV UHFHLYHG SUHVVXUH LQMHFWLRQV RI D VROXWLRQ RI b +53 DQG b :*$+53 XVLQJ D MLO +DPLOWRQ V\ULQJH RU D QLWURJHQ EXUVW SLFRVSULW]HU 5HLHU HW DO nDf Ef 7ZR UDWV UHFHLYHG LRQWRSKRUHWLF LQMHFWLRQV RI b :*$+53 Ff RQH UDW UHFHLYHG D SOHGJHW RI *HOIRDP VRDNHG LQ b +53 DQG b :*$+53 DQG Gf +53 DQG :*$+53 ZHUH DSSOLHG WR WKH UHPDLQLQJ VL[ UDWV XVLQJ FU\VWDOV GLVVROYHG RQWR WKH HQG RI D WXQJVWHQ ZLUH +RXOH DQG 5HLHU nf )RU WKH UHFLSURFDO VWXG\ +53 DQG :*$+53 ZHUH DSSOLHG WR WKH KRVW VSLQDO FRUG ZLWK D WXQJVWHQ ZLUH Gf DERYH Q OOf ,Q RUGHU WR H[DPLQH WKH +53 WUDQVSRUW FKDUDFWHULVWLFV

PAGE 67

XVLQJ WKLV PHWKRG D VLPLODU WXQJVWHQ ZLUH ZDV SODFHG LQWR RQH QRUPDO UDW DW WKH 7 YHUWHEUDO OHYHO +53 KLVWRFKHPLVWU\ $IWHU DOORZLQJ KRXUV IRU WUDQVSRUW RI WKH WUDFHU WKH UHFLSLHQWV FRQWDLQLQJ +53 DQGRU :*$+53 LQMHFWLRQV ZHUH GHHSO\ DQHVWKHWL]HG ZLWK VRGLXP SHQWREDUELWDO DQG SHUIXVHG WUDQVFDUGLDOO\ ZLWK PO KHSDULQL]HG b 1D&O IROORZHG E\ PO IL[DWLYH b SDUDIRUPDOGHK\GH b JOXWDUDOGHK\GH LQ 0 6RUHQVRQnV SKRVSKDWH EXIIHUf 7LVVXH EORFNV LQFOXGLQJ WKH WUDQVSODQW DQG PP RI KRVW VSLQDO FRUG URVWUDO DQG FDXGDO WR WKH JUDIW ZHUH UHPRYHG 9LEUDWRPH VHFWLRQV XUQf ZHUH FXW LQ WKH VDJLWWDO RU KRUL]RQWDO SODQH 7KH VHFWLRQV ZHUH UHDFWHG ZLWKLQ KRXUV DFFRUGLQJ WR WKH WHWUDPHWK\O EHQ]LGLQH 70%f SURWRFRO RI GH 2OPRV HW DO nf 6HFWLRQV ZHUH WKHQ PRXQWHG RQWR JHODWLQFRDWHG VOLGHV DQG VHOHFWHG VOLGHV ZHUH FRXQWHUVWDLQHG ZLWK b 1HXWUDO 5HG WR UHYHDO WKH F\WRDUFKLWHFWXUH RI WKH KRVW DQG JUDIW WLVVXHV )OXRUR*ROG )*f 5HWURJUDGH ODEHOLQJ )RU WKH VHFRQG VHW RI H[SHULPHQWV D b VROXWLRQ RI )* )OXRURFKURPH ,QF (QJOHZRRG &2f ZDV PDGH LQ b 1D&O DQG WKH VROXWLRQ ZDV WKHQ GUDZQ LQWR JODVV SLSHWWHV /WP WLS GLDPHWHUf $IWHU D GXUDO LQFLVLRQ ZDV PDGH ZLWK WKH EHYHOHG HQG RI D JDXJH QHHGOH WKH )* VROXWLRQ ZDV LQMHFWHG LQWR WKH WUDQVSODQWV Q f RU WKH KRVW VSLQDO FRUG Q f XVLQJ D UDSLG QLWURJHQ EXUVW 3LFR VSULW]HUf DSSOLHG WR WKH HQG RI WKH JODVV PLFURSLSHWWH 7KH

PAGE 68

DSSUR[LPDWH LQMHFWHG YROXPH RI WKH )* VROXWLRQ ZDV HVWLPDWHG E\ PHDVXULQJ WKH GLDPHWHU RI WKH KHPLVSKHUH HMHFWHG RQWR D SDUDILOP VKHHW DQG XVLQJ WKH DSSUR[LPDWLRQ Y UGff f 9ROXPHV RI LO ZHUH LQMHFWHG LQWR WKH WUDQVSODQWV DQG , LQWR WKH KRVW WLVVXH 2QH QRUPDO UDW DOVR UHFHLYHG DQ LQMHFWLRQ RI QO RI )* VROXWLRQ DW WKH 7 YHUWHEUDO OHYHO IRU FRPSDULVRQ 7LVVXH SURFHVVLQJ 7KH )* FRQWDLQLQJ WLVVXH ZDV SURFHVVHG DV GHVFULEHG E\ 6FKPXHG DQG )DOORQ nf $W GD\V DIWHU WKH LQMHFWLRQ WKH UDWV ZHUH SHUIXVHG ZLWK VDOLQH IROORZHG E\ IL[DWLYH FRQWDLQLQJ b SDUDIRUPDOGHK\GH DQG b JOXWDUDOGHK\GH LQ 0 SKRVSKDWH EXIIHU 6SLQDO FRUG EORFNV FRQWDLQLQJ WKH WUDQVSODQW DQG PP RI WKH VXUURXQGLQJ URVWUDO DQG FDXGDO VSLQDO FRUG ZHUH UHPRYHG DQG SRVWIL[HG LQ WKH VDPH IL[DWLYH IRU KUV WR RYHUQLJKW DW r& 9LEUDWRPH VHFWLRQV RI P ZHUH FXW LQ WKH VDJLWWDO SODQH ,Q DGGLWLRQ HYHU\ VL[WK VHFWLRQ ZDV VDYHG LQ 0 3%6 IRU VXEVHTXHQW LPPXQRF\WRFKHPLFDO GHWHFWLRQ RI *)$3 VHH EHORZf ,Q RI WKH UDWV WKDW UHFHLYHG ODUJHU LQMHFWLRQV RI )* LQWR WKH WUDQVSODQWV VL[ DGGLWLRQDO WLVVXH EORFNV ZHUH DOVR VHFWLRQHG 7KHVH LQFOXGHG FURVV VHFWLRQV RI WKH KRVW VSLQDO FRUG PP URVWUDO DQG FDXGDO WR WKH WUDQVSODQW KRUL]RQWDO VHFWLRQV IURP FHUYLFDO DQG WKRUDFLF VSLQDO FRUG DQG VHFWLRQV RI KRVW EUDLQVWHP DQG EUDLQ 7KH GRUVDO URRW JDQJOLD IURP WKHVH UHFLSLHQWV ZHUH HPEHGGHG LQ SDUDIILQ DQG VHFWLRQHG DW P 7KH 9LEUDWRPH DQG SDUDIILQ VHFWLRQV ZHUH

PAGE 69

PRXQWHG GLUHFWO\ RQWR JHODWLQFRDWHG VOLGHV 6OLGHV IURP SDUDIILQ EORFNV ZHUH KHDWHG WR r& IRU KRXUV DQG GHSDUDIILQL]HG $OO )* VOLGHV ZHUH FOHDUHG LQ [\OHQH FRYHUVOLSSHG ZLWK )OXRURPRXQW *XUU %LRPHGLFDO 6SHFLDOWLHV 6DQWD 0RQLFD &$f DQG YLHZHG RQ D =HLVV $[LRSKRW PLFURVFRSH ZLWK IOXRUHVFHQW 89 LOOXPLQDWLRQ 3KDVHROXV YXOJDULV OHXFRDTTOXWLQLQ 3+$/f $QWHURJUDGH ODEHOLQJ DQG WLVVXH VHFWLRQLQJ 7R H[DPLQH WKH SDWWHUQV RI D[RQDO HORQJDWLRQ LQWR JUDIW DQG KRVW WLVVXHV DQWHURJUDGHO\ILOOHG D[RQV ZHUH LGHQWLILHG E\ LPPXQRF\WRFKHPLFDO GHWHFWLRQ RI 3+$/ 9HFWRU /DERUDWRULHV ,QF %XUOLQJKDP &$f 7KH WUDFHU ZDV DSSOLHG E\ D PRGLILFDWLRQ RI WKH PHWKRGV RI *HUIHQ DQG 6DZFKHQNR nf 7KH 3+$/ ZDV GLVVROYHG WR b LQ P0 SKRVSKDWH EXIIHU S+ f *ODVV PLFURSLSHWWHV ZHUH FOHDQHG ZLWK DFHWRQH DQG b HWKDQRO DQG EURNHQ WR D WLS GLDPHWHU RI LP $IWHU H[SRVLQJ WKH WUDQVSODQW Q f RU KRVW VSLQDO FRUG Q f WKH WUDFHU ZDV DSSOLHG WR WKH DSSURSULDWH VLWH E\ LRQWRSKRUHVLV IRU PLQXWHV XVLQJ D $ LQWHUUXSWHG SRVLWLYH FXUUHQW VHF RQ VHF RIIf $IWHU DOORZLQJ GD\V IRU WUDQVSRUW RI WKH 3+$/ WKH UHFLSLHQWV ZHUH SHUIXVHG DV DERYH ZLWK IL[DWLYH FRQWDLQLQJ b SDUDIRUPDOGHK\GH DQG b JOXWDUDOGHK\GH 7KH FRUG EORFNV LQFOXGLQJ WKH WUDQVSODQW DQG PP RI WKH VXUURXQGLQJ URVWUDO DQG FDXGDO VSLQDO FRUG ZHUH UHPRYHG DQG SRVWIL[HG RYHUQLJKW DW r& 6DJLWWDO VHFWLRQV RI WKHVH

PAGE 70

EORFNV ZHUH FXW DW QP RQ D 9LEUDWRPH DQG VWRUHG LQ 0 SRWDVVLXP SKRVSKDWH EXIIHUHG VDOLQH .3%6f (YHU\ VL[WK VHFWLRQ ZDV VDYHG IRU LPPXQRF\WRFKHPLFDO VWDLQLQJ ZLWK DQWLERGLHV WR *)$3 VHH EHORZf ,PPXQRF\WRFKHPLFDO GHWHFWLRQ RI 3+$/ 7KH UHPDLQLQJ IUHHIORDWLQJ 9LEUDWRPH VHFWLRQV ZHUH SURFHVVHG IRU WKH LGHQWLILFDWLRQ RI FHOOV DQG SURFHVVHV FRQWDLQLQJ 3+$/ 7KH VHFWLRQV ZHUH ILUVW ZDVKHG LQ 0 .3%6 DQG LQFXEDWHG IRU KUV LQ D SUHEORFNLQJ EDWK FRQWDLQLQJ b QRUPDO UDEELW VHUXP DQG b 7ULWRQ ; $OO WKH VHFWLRQV ZHUH WKHQ LQFXEDWHG LQ JRDW DQWL3+$/ 9HFWRUf GLOXWHG LQ .3%6 IRU KRXUV DW r& DQG DGGLWLRQDO KRXUV DW URRP WHPSHUn DWXUH 7KH VHFWLRQV ZHUH UHZDVKHG DQG WKHQ SURFHVVHG ZLWK ELRWLQ\ODWHG UDEELW DQWLJRDW ,J* f DQG 9HFWRU $YLGLQ %LRWLQSHUR[LGDVH &RPSOH[ $%&f DV SHU WKH VXSSOLHUnV LQVWUXFWLRQV 7KH ILQDO SHUR[LGDVH FRQMXJDWH ZDV UHDFWHG ZLWK + LQ WKH SUHVHQFH RI b '$% 7KH '$% UHDFWLRQ ZDV GRQH HLWKHU ZLWK WKH DGGLWLRQ RI b QLFNHO DPPRQLXP VXOIDWH EODFN UHDFWLRQ SURGXFWf RU LQ WKH DEVHQFH RI QLFNHO EURZQ UHDFWLRQ SURGXFWf 7KH QLFNHOHQKDQFHG VHFWLRQV ZHUH FRXQWHUVWDLQHG ZLWK b &UHV\O 9LROHW RU b 1HXWUDO 5HG SULRU WR FRYHUVOLSSLQJ +LVWRORJLFDO DQDO\VLV RI DQDWRPLFDO WUDFHUV 0RXQWHG VHULDO VHFWLRQV FRQWDLQLQJ WKH WUDFHU LQMHFWLRQ VLWHV ZHUH H[DPLQHG WR GHWHUPLQH WKH ORFDWLRQ RI WKH LQMHFWLRQ VLWH DQG WKH H[WHQW RI WUDFHU GLIIXVLRQ UHODWLYH WR

PAGE 71

WKH KRVWJUDIW LQWHUIDFH (DFK VSHFLPHQ ZDV WKHQ DFFHSWHG RU UHMHFWHG IURP WKH VWXG\ DFFRUGLQJ WR VSHFLILF WUDQVSRUW DQG GLIIXVLRQ FULWHULD DV GHVFULEHG LQ 5HVXOWV 5HWURJUDGHO\ ILOOHG FHOOV ZHUH LGHQWLILHG DQG PDQXDOO\ FRXQWHG LQ VXFFHVVLYH PP ILHOGV DW [ &HOOV ZKLFK GHPRQVWUDWHG QRQVSHFLILF IOXRUHVFHQFH ZKHQ H[SRVHG WR UKRGDPLQH QPf RU IOXRUHVFHLQ QPf PLFURVFRSH ILOWHUV ZHUH QRW FRXQWHG (DFK ILHOG ZDV FRXQWHG WLPHV DQG WKH PHGLDQ YDOXH ZDV DFFHSWHG $OO FHOO FRXQWV ZHUH FRUUHFWHG DFFRUGLQJ WR FODVVLFDO PHWKRGV $EHUFURPELH nf 7RWDO FHOO QXPEHU ZDV REWDLQHG E\ DVVXPLQJ DQ DYHUDJH FHOO GLDPHWHU RI P IRU JUDIW FHOOV DQG QP IRU KRVW QHXURQV 7KH GLVWULEXWLRQ RI ODEHOHG FHOOV DQG D[RQV ZDV GHWHUPLQHG IURP GUDZLQJ WXEH WUDFLQJV RI GDUNILHOG RU EULJKWILHOG LPDJHV +53 DQG 3+$/f RU IURP SKRWRJUDSKLF PRQWDJHV RI IOXRUHVFHQFH PLFURJUDSKV )*f 7R GHWHUPLQH WKH GLVWDQFHV RI DQWHURJUDGHO\ ODEHOHG D[RQDO SURMHFWLRQV D GLJLWL]LQJ WDEOHW DQG PRUSKRPHWU\ VRIWZDUH 9LGHRSODQ .RQWURQ )5*f ZDV FDOLEUDWHG IRU WKH DSSURSULDWH PDJQLILFDWLRQ 0HDVXUHPHQWV ZHUH WDNHQ IURP WKH GUDZLQJ WXEH LOOXVWUDWLRQV RU SKRWRPLFURJUDSKV 6LPLODU PHWKRGV ZHUH XVHG WR GRFXPHQW WKH GLVWDQFHV EHWZHHQ WKH LQMHFWLRQ VLWH DQG WKH RXWHUPRVW ]RQH RI WUDFHU GLIIXVLRQ DV ZHOO DV WKH UHODWLRQVKLS RI WKHVH UHJLRQV WR WKH KRVWJUDIW LQWHUIDFH

PAGE 72

,PPXQRFYWRFKHPLFDO VWDLQLQJ DQG DQDO\VLV RI *)$3 $ VHULHV RI VHFWLRQV MXP DSDUWf IURP UHFLSLHQWV ZLWK )* RU 3+$/ LQMHFWLRQV ZDV LQFXEDWHG LQ UDEELW SRO\FORQDO DQWLVHUXP SURGXFHG DJDLQVW *)$3 JLIW RI 'U /DZUHQFH ) (QJ 9$ 0HGLFDO &HQWHU 3DOR $OWR &$f 7KH DQWLVHUXP ZDV GLOXWHG DQG VHFWLRQV ZHUH LQFXEDWHG RYHUQLJKW DW r& 'HWHFWLRQ RI WKH SULPDU\ DQWLERG\ ZDV SHUIRUPHG DFFRUGLQJ WR WKH SHUR[LGDVH DQWLSHUR[LGDVH PHWKRG 6WHUQEHUJHU nf DV GHVFULEHG EHORZ 7UDFLQJV RI WKH URVWUDO DQG FDXGDO LQWHUIDFH UHJLRQV IRU HDFK VHFWLRQ ZHUH PDGH XVLQJ D GUDZLQJ WXEH GHOLQHDWLQJ WKH UHJLRQV FRQWDLQLQJ GHQVH *)$3 VWDLQLQJ EHWZHHQ KRVW DQG JUDIW WLVVXH )RU VSHFLPHQV ZLWK )* LQMHFWLRQV WKH OHQJWKV RI WKH LQWHUIDFH DQG WKH UHJLRQV FRQWDLQLQJ JOLDO VFDU IRUPDWLRQ ZHUH PHDVXUHG XVLQJ D GLJLWL]LQJ SDG DQG 9LGHRSODQ VRIWZDUH 7KH FRPSRVLWH )XVLRQ ,QGH[ ),f IRU HDFK LQWHUIDFH UHJLRQ ZDV GHILQHG DV WKH DYHUDJH SHUFHQWDJH RI WKH LQWHUIDFH ZKLFK ZDV GHYRLG RI GHQVH JOLDO VFDUULQJ +RXOH DQG 5HLHU nf 7KH GHQVLW\ RI JOLDO VWDLQLQJ ZDV GHWHUPLQHG IRU ERWK JUDIW DQG VXUURXQGLQJ KRVW WLVVXHV LQ UHFLSLHQWV $ SURJUDP ZDV GHYHORSHG XVLQJ WKH =HLVV ,%$6 LPDJH DQDO\VLV V\VWHP .RQWURQ )5*f DQG D KLJK UHVROXWLRQ YLGHR FDPHUD '$*( ,QF &&'f $W D YLHZLQJ PDJQLILFDWLRQ RI [ IRXU SDLUV RI LPDJHV IURP HDFK *)$3 VWDLQHG VHFWLRQ HDFK SDLU LQFOXGLQJ D JUDIW UHJLRQ DQG KRVW JUD\ PDWWHU UHJLRQf ZHUH GLJLWL]HG DQG FRQYHUWHG WR ELQDU\ LPDJHV 7KH ILUVW ILHOG RI

PAGE 73

HDFK SDLU ZDV VHJPHQWHG LQWHUDFWLYHO\ E\ WKH XVHU WR GLVWLQJXLVK JOLDO SURFHVVHV IURP EDFNJURXQG DV GHVFULEHG E\ %MRUNOXQG+ HW DO nf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nV W WHVW IRU SDLUHG VDPSOHV 6SHQFH HW DO nf ,PPXQRVWDLQLQT )RU 6SHFLILF 3RSXODWLRQV RI +RVW )LEHUV ,PPXQRKLVWRFKHPLFDO SURFHGXUHV $GMDFHQW VHULHV RI VHFWLRQV IURP WUDQVSODQW UHFLSLHQWV ZHUH VWDLQHG ZLWK SRO\FORQDO DQWLVHUD UDLVHG DJDLQVW QHXURWUDQVPLWWHUV V\QWKHWLF HQ]\PHV RU SHSWLGHV IRXQG LQ VSHFLILF SRSXODWLRQV RI KRVW ILEHUV 6HH 7DEOH f 2I WKLV JURXS UHFLSLHQWV ZHUH VHOHFWHG IURP WUDFHU VSHFLPHQV ZLWK XQDFFHSWDEOH RU IDLOHG LQMHFWLRQV $OO RI WKH UHFLSLHQWV ZHUH SHUIXVHG DV GHVFULEHG DERYH ZLWK IL[DWLYH FRQWDLQLQJ b SDUDIRUPDOGHK\GH DQG b JOXWDUDOGHK\GH LQ 0 6RUHQVRQnV SKRVSKDWH EXIIHU S+ f 6HFWLRQV FRQWDLQLQJ WKH JUDIW DQG VXUURXQGLQJ KRVW VSLQDO FRUG ZHUH

PAGE 74

FXW DW P RQ D 9LEUDWRPH DQG VWRUHG LQ 0 SKRVSKDWH EXIIHUHG VDOLQH SULRU WR VWDLQLQJ 7$%/( /,67 2) $17,6(5$ 6285&( $1' ',/87,21 $QWLVHUD 6RXUFH $QWLERG\ GLOXWLRQ +7 2; 7+ &*53 ,QFVWDU &RUS RYHUQLJKW ,QFVWDU &RUS RYHUQLJKW (XJHQHWHFK,QF RYHUQLJKW 3HQLQVXOD /DEV KRXUV ,PPXQRF\WRFKHPLFDO VWDLQLQJ ZDV SHUIRUPHG RQ DGMDFHQW VHULHV RI VHFWLRQV XVLQJ DQWLVHUD GLUHFWHG DJDLQVW WKH IROORZLQJ +7VHURWRQLQ 2;R[\WRFLQ 7+W\URVLQH K\GUR[\ODVH &*53&DOFLWRQLQ JHQHUHODWHG SHSWLGH )UHH IORDWLQJ VHFWLRQV ZHUH SURFHVVHG XVLQJ WKH 3$3 LPPXQRF\WRFKHPLFDO SURFHGXUH 6WHUQEHUJHU nf 7KH SULPDU\ DQWLVHUD XVHG LQ WKLV VWXG\ ZHUH DOO UDLVHG LQ UDEELW 7DEOH f DQG GLOXWHG LQ KLJK VDOW EXIIHU FRQWDLQLQJ b 7ULWRQ ; 7+6%f $IWHU WKH VHFWLRQV ZHUH UHPRYHG IURP WKH SULPDU\ DQWLERG\ VROXWLRQ WKH\ ZHUH ZDVKHG WLPHV LQ 7+6% LQFXEDWHG LQ UDEELW DQWLJRDW ,J* DW IRU PLQ WR KU ZDVKHG DJDLQ LQ 7+6% LQFXEDWHG LQ 5DEELW 3$3 IRU PLQ DQG ILQDOO\ ZDVKHG LQ SKRVSKDWH EXIIHUHG VDOLQH 3%6f RU DPPRQLXP SKRVSKDWH EXIIHU 7KH SHUR[LGDVH ZDV YLVXDOL]HG ZLWK b '$% DQG b + 6WDLQLQJ RI ILEHUV ZLWK DQWL+7 DQG DQWL7+ ZHUH GRQH LQ WKH SUHVHQFH RI b QLFNHO DPPRQLXP SKRVSKDWH WR SURGXFH D EODFN UHDFWLRQ SURGXFW 6HFWLRQV ZHUH WKHQ PRXQWHG RQ JHODWLQ FRDWHG VOLGHV 6RPH VOLGHV ZHUH FRXQWHUVWDLQHG ZLWK b 1HXWUDO

PAGE 75

5HG RU b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b RI WKH UHFLSLHQW UDWV 1HDUO\ DOO RI WKH JUDIWV ILOOHG WKH OHVLRQ FDYLW\ DQG VKRZHG JURVV DSSRVLWLRQ ZLWK WKH VXUURXQGLQJ KRVW WLVVXHV 7KH FHOOXODU RUJDQL]DWLRQ DQG YDULDELOLW\ ZLWKLQ WKH KRVW JUDIW LQWHUIDFH ZDV VLPLODU WR WKDW GHVFULEHG LQ SUHYLRXV VWXGLHV IURP WKLV ODERUDWRU\ 5HLHU HW DO nD +RXOH DQG 5HLHU nf :LWK UHJDUG WR WKH LQWHUIDFH WKRVH VHFWLRQV FRXQWHUVWDLQHG ZLWK &UHV\O 9LROHW RU 1HXWUDO 5HG RIWHQ FRQWDLQHG UHJLRQV EHWZHHQ WKH KRVW DQG JUDIW WLVVXH WKDW ZHUH RFFXSLHG E\ VPDOO GHQVHO\ SDFNHG FHOO QXFOHL UHVHPEOLQJ JOLDO FHOOV ,Q FRQWUDVW RWKHU UHJLRQV RI WKH LQWHUIDFH ZHUH GHYRLG RI DQ REYLRXV FHOOXODU ERXQGDU\ EHWZHHQ WKH WZR

PAGE 76

WLVVXHV ,Q WKHVH PRUH LQWHJUDWHG DUHDV WKH RQO\ GLVWLQFWLRQ EHWZHHQ KRVW DQG JUDIW ZDV D WUDQVLWLRQ LQ WKH JHQHUDO F\WRDUFKLWHFWXUDO RUJDQL]DWLRQ $ VLPLODU UDQJH RI KRVWJUDIW IXVLRQ ZDV REVHUYHG LQ *)$3 VWDLQHG VHFWLRQV 1HXURDQDWRPLFDO 7UDFLQJ :LWK +53 DQG :*$+53 ,QMHFWLRQV LQWR WUDQVSODQWV 6ROXWLRQV RI +53 DQG :*$+53 ZHUH LQMHFWHG RU DSSOLHG WR WUDQVSODQWV 7DEOH f 7KH VXUYLYLQJ JUDIWV ZHUH FODVVLILHG EDVHG XSRQ WKH KLVWRORJLFDO DQDO\VLV RI WKH LQMHFWLRQ DQG WKH H[WHQW RI WUDFHU GLIIXVLRQ 7KH LQMHFWLRQ VLWHV ZHUH H[DPLQHG XQGHU GDUNILHOG LOOXPLQDWLRQ DQG WKH H[WHQW RI HDFK ZDV GHILQHG DV WKH DUHD FRQWDLQLQJ D SXUSOH RSDTXH FRUH DQG WKH HQWLUH VXUURXQGLQJ UHJLRQ RI RUDQJH 70% UHDFWLRQ SURGXFW )LJ DEf 7KH LQMHFWLRQ VLWH ZDV UHVWULFWHG VROHO\ WR WKH WUDQVSODQW LQ VL[ RI WKH UHFLSLHQWV 7DEOH *URXSV $ %f 7KH WZR VSHFLPHQV FODVVLILHG LQWR *URXS $ FRQWDLQHG WKH VPDOOHVW LQMHFWLRQ VLWHV ZKLFK H[WHQGHG OHVV WKDQ PP LQ PD[LPXP GLDPHWHU :KLOH QR ODEHOHG FHOOV RU D[RQV ZHUH REVHUYHG LQ WKH KRVW VSLQDO FRUG WKHVH VPDOO LQMHFWLRQV LOOXVWUDWHG WKH SUHVHQFH RI LQWULQVLF JUDIW SURMHFWLRQV )LJ ODf 7KH PDMRULW\ RI UHWURJUDGHO\ ODEHOHG FHOOV LQ WKHVH VSHFLPHQV ZHUH ORFDWHG ZLWKLQ PP RI WKH FHQWHU RI WKH LQMHFWLRQ VLWH KRZHYHU DGGLWLRQDO UHWURJUDGHO\ ILOOHG QHXURQV ZHUH IRXQG WKURXJKRXW WKH JUDIWV ,Q WKH IRXU RWKHU UHFLSLHQWV 7DEOH *URXS %f WKH LQMHFWLRQ VLWHV ZHUH

PAGE 77

7$%/( +53:*$+53 ,1-(&7,216 ,172 )6& 75$163/$176 &RGH 3RVW*UDIWf ,QWHUYDOf 0HWKRG *URXSE ,QWULQ SURM (II F SURM $II G SURM +*3 ZNf 3LFRVSULW] $ +*3 ZNf ,RQWRSKRUHVLV $ +*3 ZNf 3LFRVSULW] % +*3 ZNf 7XQJVWHQ ZLUH % +*3 ZNf 7XQJVWHQ ZLUH % +*3 ZNf 7XQJVWHQ ZLUH % +*3 ZNf 3LFRVSULW] & +*3 ZNf +DPLOWRQ & +* PRf 7XQJVWHQ ZLUH & +*3 ZNf +DPLOWRQ & ,QGLFDWHV HYLGHQFH RI SURMHFWLRQV IURP ODEHOHG SURILOHV SUHVHQW LQ KRVW RU JUDIW WLVVXH D 7KH WUDFHUV ZHUH DSSOLHG XVLQJ RQH RI ILYH SURFHGXUHV VHH 0HWKRGVf E 5HFLSLHQWV LQFOXGHG LQ DQDO\VLV ZHUH FODVVLILHG DFFRUGLQJ WR WKH H[WHQW RI WUDFHU GLIIXVLRQ DV IROORZV $ ,QMHFWLRQ VLWH PP LQ GLDPHWHU DQG FRQILQHG WR JUDIW % ,QMHFWLRQ ODUJHU WKDQ PP DQG FRQILQHG WR JUDIW & ,QMHFWLRQ VLWH ZLWKLQ JUDIW GLIIXVHG LQWR RU VOLJKWO\ RYHU LQWHUIDFH F (IIHUHQW SURMHFWLRQV RI JUDIW D[RQV $QWHURJUDGH D[RQ ODEHO H[WHQGHG LQWR KRVW VSLQDO FRUG G $IIHUHQW SURMHFWLRQV IURP KRVW QHXURQV 5HWURJUDGHO\ ODEHOHG FHOOV IRXQG LQ KRVW VSLQDO FRUG

PAGE 78

)LJXUH +53 DQG :*$+53 WUDFLQJ UHYHDOHG LQWULQVLF LQWHUDFWLRQV DQG VRPH SURMHFWLRQV RI JUDIW DQG KRVW D[RQV Df 'UDZLQJ WXEH WUDFLQJV RI VHTXHQWLDO VDJLWWDO VHFWLRQV WKURXJK D JUDIW ZLWK D VPDOO *URXS $f LQMHFWLRQ 7KH FHQWHU RI WKH LQMHFWLRQ VLWH LV VKRZQ DV VROLG EODFN DQG WKH DUHD FRQWDLQLQJ GHQVH UHDFWLRQ SURGXFW ZLWK QR GLVFHUQDEOH FHOOV DQG D[RQV LV UHSUHVHQWHG E\ WKH KDWFKHG UHJLRQ 7KH DUHD RXWOLQHG E\ D GRWWHG OLQH UHSUHVHQWV D KLJK GHQVLW\ RI ODEHOHG FHOOV DQG D[RQV DQG HDFK LQGLYLGXDO FHOO ZLWKLQ WKH JUDIW LV LQGLFDWHG E\ ODUJHU GRWV Ef /DEHOHG FHOOV DQG D[RQV ZHUH GLVWULEXWHG WKURXJKRXW D WUDQVSODQW W +*3f WR WKH KRVWJUDIW LQWHUIDFH DUURZKHDGVf IROORZLQJ D ODUJHU +53:*$+53 LQMHFWLRQ LQWR WKH GRUVDO UHJLRQ RI D JUDIW Ff/DEHOHG JUDIW D[RQV FRXUVHG SDUDOOHO WR WKLV UHJLRQ RI WKH KRVWJUDIW LQWHUIDFH EXW GR QRW SHQHWUDWH WKH KRVW VSLQDO FRUG Kf GJf $[RQDO SURMHFWLRQV IRUPHG EHWZHHQ KRVW DQG JUDIW WLVVXHV *UDIW HIIHUHQW SURMHFWLRQV ZHUH LGHQWLILHG E\ UHWURJUDGH WUDQVSRUW LQWR WUDQVSODQW QHXURQV IROORZLQJ LQMHFWLRQV LQWR WKH KRVW VSLQDO FRUGGf DQG DQWHURJUDGHO\ ODEHOHG D[RQV ZKLWH DUURZVf H[WHQGLQJ LQWR WKH KRVW VSLQDO FRUG DIWHU DQ LQMHFWLRQ LQWR WKH WUDQVSODQW Hf If ([DPSOH RI VKRUWGLVWDQFH LQJURZWK RI KRVW D[RQV LQWR D WUDQVSODQW IROORZLQJ DQ LQMHFWLRQ PDGH PP URVWUDO WR D JUDIW Jf ,OOXVWUDWLRQ RI WKH SRWHQWLDO IRU JUHDWHU D[RQDO LQWHUDFWLRQV IROORZLQJ DQ LQMHFWLRQ ZKLFK GLIIXVHG DFURVV WKH GRUVDO UHJLRQ RI WKH KRVWJUDIW LQWHUIDFH VSHFLPHQ ++f 1RWH WKDW UHWURJUDGHO\ ILOOHG QHXURQV rf PD\ UHSUHVHQW LQWULQVLF SURMHFWLRQV ODEHOHG E\ WUDFHU GLIIXVLRQ +RZHYHU ODEHOHG ILEHUV FDQ EH VHHQ LQ WKLV YHQWUDO VHFWLRQ ZKHUH WKH GLIIXVLRQ GRHV QRW FRQIXVH WKH KRVWJUDIW ERUGHU $[RQV H[WHQGHG DFURVV WKH LQWHUIDFH UHJLRQ LH DUURZKHDGf EHWZHHQ KRVW KHDYLO\ ODEHOHGf DQG JUDIW OLJKWO\ ODEHOHGf WLVVXHV 6FDOH LQ D PP EF QP GJ P

PAGE 80

ODUJHU WKDQ PP LQ GLDPHWHU EXW ZHUH VWLOO FRQILQHG WR WKH WUDQVSODQWV $Q H[WHQVLYH QHWZRUN RI LQWULQVLF JUDIW SURMHFWLRQV ZDV DJDLQ HYLGHQW %RWK ODEHOHG FHOOV DQG D[RQDO SURILOHV ZHUH REVHUYHG WKURXJKRXW WKH WUDQVSODQWV DQG XS WR WKH LQWHUIDFH LQ DOO GLUHFWLRQV )LJ EFHf 7KH WUDQVSODQWV LQ *URXS % DOVR VXJJHVWHG WKH SUHVHQFH RI D[RQDO SURMHFWLRQV EHWZHHQ KRVW DQG JUDIW WLVVXHV 6LPLODU WR ILQGLQJV IURP SUHOLPLQDU\ VWXGLHV 5HLHU HW DO nDf UHWURJUDGHO\ ILOOHG QHXURQV ZHUH RFFDVLRQDOO\ IRXQG ZLWKLQ WKH KRVW VSLQDO FRUG (IIHUHQW SURMHFWLRQV IURP JUDIW QHXURQV ZHUH DOVR REVHUYHG DV DQWHURJUDGHO\ILOOHG D[RQV FRXOG EH IROORZHG DFURVV WKH LQWHUIDFH LQWR WKH KRVW LQ WZR UHFLSLHQWV )LJ OHf 8QIRUWXQDWHO\ WKH SRVVLELOLW\ RI DGGLWLRQDO ODEHOHG D[RQV RULHQWHG SHUSHQGLFXODU WR WKH SODQH RI VHFWLRQ FRXOG QRW EH DVVHVVHG ,Q FRQWUDVW WKHUH ZHUH VRPH UHJLRQV RI HDFK KRVWJUDIW LQWHUIDFH ZKHUH WKH WZR WLVVXHV DSSHDUHG WR EH VHSDUDWHG E\ D JOLDO SDUWLWLRQ ,Q WKHVH UHJLRQV D[RQV FRXUVHG SDUDOOHO WR WKH LQWHUIDFH EXW WKH\ GLG QRW H[WHQG LQWR WKH KRVW VSLQDO FRUG )LJ Ff 7KH UHFLSLHQWV LQ *URXS & KDG LQMHFWLRQ VLWHV ZKLFK ZHUH QRW FRQILQHG WR WKH JUDIW ,Q HDFK FDVH KRZHYHU WKH RXWHU ]RQH RI 70% UHDFWLRQ SURGXFW H[WHQGHG EH\RQG HLWKHU WKH URVWUDO RU FDXGDO ERUGHU RI WKH JUDIW ZKLOH WKH RSDTXH FHQWHU ZDV FRQILQHG WR WKH JUDIW $W WKRVH UHJLRQV ZKHUH WKH LQMHFWLRQ H[WHQGHG RYHU WKH LQWHUIDFH ODEHOHG D[RQV DQG KRVW QHXURQV ZHUH IRXQG ZLWKLQ PP RI WKH KRVWJUDIW LQWHUIDFH

PAGE 81

EXW IHZ ODEHOHG FHOOV ZHUH REVHUYHG IDUWKHU DZD\ 7KLV ODEHOLQJ SDWWHUQ GLIIHUHG IURP WKH SDWWHUQ REVHUYHG IROORZLQJ SODFHPHQW RI +53 DQG :*$+53 LQWR WKH QRUPDO VSLQDO FRUG VHH 0HQHWUH\ HW DO nf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f LQMHFWLRQV WKDW H[WHQGHG WR ZLWKLQ PP RI WKH LQWHUIDFH UHJLRQ 2I WKH VHYHQ VSHFLPHQV IRXU FRQWDLQHG UHWURJUDGHO\ ILOOHG QHXURQV ZLWKLQ WKH WUDQVSODQWV )LJ Gf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

PAGE 82

7$%/( +53:*$+53 ,1-(&7,216 ,172 +267 63,1$/ &25' &RGH ,6D 3RVW*UDIWf ,QWHUYDOf 'LVWE F,QW PPf 'LVWF H,QW PPf (II G SURM $II H SURM ++ PRf URVW FDXG f§ f§ ++ PR fURVW ++ PRf URVW f§ f§ FDXG f§ ++ PRf URVW f§ FDXG f§ ++ PRf URVW FDXG f§ ++ PRf URVW f§ f§ FDXG f§ ++ PRf URVW f§ FDXG f§ ,QGLFDWHV UHWURJUDGHO\ ILOOHG FHOOV RU DQWHURJUDGHO\ ILOOHG D[RQV SUHVHQW ZLWKLQ WKH WUDQVSODQW D ,QMHFWLRQ 6LWH ,QMHFWLRQ SODFHG HLWKHU URVWUDO URVWf RU FDXGDO FDXGf WR KRVWJUDIW LQWHUIDFH E 0HDVXUHG GLVWDQFH IURP FHQWHU RI LQMHFWLRQ VLWH WR WKH KRVWJUDIW LQWHUIDFH F 0HDVXUHG GLVWDQFH IURP WKH HGJH RI +53 UHDFWLRQ SURGXFW RU GLIIXVLRQ WR WKH KRVWJUDIW LQWHUIDFH G (IIHUHQW SURMHFWLRQV RI WUDQVSODQW QHXURQV 5HWURJUDGHO\ ODEHOHG FHOOV ZLWKLQ WKH JUDIW H $IIHUHQW SURMHFWLRQV LQWR WKH JUDIW $QWHURJUDGHO\ ODEHOHG D[RQV IURP WKH KRVW VSLQDO FRUG

PAGE 83

DQG YHU\ VSDUVH D[RQDO SURILOHV LQ WKH WUDQVSODQW )LJ If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f f )OXRUR*ROG ,QMHFWLRQV 'LVWULEXWLRQ RI &HOOV ,QMHFWLRQV LQWR WKH KRVW VSLQDO FRUG )OXRUR*ROG LQMHFWLRQV ZHUH PDGH LQWR WKH KRVW VSLQDO FRUG RI UHFLSLHQWV DW GLVWDQFHV UDQJLQJ IURP PP IURP WKH KRVWJUDIW LQWHUIDFH ,QMHFWLRQV LQ VHYHQ RI WKH UDWV PHW WKUHH FULWHULD f WKH LQMHFWLRQV ZHUH FRQILQHG WR WKH KRVW VSLQDO FRUG f WKH\ VKRZHG QR HYLGHQFH RI GLIIXVLRQ LQWR WKH FHUHEUDO VSLQDO IOXLG DV VHHQ E\ D KLJK GHJUHH RI QRQVSHFLILF IOXRUHVFHQFH WKURXJKRXW WKH VSLQDO FRUGf RU FHQWUDO FDQDO UHJLRQ DQG f WKH\ ZHUH ODUJH HQRXJK WR ODEHO

PAGE 84

D KLJK SHUFHQWDJH RI KRVW QHXURQV DGMDFHQW WR WKH JUDIW 5HVXOWV IURP WKHVH DQLPDOV DUH VXPPDUL]HG LQ 7DEOH 7$%/( ,1-(&7,216 2) )/8252*2/' ,172 7+( +267 63,1$/ &25' &RGH ,6 3RVW*UDIWfPPf ,QWHUYDOf KRVWf ODEHOHG FHOOV LQ JUDIW )XVLRQ ,QGH[ bVGf *OLDOF 5DWLR *+ )*& PRf PP URVW bsf r )*& PRf PP URVW bsf )*& PRf PP URVW bsf r )*& PRf PP URVW bsf r )*& PRf PP FDXG bsf r )*&Pf PP FDXG bsf )*& ZNf PP FDXG bsf D 7RWDO QXPEHU RI UHWURJUDGHO\ ILOOHG FHOOV ZLWKLQ WKH WUDQVSODQW &HOO FRXQWV FRUUHFWHG DFFRUGLQJ WR WKH PHWKRG RI $EHUFURPELH f E &RPSRVLWH )XVLRQ ,QGH[ ),f SHUFHQWDJH RI WKH KRVWJUDIW LQWHUIDFH FORVHVW WR LQMHFWLRQf ZKLFK LV GHYRLG RI GHQVH JOLDO VFDU DYHUDJH RI VHFWLRQVWUDQVSODQWf F 5DWLR RI WKH SHUFHQW DUHD RFFXSLHG E\ JOLDO HOHPHQWV r LQGLFDWHV WKDW WKH JOLDO GHQVLW\ ZLWKLQ WKH JUDIW ZDV VLJQLILFDQWO\ KLJKHU WKDQ WKDW LQ WKH VXUURXQGLQJ KRVW WLVVXH S f 7KH LQWHUIDFH EHWZHHQ WKH KRVW JUD\ PDWWHU DQG JUDIW WLVVXH ZDV FKDUDFWHUL]HG E\ D VKDUS GHFUHDVH LQ WKH GHQVLW\ RI ODEHOHG QHXURQV EHWZHHQ DGMRLQLQJ KRVW DQG WUDQVSODQWHG WLVVXH )LJ Df ,Q DOO VHYHQ JUDIWV WKHUH ZHUH UHJLRQV RI IXVLRQ EHWZHHQ KRVW DQG JUDIW WLVVXHV DV LGHQWLILHG ZLWK *)$3 VWDLQLQJ VHH EHORZ )LJ Ef 6L[ RI WKH JUDIWV FRQWDLQHG UHWURJUDGHO\ ODEHOHG FHOOV 7KHVH LQFOXGHG WKUHH

PAGE 85

WUDQVSODQWV )*&f HDFK ZLWK IHZHU WKDQ UHWURJUDGHO\ ILOOHG QHXURQV ZLWKLQ WKH JUDIW DQG WKUHH RWKHUV )*&f HDFK ZLWK PRUH WKDQ ODEHOHG FHOOV ,Q WKLV VDPSOH WKH GLIIHUHQFHV EHWZHHQ WKH WZR JURXSV GLG QRW FRUUHVSRQG ZLWK WKH SRVWJUDIWLQJ LQWHUYDO RU WKH GLVWDQFH EHWZHHQ WKH LQMHFWLRQ VLWH DQG WKH KRVWJUDIW LQWHUIDFH +RZHYHU PRUH ODEHOHG FHOOV ZHUH IRXQG LQ WKH WKUHH UHFLSLHQWV ZLWK )* SODFHG FDXGDO WR WKH JUDIW WKDQ WKRVH ZLWK LQMHFWLRQV SODFHG DW URVWUDO OHYHOV 7UDQVSODQW QHXURQV ZKLFK SURMHFWHG LQWR WKH KRVW VSLQDO FRUG ZHUH GLVWULEXWHG WKURXJKRXW WKH JUDIW WLVVXH )LJ Hf 0RVW RI WKHVH UHWURJUDGHO\ ODEHOHG QHXURQV ZHUH PXOWLSRODU DQG VPDOO PHDVXULQJ >LP LQ GLDPHWHU )LJ Ff DOWKRXJK ODUJHU FHOOV ZHUH REVHUYHG RFFDVLRQDOO\ )LJ Gf +LVWRJUDPV ZHUH PDGH IRU HDFK RI WKHVH VL[ JUDIWV WR VKRZ WKH GLVWULEXWLRQ RI WKH ODEHOHG FHOOV DV D IXQFWLRQ RI GLVWDQFH IURP WKH KRVWJUDIW LQWHUIDFH )LJ f ,Q WKH UHFLSLHQWV ZLWK IHZ ODEHOHG FHOOV WRS RI )LJ f WKHUH ZHUH PRUH FHOOV ZLWKLQ WKH ILUVW PLOOLPHWHU RI WKH WUDQVSODQW DQG D GHFUHDVH LQ WKH GHQVLW\ RI ODEHOHG FHOOV ZLWK GLVWDQFH IURP WKH KRVWJUDIW LQWHUIDFH +RZHYHU ODEHOHG QHXURQV ZHUH PRUH HYHQO\ GLVWULEXWHG LQ JUDIWV FRQWDLQLQJ PDQ\ IOXRUHVFHQW FHOOV UHJDUGOHVV RI WKH DEVROXWH OHQJWK RI WKH WUDQVSODQWV

PAGE 86

)LJXUH ,GHQWLILFDWLRQ RI JUDIW QHXURQV SURMHFWLQJ LQWR WKH KRVW VSLQDO FRUG E\ UHWURJUDGH WUDQVSRUW RI )OXRUR*ROG Df 7KH LQWHUIDFH EHWZHHQ KRVW Kf DQG WUDQVSODQW Wf LV FKDUDFWHUL]HG E\ D PDUNHG GHFUHDVH LQ GHQVLW\ RI ODEHOHG QHXURQV Ef 6HFWLRQ DGMDFHQW WR WKDW VKRZQ LQ Df DIWHU VWDLQLQJ ZLWK DQWL*)$3 $ JOLDO VFDU LV SUHVHQW DORQJ WKH GRUVDO UHJLRQ RI WKLV KRVWJUDIW LQWHUIDFH ERWWRP KDOI RI ILJXUHf ZKLOH WKH KRVW DQG JUDIW WLVVXHV DUH ZHOO IXVHG LQ WKH YHQWUDO UHJLRQ EHWZHHQ DUURZKHDGVf Ff +LJKHU PDJQLILFDWLRQ RI W\SLFDO UHWURJUDGHO\ ODEHOHG FHOOV ZLWKLQ WKLV JUDIW Gf ([DPSOH RI DQ RFFDVLRQDO ODUJH JUDIW QHXURQ WKDW SURMHFWHG LQWR WKH KRVW VSLQDO FRUG Hf 3KRWRPRQWDJH RI D VDJLWWDO VHFWLRQ IURP VSHFLPHQ )*& WR LOOXVWUDWH WKH GLVWULEXWLRQ RI ODEHOHG QHXURQV WKURXJKRXW D WUDQVSODQW IROORZLQJ D )* LQMHFWLRQ LQWR WKH KRVW VSLQDO FRUG Kf 7KH KRVWJUDIW LQWHUIDFH LV PDUNHG E\ D ZKLWH GRWWHG OLQH 6FDOH LQ DEH P FG [P

PAGE 88

)LJXUH ,QGLYLGXDO KLVWRJUDPV VKRZ WKH GLVWULEXWLRQ RI ODEHOHG FHOOV ZLWKLQ VL[ WUDQVSODQWV ZLWK UHVSHFW WR WKH KRVWJUDIW LQWHUIDFH FORVHVW WR WKH LQMHFWLRQ VLWH 7KH FRUUHFWHG QHXURQ WRWDO LV LQ SDUHQWKHVHV DERYH HDFK JUDIW (DFK EDU UHSUHVHQWV WKH QXPEHU RI QHXURQV FRXQWHG ZLWKLQ VXFFHVVLYH PP VHJPHQWV RI WKH JUDIW 1RWH WKDW WKH ODVW EDU LQ HDFK JUDSK PD\ UHSUHVHQW OHVV WKDQ D IXOO PLOOLPHWHU RI JUDIW WLVVXH

PAGE 89

1XPEHUV RI /DEHOHG &HOOV ',675,%87,21 2) 5(752*5$'(/< /$%(/(' 75$163/$17 1(85216 )2//2:,1* )/8252*2/' ,1-(&7,216 ,172 7+( +267 63,1$/ &25' )*& f WR 92 r r )*& f )*& f )*& RWR X r }X! }DMR )*& f /2 'LVWDQFH IURP +RVW*UDIW ,QWHUIDFH PPf

PAGE 90

,QMHFWLRQV RI )* LQWR WUDQVSODQWV ,Q WHQ RI WKH UHFLSLHQWV ZLWK )* LQMHFWLRQV LQWR WKH JUDIWV WKH H[WHQW RI WUDFHU GLIIXVLRQ ZDV FRQILQHG WR WKH WUDQVSODQW :KLOH VHYHQ JUDIWV KDG VRPH FHOOV ODEHOHG LQ WKH KRVW VSLQDO FRUG WKH QXPEHUV RI ODEHOHG FHOOV UDQJHG IURP IHZ KRVW QHXURQV WR PRUH WKDQ KRVW VSLQDO FRUG FHOOV DQG GRUVDO URRW JDQJOLRQ '5*f QHXURQV )LJXUH VXPPDUL]HV WKH ORFDWLRQ DQG H[WHQW RI WKH JUDIW LQMHFWLRQV DQG WKH GLVWULEXWLRQ RI ODEHOHG KRVW QHXURQV )LYH RI WKHVH )* LQMHFWLRQV ZHUH PP LQ GLDPHWHU )*& D + + + + )LJ Df ,Q WKHVH VSHFLPHQV QHDUO\ DOO RI WKH ODEHOHG QHXURQV ZHUH IRXQG ZLWKLQ WKH JUDIW LWVHOI 7KH LQWULQVLF QHXURQV ZHUH FRQFHQWUDWHG LQ WKH UHJLRQ QHDUHVW WKH LQMHFWLRQ VLWH +RZHYHU VRPH UHWURJUDGHO\ ODEHOHG FHOOV ZHUH IRXQG LQ DOO UHJLRQV RI WKHVH WUDQVSODQWV )LJ Ef /DEHOHG QHXURQV ZHUH DOVR IRXQG LQ WKH DGMDFHQW KRVW VSLQDO FRUG LQ WZR RI WKHVH UHFLSLHQWV ,Q ERWK FDVHV WKH ODEHOHG FHOOV ZHUH ORFDWHG ZLWKLQ PP RI WKH LQWHUIDFH ]RQH ZKLFK ZDV DGMDFHQW WR WKH LQMHFWLRQ VLWH DQG GLVWULEXWHG ZLWKLQ WKH PHGLDO RU ODWHUDO LQWHUPHGLDWH JUD\ 7KH JUHDWHVW QXPEHU RI ODEHOHG KRVW QHXURQV ZHUH IRXQG LQ WKH UHFLSLHQW ZLWK DQ LQMHFWLRQ ORFDWHG ZLWKLQ PP RI WKH KRVWJUDIW LQWHUIDFH +f ,Q WKH UHPDLQLQJ WUDQVSODQWV IURP WKLV JURXS )*& +f WKH LQMHFWLRQ VLWH ZDV ODUJHU WKDQ PP LQ

PAGE 91

)LJXUH 'LVWULEXWLRQ RI LQMHFWLRQ VLWHV DQG ODEHOHG QHXURQV IROORZLQJ LQMHFWLRQV RI )OXRUR*ROG LQWR WUDQVSODQWV 7KH FHQWHU SDQHO FRQWDLQV IUHHKDQG GUDZLQJV RI D UHSUHVHQWDWLYH VHFWLRQ ZLWK LQGLYLGXDO GRWV UHSUHVHQWLQJ FHOOV DQG WKHLU DSSUR[LPDWH ORFDWLRQV ZLWKLQ WKH JUDIW 1XPEHUV RI FHOOV URVWUDO DQG FDXGDO WR WKH JUDIW DUH LQGLFDWHG DFFRUGLQJ WR QXPEHUV ZLWKLQ D UDQJH RI GLVWDQFHV IURP WKH LQWHUIDFH '5* GRUVDO URRW JDQJOLD WRWDOV /HIW DQG 5LJKW DOO JUDIWV ZHUH PDGH RQ WKH OHIWf

PAGE 92

$QLPDO 3RVWJ &HO ,V 5RVWUDO ,f 'LVWDQFH LQ WR 7UDQVSODQW PPf *OLDO VFDU ),;M )*&D PRf )*&+ PRf ; )*&+ PRf ; )*&+ PR f ; )*&+ PRf ; )*&0 PR f ; )*& PRf ; )*& PRf ; )*& PRf ; )*& B B ; ZNf *OLDO VFDU ),;f &HOOV &DXGDO 'LVWDQFH LQ WR 7UDQVSODQW PPf '5* / 5 ; ; ; ; ; ; ; ; ; B B B B MAQUL 1XPEHUV LQ HDFK FROXPQ UHSUHVHQW WKH QXPEHU RI FHOOV FRXQWHG LQ HDFK UHJLRQ 6FDOH FRUUHVSRQGV WR DSSUR[ PP )XVLRQ ,QGH[ ; RI OLQHDU KRVWJUDIW LQWHUIDFH ZKLFK LV GHYLRG RI JOLDO VFDU DYHUDJH RI VHFWLRQV SHU DQLPDOf

PAGE 93

GLDPHWHU EXW ZDV VWLOO FRQILQHG WR WKH JUDIW $OO RI WKHVH UHFLSLHQWV FRQWDLQHG VRPH UHWURJUDGHO\ ODEHOHG KRVW QHXURQV $JDLQ WKH JUHDWHVW QXPEHUV RI KRVW QHXURQV ZHUH IRXQG ZKHQ WKH LQMHFWLRQ VLWH ZDV ZLWKLQ PP RI HLWKHU WKH URVWUDO RU FDXGDO LQWHUIDFH ,Q DGGLWLRQ WKH PDMRULW\ RI ODEHOHG KRVW QHXURQV ZHUH IRXQG LPPHGLDWHO\ DGMDFHQW WR WKH WUDQVSODQWV ,Q WKH EHVW FDVH )*& )LJV FLf WKH )* LQMHFWLRQ H[WHQGHG WR PP IURP WKH FDXGDO LQWHUIDFH 5HWURJUDGHO\ ODEHOHG QHXURQV ZHUH IRXQG WKURXJKRXW KRVW VSLQDO FRUG FDXGDO WR WKH JUDIW ZKLOH IHZ QHXURQV ZHUH IRXQG URVWUDO WR WKH JUDIW :LWKLQ WKH VDFUDO VSLQDO FRUG )LJ K LH PP DZD\f ODEHOHG FHOOV ZHUH GLVWULEXWHG WKURXJKRXW WKH GRUVDO DQG LQWHUPHGLDWH JUD\ UHJLRQV ODPLQDH ,9,,f DV ZHOO DV UHJLRQV RI WKH YHQWUDO KRUQ ,Q DGGLWLRQ PRUH FHOOV ZHUH ORFDWHG LSVLODWHUDO WKDQ FRQWUDODWHUDO WR WKH JUDIW ,Q WKLV DQLPDO D ODUJH QXPEHU RI LSVLODWHUDO '5* FHOOV ZHUH IRXQG DV ZHOO )LJ Lf +RZHYHU VHFWLRQV IURP FHUYLFDO DQG WKRUDFLF VSLQDO FRUG DV ZHOO DV EUDLQVWHP DQG EUDLQ FRQWDLQHG QR ODEHOHG FHOOV LQ DQ\ RI WKH UHFLSLHQWV 7KH DEVHQFH RI UHWURJUDGH ODEHOLQJ RI GHVFHQGLQJ ILEHUV ZDV LQ FRQWUDVW ZLWK WKH SDWWHUQ REVHUYHG IROORZLQJ D VLPLODU LQMHFWLRQ LQWR D QRUPDO UDW &RPSDULVRQ RI )* DQG +53:*$+53 LQMHFWLRQV LQ QRUPDO UDWV 7R FRPSDUH WKH SDWWHUQV RI UHWURJUDGH FHOO ODEHOLQJ XVLQJ WKH WZR WUDFHUV DQG WKH SUHVHQW LQMHFWLRQ WHFKQLJXHV HDFK RI WKHVH WUDFHUV ZDV DOVR LQMHFWHG LQWR D QRUPDO UDW DW WKH 7

PAGE 94

)LJXUH 5HWURJUDGHO\ ODEHOHG KRVW QHXURQV IROORZLQJ )* LQMHFWLRQV LQWR WUDQVSODQWV DEf ,QWULQVLF ODEHOHG QHXURQV IROORZLQJ D VPDOO )* LQMHFWLRQ PP GLDPHWHUf LQWR D WUDQVSODQW Wf Df 7KH PDMRULW\ RI ODEHOHG QHXURQV ZHUH ORFDWHG LQ WKH LPPHGLDWH YLFLQLW\ RI WKH LQMHFWLRQ ZKLFK ZDV DGMDFHQW WR WKH URVWUDO KRVWJUDIW LQWHUIDFH DUURZKHDGVf &HOOV ZHUH DOVR IRXQG DW WKH IDU HQG RI WKH JUDIW Ef DW D GLVWDQFH RI PP IURP WKH LQMHFWLRQ FLf 'LVWULEXWLRQ RI UHWURJUDGHO\ ODEHOHG FHOOV IROORZLQJ D ODUJHU LQMHFWLRQ GRWWHG OLQH LQ Ff QHDU WKH FDXGDO ERUGHU DUURZKHDGVf RI VSHFLPHQ )*& Gf 0RVW IOXRUHVFHQW ODEHOHG FHOOV ZKLWH DUURZVf ZHUH IRXQG LPPHGLDWHO\ DGMDFHQW WR WKH KRVWJUDIW LQWHUIDFH ,QVHW 9HULILFDWLRQ RI WKH JUDIW ERUGHU ZDV REWDLQHG LQ HDFK FDVH E\ YLHZLQJ WKH LQWHUIDFH UHJLRQ ZLWK GDUNILHOG RSWLFV XVLQJ WKH ORFDWLRQ RI EORRG YHVVHOV rf DV ODQGPDUN SRLQWV Hf *)$3 VWDLQLQJ RI RQH VHFWLRQ IURP WKLV VSHFLPHQ LOOXVWUDWHV D KLJK GHJUHH RI IXVLRQ EHWZHHQ KRVW DQG JUDIW EHWZHHQ DUURZKHDGVf If $ SDWFK RI UHWURJUDGHO\ ODEHOHG QHXURQV IRXQG DSSUR[LPDWHO\ PP FDXGDO WR WKH WUDQVSODQW Jf 7UDQVYHUVH VHFWLRQ RI WKH VDFUDO VSLQDO FRUG FRQWDLQV IRXU UHWURJUDGHO\ ODEHOHG QHXURQV Kf &RPSRVLWH GUDZLQJ RI VHFWLRQV IURP WKH KRVW VDFUDO VSLQDO FRUG PP FDXGDO WR WKH WUDQVSODQW /HIW LQ WKH ILJXUH LV LSVLODWHUDO WR WKH JUDIW ULJKW LV FRQWUDODWHUDO 7KH SKRWRJUDSK LQ J ZDV REWDLQHG IURP WKH UHJLRQ HQFORVHG LQ WKH ER[ Lf /DEHOHG GRUVDO URRW JDQJOLRQ QHXURQV LSVLODWHUDO WR WKH WUDQVSODQW 6FDOH LQ DFGH XUQ EIJL P

PAGE 96

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f 7KH LQMHFWLRQ UHJLRQ ZDV HDVLO\ GHILQHG E\ WKH SUHVHQFH RI GDUNO\ ILOOHG SHULNDU\D ,Q VRPH VSHFLPHQV WKH LQMHFWLRQ VLWH FRQWDLQHG RQO\ D VPDOO QXPEHU RI ODEHOHG QHXURQV )LJ DEf /DUJHU LQMHFWLRQ VLWHV H[WHQGHG XS WR PP LQ GLDPHWHU 3URMHFWLRQV RI WUDQVSODQW QHXURQV 2I WKH UHFLSLHQWV ZLWK 3+$/ LQMHFWLRQV LQWR WKH WUDQVSODQWV ILYH KDG DFFHSWDEOH D[RQDO ODEHOLQJ 7DEOH WRSf $OO RI WKHVH LQMHFWLRQV UHYHDOHG DQ H[WHQVLYH QHWZRUN RI D[RQDO SURMHFWLRQV ZLWKLQ WKH JUDIW $[RQV QHDU WKH LQMHFWLRQ VLWH H[KLELWHG DEXQGDQW EUDQFKLQJ DQG WKH QHDUE\ QHXURQV ZHUH VXUURXQGHG E\ WHUPLQDO HQODUJHPHQWV 6XUYH\ HOHFWURQ PLFURJUDSKV RI RQH VXFK JUDIW VKRZHG ODEHOHG WHUPLQDO ERXWRQV WKURXJKRXW WKH JUDIW QHXURSLO -DNHPDQ DQG 5HLHU XQSXEOLVKHG REVHUYDWLRQVf :KLOH WKH JUHDWHVW GHQVLW\

PAGE 97

7$%/( ,1-(&7,216 2) 3+$/ ,172 )6& 75$163/$176 25 7+( +267 63,1$/ &25' &RGH ,6D 5HJLRQ $[RQDO 3URMHFWLRQV 3RVW*UDIWf ,QWHUYDOf 3+$/ PRf*UDIW 9HQWUDO (IIHUHQW D[RQV FDQ EH WUDFHG DFURVV LQWHUIDFH ,QMXU\ ILOOHG KRVW D[RQV DOVR HYLGHQW 3+$/ ZNf*UDIW GRUVDO PHGLDO (IIHUHQW D[RQV LQ URVWUDO GRUVDO KRUQ DQG LQWHUPHGLDWH JUD\ UHJLRQV RI KRVW 3+$/ PRf *UDIW GRUVDO (IIHUHQW D[RQV SUHVHQW LQ PHGLDO GRUVDO KRUQ GRUVDO WUDFWV DQG LQWHUPHGLDWH JUD\ DQG LQ URVWUDO LQWHUPHGLDWH JUD\ UHJLRQV 3+$/ PRf *UDIW GRUVDO ODWHUDO (IIHUHQW D[RQV LQQHUYDWH KRVW ODWHUDO PRWRQHXURQV 3+$/O2ZNf *UDIW FDXGDO 1R (IIHUHQW D[RQV 3+$/ ZNf +RVW PP FDXGDO $IIHUHQW D[RQV H[WHQGLQJ PP LQWR JUDIW 3+$/ PRf +RVW PP FDXGDO $IIHUHQW D[RQV H[WHQGLQJ PP LQWR JUDIW 3+$/ZNf +RVW PP URVWUDO $OO D[RQV VWRS LQWHUIDFH D W 3+$/PRf +RVW PP FDXGDO 0DQ\ FHOOV DQG WKURXJKRXW JUDIWr PRVW PP RI LQWHUIDFH D[RQV ZLWKLQ 3+$/ PRf +RVW PP 1R DIIHUHQW D[RQV D ,QMHFWLRQ VLWH ,RQWRSKRUHWLF LQMHFWLRQ LQWR JUDIW RU KRVW VSLQDO FRUG E 5HJLRQ RI JUDIW ZLWK LQMHFWLRQ RU GLVWDQFH DQG GLUHFWLRQ EHWZHHQ LQMHFWLRQ LQ KRVW VSLQDO FRUG DQG WKH KRVWJUDIW LQWHUIDFH r (YLGHQFH RI ERWK DQWHURJUDGH DQG UHWURJUDGH WUDQVSRUW

PAGE 98

RI ILEHUV ZDV IRXQG ZLWKLQ WKH LQMHFWLRQ UHJLRQ ODEHOHG D[RQV ZHUH IRXQG LQ DOO DUHDV RI WKH JUDIWV 7KUHH JHQHUDO D[RQDO SURMHFWLRQ SDWWHUQV ZHUH VHHQ ZLWKLQ WKH WUDQVSODQWV :LWKLQ JUDIW UHJLRQV FRQWDLQLQJ GHQVHO\ SDFNHG QHXURQDO FHOO ERGLHV WKH ODEHOHG D[RQV EUDQFKHG H[WHQVLYHO\ )LJV EFf ,Q FRQWUDVW ZKHUH IHZ SHULNDU\D ZHUH IRXQG ODEHOHG D[RQV UHPDLQHG PRVWO\ XQEUDQFKHG DQG IROORZHG D UHODWLYHO\ VWUDLJKW WUDMHFWRU\ )LJ Ef )LQDOO\ DW WKH LQWHUIDFH UHJLRQV LQGLYLGXDO D[RQV RIWHQ FRXUVHG SDUDOOHO WR WKH KRVWJUDIW ERUGHU DQG RFFDVLRQDOO\ H[WHQGHG LQWR WKH KRVW QHXURSLO )LJ Gf /DEHOHG D[RQV FRXOG EH IROORZHG LQWR WKH KRVW VSLQDO FRUG LQ IRXU RI WKHVH JUDIWV 7KH SDWWHUQ RI D[RQDO RXWJURZWK ZDV VOLJKWO\ GLIIHUHQW IRU HDFK VSHFLPHQ ,Q RQH FDVH 3+$/f WKH LQMHFWLRQ ZDV SODFHG YHQWURPHGLDOO\ ZLWKLQ WKH WUDQVSODQW DQG UHVXOWHG LQ LQMXU\ WR ILEHUV LQ WKH YHQWUDO ZKLWH PDWWHU RI WKH KRVW 7KH DSSHDUDQFH RI WKHVH LQMXU\ILOOHG D[RQV ZDV GLVWLQFW 7KHVH D[RQV ZHUH YHU\ KHDYLO\ ODEHOHG DQG WKH\ H[KLELWHG EXOERXV WHUPLQDO VZHOOLQJV 6LPLODU SURILOHV ZHUH QRW IRXQG LQ DQ\ RI WKH RWKHU DQLPDOV LQ WKLV JURXS )ROORZLQJ LQMHFWLRQV LQWR WZR RWKHU JUDIWV 3+$/ f D KLJK GHQVLW\ RI ODEHOHG D[RQV ZHUH VHHQ ZLWKLQ WKH WUDQVSODQWV HVSHFLDOO\ QHDU WKH GRUVDO UHJLRQ RI WKH KRVW JUDIW LQWHUIDFH /DEHOHG ILEHUV H[WHQGHG DFURVV WKH LQWHUIDFH DQG LQWR WKH DGMDFHQW URVWUDO RU FDXGDO GRUVDO KRUQ ODPLQDH ,,,,f RI WKH KRVW )LJ Df

PAGE 99

)LJXUH ,QWULQVLF 3+$/ ODEHOHG SURILOHV IROORZLQJ D VPDOO LQMHFWLRQ LQWR WUDQVSODQW 3+$/ Df 6DJLWWDO VHFWLRQ SURYLGLQJ RULHQWDWLRQ 7KH LQMHFWLRQ ZDV SODFHG LQ WKH YHQWURPHGLDO UHJLRQ RI WKH WUDQVSODQW Wf GRXEOH DUURZKHDGVf Ef (QODUJHPHQW IURP D QHDUE\ VHFWLRQ RI WKH VDPH WUDQVSODQW /DEHOHG D[RQV ZLWKLQ DFHOOXODU UHJLRQV H[KLELW OLWWOH EUDQFKLQJ DUURZVf ZKLOH H[WHQVLYH EUDQFKLQJ LV REVHUYHG DURXQG FRXQWHUVWDLQHG FHOO ERGLHV DUURZKHDGVf Ff :LWKLQ WKH WUDQVSODQW EXW IDUWKHU IURP WKH LQMHFWLRQ VLWH ODEHOHG D[RQV H[KLELW DQ H[WHQVLYH SDWWHUQ RI SURMHFWLRQV Gf (QODUJHPHQW RI WKH LQWHUIDFH UHJLRQ RXWOLQHG LQ WKH ER[ LQ Df /DEHOHG D[RQV DUURZKHDGVf ZLWKLQ WKH WUDQVSODQW WUDYHO SDUDOOHO WR WKH KRVW JUDIW LQWHUIDFH 2QH D[RQ WKHQ WXUQV DEUXSWO\ WR HQWHU WKH DGMDFHQW KRVW VSLQDO FRUG Kf 6FDOH LQ D P EFG P

PAGE 100

Y r rZ I b

PAGE 101

)LJXUH ([DPSOHV RI HIIHUHQW SURMHFWLRQV LQWR WKH KRVW VSLQDO FRUG IROORZLQJ 3+$/ LQMHFWLRQV LQWR WUDQVSODQWV Df ,QWHUIDFH EHWZHHQ WUDQVSODQW Wf 3+$/ DQG WKH GRUVDO KRUQ RI WKH KRVW VSLQDO FRUG /DEHOHG D[RQV FURVV WKH LQWHUIDFH DQG H[WHQG ORQJLWXGLQDOO\ ZLWKLQ WKH URVWUDO GRUVDO KRUQ K'+f Ef /DEHOHG D[RQV DSSUR[LPDWHO\ PP FDXGDO WR D WUDQVSODQW ZLWK D 3+$/ LQMHFWLRQ 2FFDVLRQDO D[RQ FROODWHUDOV H[WHQGHG LQWR WKH GHHSHU OD\HUV RI WKH GRUVDO KRUQ DUURZKHDGVf Ff ([DPSOH RI DQ D[RQ WKDW H[WHQGHG IURP D WUDQVSODQW Wf LQWR WKH KRVW LQWHUPHGLDWH JUD\ UHJLRQ K*f DQG EUDQFKHG DURXQG WKH KRVW QHXURQV Gf /DEHOHG D[RQV LQ VSHFLPHQ 3+$/ SURMHFW RXW RI WKH WUDQVSODQW DQG LQWR WKH QHDUE\ PRWRQHXURQ SRRO FDXGDO WR WKH JUDIW 1XPHURXV ERXWRQn OLNH SURILOHV DUH SUHVHQW ZLWKLQ WKH VXUURXQGLQJ WLVVXH DUURZKHDGVf 6FDOH ]P LQ DOO ILJXUHV

PAGE 103

:LWKLQ WKH KRVW WKH HIIHUHQW D[RQV ZHUH IRXQG LQ ERWK P\HOLQDWHG DQG XQP\HOLQDWHG UHJLRQV 7KHVH ODEHOHG ILEHUV FRQWLQXHG DORQJ D SULPDULO\ ORQJLWXGLQDO WUDMHFWRU\ DQG VRPH FRXOG EH IROORZHG DV IDU DV PP IURP WKH LQWHUIDFH &ROODWHUDOV IURP ZHUH DOVR REVHUYHG H[WHQGLQJ LQWR GHHSHU UHJLRQV RI WKH KRVW JUD\ PDWWHU )LJ Ef ,Q DGGLWLRQ VRPH D[RQDO RXWJURZWK RFFXUUHG DFURVV WKH LQWHUIDFH ZKHUH WKH JUDIW WLVVXH ZDV DSSRVHG WR WKH LQWHUPHGLDWH JUD\ UHJLRQV RI WKH KRVW )LJ Ff 6RPH RI WKHVH D[RQV FRXOG EH IROORZHG IRU VHYHUDO KXQGUHG PLFURQV EHIRUH WHUPLQDWLQJ LQ WKH KRVW YHQWUDO KRUQ 7KH WZR UHPDLQLQJ UHFLSLHQWV IURP WKLV JURXS KDG ORQJHU PPf WUDQVSODQWV ,Q RQH VSHFLPHQ 3+$/f D ODUJH LQMHFWLRQ ZDV PDGH QHDU WKH FDXGDO HQG RI WKH JUDIW ZKHUH WKH WUDQVSODQW ZDV ZHOO DSSRVHG WR KRVW PRWRQHXURQV 1XPHURXV ODEHOHG D[RQV FRXOG EH IROORZHG DFURVV WKH FDXGDO JUDIWKRVW ERUGHU DQG LQWR WKH PRWRQHXURQ SRROV DGMDFHQW WR WKH JUDIW )LJ Gf :KLOH ODEHOHG ILEHUV ZHUH DOVR IRXQG DW WKH URVWUDO SROH RI WKLV JUDIW QR D[RQV H[WHQGHG LQWR WKH KRVW VSLQDO FRUG DFURVV WKH URVWUDO LQWHUIDFH 7KH RWKHU VSHFLPHQ IURP WKLV JURXS 3+$/f KDG D VPDOO LQMHFWLRQ LQ WKH YHQWUDO UHJLRQ RI WKH JUDIW %RWK URVWUDO DQG FDXGDO LQWHUIDFHV RI WKLV JUDIW ZHUH SRRUO\ DSSRVHG WR WKH KRVW QHXURSLO DQG QR ODEHOHG D[RQV H[WHQGHG LQWR WKH UHFLSLHQW VSLQDO FRUG

PAGE 104

3URMHFWLRQV RI ORFDO KRVW QHXURQV 6XFFHVVIXO LRQWRSKRUHWLF LQMHFWLRQV RI 3+$/ ZHUH PDGH LQWR WKH KRVW VSLQDO FRUG DW PP IURP WKH LQWHUIDFH UHJLRQ LQ ILYH UDWV ,Q HDFK FDVH WKH KRVW WLVVXHV FRQWDLQHG D GHQVH ILEHU SOH[XV WKURXJKRXW WKH JUD\ PDWWHU )LJV DFf DQG ORQJ D[RQDO SURMHFWLRQV ZHUH REVHUYHG ZLWKLQ WKH GRUVDO DQG YHQWUDO P\HOLQDWHG WUDFWV 7KH SUHVHQFH RI LQMXUHG D[RQV DQG UHVXOWLQJ LQMXU\ILOO ODEHOLQJ SDWWHUQV ZHUH FRPPRQ IHDWXUHV DPRQJ WKHVH UHFLSLHQWV (YLGHQFH RI KRVW DIIHUHQW LQJURZWK ZDV REWDLQHG LQ IRXU VSHFLPHQV 7KH SDWWHUQ RI KRVW ILEHU LQJURZWK ZDV VLPLODU IRU RI WKH 3+$/DQG )LJV DEf $ KLJK GHQVLW\ RI ODEHOHG D[RQV ZDV VHHQ XS WR WKH KRVWJUDIW LQWHUIDFH )URP WKLV SRLQW D IHZ ODEHOHG ILEHUV H[WHQGHG LQWR WKH WUDQVSODQW 7KH ODEHOHG D[RQV ZLWKLQ WKHVH JUDIWV WHUPLQDWHG VKRUWO\ DIWHU HQWHULQJ WKH JUDIW DQG QR ILEHUV ZHUH IRXQG DW GLVWDQFHV JUHDWHU WKDQ PP DZD\ IURP WKH LQWHUIDFH $W WKH HQGV RI VRPH RI WKHVH D[RQV ERXWRQOLNH SURILOHV ZHUH REVHUYHG LQ GLUHFW DSSRVLWLRQ WR &UHV\O 9LROHW VWDLQHG FHOOV ,Q WKH IRXUWK VSHFLPHQ 3+$/f D ODUJH LQMHFWLRQ ZDV PDGH LQ WKH KRVW VSLQDO FRUG PP IURP WKH KRVWJUDIW LQWHUIDFH 7KH SUHVHQFH RI VHYHUDO OLJKWO\ ILOOHG QHXURQDO SHULNDU\D IDU IURP WKH LQMHFWLRQ VLWH VXJJHVWHG WKDW VXEVWDQWLDO UHWURJUDGH WUDQVSRUW KDG RFFXUUHG 6KX DQG 3HWHUVRQ nf %RWK ODEHOHG D[RQV DQG FHOOV ZHUH SUHVHQW

PAGE 105

)LJXUH 3+$/ ODEHOHG SURILOHV IROORZLQJ LQMHFWLRQV LQWR WKH KRVW VSLQDO FRUG Df 6KRUWGLVWDQFH LQJURZWK RI KRVW ILEHUV DFURVV WKH LQWHUIDFH GDVKHG OLQHf DQG LQWR )6& WUDQVSODQWV LQ VSHFLPHQ 3+$/ Ef +LJKHU SRZHU PLFURJUDSK RI D[RQDO LQJURZWK LQ DQRWKHU UDW 1RWH WKH D[RQDO EUDQFKLQJ DQG D[RQDO VZHOOLQJV VXUURXQGLQJ WKH WUDQVSODQW QHXURQV Ff ,Q VSHFLPHQ 3+$/ QR D[RQV FURVVHG WKH LQWHUIDFH IROORZLQJ D ODUJH 3+$/ LQMHFWLRQ PP IURP WKH KRVWJUDIW LQWHUIDFH Gf )ROORZLQJ D VLPLODU LQMHFWLRQ LQ VSHFLPHQ 3+$/ ODUJH QXPEHUV RI DQWHURJUDGHO\ ILOOHG D[RQV DQG UHWURJUDGHO\ ILOOHG FHOOV ZLWKLQ WKH JUDIW VXJJHVWHG D JUHDW GHDO RI LQWHUDFWLRQ EHWZHHQ WKH WZR WLVVXHV 1RWLFH WKH D[RQDO SURILOHV FURVVLQJ EHWZHHQ WKH WZR WLVVXHV LQ D UHJLRQ RI IXVLRQ EHWZHHQ KRVW DQG JUDIW DUURZVf 6FDOH LQ DFG LP E ]P

PAGE 107

ZLWKLQ WKLV JUDIW DQG ODEHOHG D[RQV ZHUH REVHUYHG FRXUVLQJ ZLWKLQ UHJLRQV RI IXVLRQ EHWZHHQ WKH KRVW DQG JUDIW WLVVXHV )LJ Gf :KLOH WKH JUHDWHVW FRQFHQWUDWLRQ RI WKHVH SURILOHV ZDV IRXQG ZLWKLQ PP RI WKH KRVWJUDIW LQWHUIDFH D VPDOO QXPEHU RI FHOOV DQG D[RQV ZHUH IRXQG LQ RWKHU UHJLRQV RI WKH WUDQVSODQW DV ZHOO $W WKH RWKHU H[WUHPH ZDV VSHFLPHQ 3+$/ ZKLFK GLG QRW FRQWDLQ DQ\ ODEHOHG DIIHUHQW SURMHFWLRQV IURP ORFDO KRVW QHXURQV )ROORZLQJ DQ LQMHFWLRQ SODFHG PP URVWUDO WR WKLV JUDIW DOO ODEHOHG ILEHUV VWRSSHG DEUXSWO\ DW WKH KRVWJUDIW LQWHUIDFH DQG VRPH HQGHG LQ ODUJH UHWUDFWLRQ WHUPLQDOV )LJ Ff /RFDO $[RQDO 3URMHFWLRQV DQG *OLDO 5HDFWLYLW\ )XVLRQ RI KRVW DQG JUDIW WLVVXHV 7KH SHUFHQWDJH RI WKH KRVWJUDIW LQWHUIDFH ZKLFK ZDV GHYRLG RI JOLDO VFDU IRUPDWLRQ VHH 0DWHULDOV DQG 0HWKRGVf ZDV KLJKO\ YDULDEOH DFURVV VHFWLRQV LQ HDFK DQLPDO FI )LJV EHf ,Q WKH PRVW GUDPDWLF H[DPSOH RI WKLV YDULDELOLW\ WKH SHUFHQWDJH RI IXVLRQ DW WKH LQWHUIDFH LQ RQH DQLPDO UDQJHG IURP b IRU D VHFWLRQ IURP WKH PHGLDO ERUGHU RI WKH WUDQVSODQW WR b ZLWKLQ D VHFWLRQ IURP ZKHUH WKH JUDIW ZDV DSSRVHG WR WKH KRVW GRUVDO DQG YHQWUDO JUD\ PDWWHU :KHQ WKH YDOXHV IURP VHFWLRQV RI HDFK )* UHFLSLHQW ZHUH DYHUDJHG D FRPSRVLWH )XVLRQ ,QGH[ ),f ZDV REWDLQHG IRU WKH URVWUDO DQG FDXGDO LQWHUIDFH UHJLRQ RI HDFK VSHFLPHQ 7KHVH YDOXHV UDQJHG IURP b WR b 7KH DYHUDJHV YDOXHV GLG QRW GLIIHU E\ PRUH WKDQ b IURP WKH YDOXHV REWDLQHG E\ GLYLGLQJ

PAGE 108

WKH WRWDO OHQJWK RI KRVWJUDIW IXVLRQ E\ WKH WRWDO LQWHUIDFH OHQJWKf ,Q WKRVH DQLPDOV ZLWK DFFHSWDEOH )* LQMHFWLRQV LQWR WKH KRVW VSLQDO FRUG Q f QR FRUUHODWLRQ ZDV IRXQG EHWZHHQ WKH FRPSRVLWH ), DQG WKH QXPEHUV RI UHWURJUDGHO\ ODEHOHG FHOOV LQ WKH WUDQVSODQWV 7DEOH f +RZHYHU DOO RI WKH JUDIWV FRQWDLQHG VRPH UHJLRQV RI IXVLRQ DORQJ WKH DSSURSULDWH LQWHUIDFH HJ )LJ Ef ,Q DGGLWLRQ WKH UHFLSLHQW ZLWK WKH IHZHVW QXPEHU RI ODEHOHG FHOOV KDG RQO\ b IXVLRQ RI WKH URVWUDO LQWHUIDFH 6WDLQLQJ ZLWK DQWLVHUD WR *)$3 ZDV DOVR FRPSOHWHG RQ RI UHFLSLHQWV ZLWK )* LQMHFWLRQV LQWR WKH JUDIWV 7KH FRPSRVLWH ), IRU WKH URVWUDO DQG FDXGDO LQWHUIDFHV RI HDFK UHFLSLHQW LV LQFOXGHG LQ )LJXUH $JDLQ HDFK RI WKHVH UHFLSLHQWV VKRZHG VRPH UHJLRQV RI IXVLRQ EHWZHHQ KRVW DQG JUDIW WLVVXHV )LJ Hf 7KH UHFLSLHQWV ZLWK WKH JUHDWHVW QXPEHU RI ODEHOHG KRVW QHXURQV KDG FRPSRVLWH ), PHDVXUHPHQWV RI b +RZHYHU QRW DOO RI WKH YDULDWLRQV LQ KRVW QHXURQ LQJURZWK FRXOG EH DFFRXQWHG IRU E\ WKH DPRXQW RI IXVLRQ DW WKH LQWHUIDFH %RWK WKH WRWDO QXPEHUV RI )* ODEHOHG FHOOV DQG WKH FRPSRVLWH ), UHIOHFW FKDUDFWHULVWLFV RI KRVWJUDIW LQWHJUDWLRQ DFURVV DQ HQWLUH LQWHUIDFH ,Q FRQWUDVW VWDLQLQJ RI DGMDFHQW VHFWLRQV ZLWK DQWLERGLHV WR *)$3 DQG 3+$/ DOORZHG D FRPSDULVRQ RI UHJLRQDO DVSHFWV RI VXFK LQWHUDFWLRQV 8VLQJ WKLV DSSURDFK D FRUUHVSRQGHQFH ZDV

PAGE 109

REVHUYHG EHWZHHQ ORFDOL]HG UHJLRQV RI WLVVXH IXVLRQ DQG WKH VLWH RI D[RQDO SURMHFWLRQV IURP WUDQVSODQW QHXURQV LQWR WKH VXUURXQGLQJ KRVW VSLQDO FRUG )LJ f ,Q FRQWUDVW IHZ D[RQV SHQHWUDWHG D JOLDO LQWHUIDFH EHWZHHQ KRVW DQG JUDIW WLVVXH RU ZLWKLQ WKH WUDQVSODQWV *OLDO UHDFWLYLW\ LQ DGMDFHQW KRVW DQG JUDIW WLVVXHV ,Q DGGLWLRQ WR WKH IRUPDWLRQ RI D JOLDO VFDU DW WKH LQWHUIDFH YDU\LQJ DPRXQWV RI DVWURF\WLF K\SHUWURSK\ ZHUH REVHUYHG ZLWKLQ WKH WUDQVSODQWV DQG WKH VXUURXQGLQJ KRVW WLVVXH 7KH DPRXQW RI JOLDO UHDFWLYLW\ ZDV H[DPLQHG LQ WKH UHFLSLHQWV ZLWK )* LQMHFWLRQV LQWR WKH KRVW VSLQDO FRUG 7DEOH f 7KHUH ZDV D ZLGH UDQJH LQ WKH GHQVLW\ RI JOLDO VWDLQLQJ ZLWKLQ HDFK RI WKH JUDIWV DV ZHOO DV VRPH YDULDELOLW\ LQ WKH VXUURXQGLQJ KRVW JUD\ PDWWHU ,Q RUGHU WR FRQWURO IRU YDULDWLRQV LQ VWDLQLQJ WKH UDWLR RI JUDIWKRVW JOLDO VWDLQLQJ ZDV GHWHUPLQHG ZLWKLQ VHFWLRQV $V GHVFULEHG LQ PHWKRGV WKH GHQVLW\ UDWLR ZDV GHWHUPLQHG IRU HDFK VHFWLRQ DV D SHUFHQWDJH RI ILHOG GHQVLWLHV LQ SDLUV REWDLQHG IURP ZLWKLQ WKH JUDIW DQG LQ WKH KRVW VSLQDO FRUG LQFOXGLQJ PP HLWKHU URVWUDO RU FDXGDO WR WKH WUDQVSODQWf 7KH DYHUDJHG UDWLR RI JUDIWKRVW JOLDO GHQVLW\ IRU WKHVH DQLPDOV UDQJHG IURP WR ,Q IRXU UHFLSLHQWV WKH JOLDO UHDFWLYLW\ LQ WKH JUDIW ZDV VLJQLILFDQWO\ KLJKHU WKDQ WKDW RI WKH VXUURXQGLQJ KRVW WLVVXH +RZHYHU WKHUH ZDV QR UHODWLRQVKLS EHWZHHQ WKH YDOXHV REWDLQHG IRU WKH JUDIWKRVW

PAGE 110

)LJXUH &RUUHVSRQGHQFH EHWZHHQ D[RQDO SURMHFWLRQV DQG JOLDO VFDU IRUPDWLRQ DW WKH KRVWJUDIW LQWHUIDFH 7KH OHIW VLGH RI WKH ILJXUH FRQWDLQV GDUNILHOG PLFURJUDSKV RI 3+$/ VWDLQHG ILEHUV IROORZLQJ DQ LQMHFWLRQ LQWR WKH GRUVDO JXDGUDQW RI WUDQVSODQW 3+$/ Wf $GMDFHQW VHFWLRQV VWDLQHG ZLWK *)$3 DUH RQ WKH ULJKW DEf $ GHQVH VFDU VHSDUDWHV KRVW IURP JUDIW LQ WKH PRVW ODWHUDO UHJLRQV DQG IHZ ILEHUV FURVV WKH LQWHUIDFH LQWR WKH KRVW WLVVXH LQ WKLV UHJLRQ 1RWH WKDW YHU\ IHZ ODEHOHG D[RQV FURVV D VPDOOHU JOLDO SDUWLWLRQ ZLWKLQ WKH WUDQVSODQW LWVHOI rf f FGf ,Q WKLV SDLU ILEHUV H[WHQG IURP WKH JUDIW LQWR WKH KRVW GRUVDOO\ FRUUHVSRQGLQJ WR D UHJLRQ RI IXVLRQ EHWZHHQ KRVW DQG JUDIW WLVVXH DUURZKHDGVf 1RWH WKDW IHZ D[RQV FURVV PRUH YHQWUDOO\ ZKHUH D GHQVH JOLDO VFDU LV SUHVHQW HIf ,Q WKH PRVW PHGLDO UHJLRQ RI WKH JUDIW ILEHUV H[WHQGHG LQWR WKH KRVW VSLQDO FRUG DFURVV WKH RQO\ SDUW RI WKH LQWHUIDFH ZKHUH D EUHDN LV IRXQG LQ WKH FRUUHVSRQGLQJ JOLDO VFDU VWDLQ 6FDOH LQ DI P

PAGE 112

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f LPPXQRUHDFWLYLWY )LEHUV H[KLELWLQJ +7 LPPXQRUHDFWLYLW\ ZHUH HYLGHQW WKURXJKRXW WKH JUD\ PDWWHU RI WKH KRVW URVWUDO WR WKH WUDQVSODQW ZKHUH WKH VWDLQLQJ SDWWHUQ ZDV LGHQWLFDO WR WKDW GHVFULEHG LQ QRUPDO UDWV 6WHLQEXVFK n %XOOLWW DQG /LJKW nf 7KHVH ILEHUV ZHUH IRXQG SUHGRPLQDQWO\ LQ IRXU UHJLRQV LQFOXGLQJ SDUWV RI WKH VXSHUILFLDO GRUVDO KRUQ WKH UHJLRQ VXUURXQGLQJ WKH FHQWUDO FDQDO ODPLQD ;f WKH LQWHUPHGLRODWHUDO FHOO FROXPQ RI WKH WKRUDFLF DQG XSSHU OXPEDU VSLQDO FRUG DQG DURXQG WKH PRWRQHXURQV LQ WKH YHQWUDO KRUQ ,Q FRQWUDVW YHU\ IHZ VWDLQHG ILEHUV ZHUH IRXQG FDXGDO WR WKH WUDQVSODQWV *UDIW WLVVXH ZDV DSSRVHG WR LQMXUHG KRVW +7 ILEHUV ZLWKLQ ERWK GRUVDO DQG YHQWUDO KDOYHV RI WKH KRVW VSLQDO FRUG 7KH YDVW PDMRULW\ RI KRVW +7 ILEHUV VWRSSHG DW WKH HGJH RI WKH JUDIWV )LJ DFf \HW PRVW RI WKHVH D[RQV PDLQWDLQHG D QRUPDO DSSHDUDQFH LQ WHUPV RI ILEHU WKLFNQHVV

PAGE 113

7$%/( ,00812&<72&+(0,&$//< 67$,1(' 6(&7,216 3RVWJUDIW ,QWHUYDO +7 7+ 2; &*53 P Z r P r r P r r r P r r 6 P 6 P 6 P 6 P 6 P r 6OO P P r 6 P 6 P 6 P 6 P 6 P 6 P r P r P r P r P r P r b JUDIWV ZD[RQDO LQJURZWK b b b b FUUDIWV ZODEHOHG FHOOV b b ORb b KRVW ILEHUV H[WHQGHG LQWR WKH WUDQVSODQW r ODEHOHG FHOOV IRXQG ZLWKLQ WKH WUDQVSODQW QR ODEHOHG KRVW ILEHUV RU FHOOV ZLWKLQ WKH WUDQVSODQW XQVWDLQHG RU SRRUO\ VWDLQHG VHULHV D 6SHFLPHQV ZLWK ODEHOHG FHOOV LQ JUDIW ZHUH QRW LQFOXGHG FDOFXODWLQJ b ,QJURZWK RI +7 FRQWDLQLQJ ILEHUV ZDV REVHUYHG LQ RI JUDIWV 7KH H[WHQW RI LQQHUYDWLRQ ZDV VLPLODU WR WKDW GHVFULEHG LQ HDUOLHU VWXGLHV 5HLHU HW DO n nDf 7KH

PAGE 114

PDMRULW\ RI WKHVH ILEHUV HQWHUHG IURP HLWKHU WKH GRUVDO RU YHQWUDO UHJLRQV RI WKH URVWUDO LQWHUIDFH )LJ E f ,Q DGGLWLRQ VRPH ILEHUV HQWHUHG WKH JUDIWV IURP WKH UHPDLQLQJ YHQWUDO ZKLWH PDWWHU LQWHUIDFH LQ PRUH PHGLDO VHFWLRQV :KLOH VRPH UHFLSLHQWV H[KLELWHG PRUH LQJURZWK IURP RQH UHJLRQ RI WKH LQWHUIDFH WKHUH ZDV QR REYLRXV WHQGHQF\ IRU D JUHDWHU LQQHUYDWLRQ IURP YHQWUDO RU GRUVDO LQWHUIDFH DFURVV DQLPDOV ,Q PDQ\ UHJLRQV RI WKH KRVWJUDIW LQWHUIDFH ZKHUH WKH WZR WLVVXHV DSSHDUHG ZHOO IXVHG +7 SURFHVVHV H[WHQGHG MXVW LQWR WKH LQWHUIDFH LQ D SDWWHUQ UHVHPEOLQJ WKH LQJURZWK RI VSLQDO FRUG D[RQV REVHUYHG IROORZLQJ ODUJH LQMHFWLRQV RI :*$ +53 LQWR QHDUE\ VHJPHQWV )LJ Df +RZHYHU DV VHHQ ZLWK )* DQG :*$+53 LQMHFWLRQV WKH GHQVLW\ RI ILEHU VWDLQLQJ UHSUHVHQWLQJ WKH LQJURZLQJ ILEHUV ZDV DOZD\V PXFK OHVV WKDQ WKDW ZLWKLQ WKH KRVW VSLQDO FRUG URVWUDO WR WKH JUDIW ,Q FRQWUDVW WR ILQGLQJV ZKHQ )6& JUDIWV KDYH EHHQ SODFHG LQWR QHZERUQ UHFLSLHQWV 5HLHU HW DO nD %UHJPDQ nf WKH YDVW PDMRULW\ RI VWDLQHG ILEHUV ZLWKLQ JUDIWV LQ WKH DGXOW ZHUH IRXQG ZLWKLQ PP RI WKH KRVWJUDIW LQWHUIDFH 1R ILEHUV ZHUH REVHUYHG JUHDWHU WKDQ PP IURP WKH QHDUHVW LQWHUIDFH WKXV QR ILEHUV H[WHQGHG DFURVV WKH JUDIWV $W VRPH UHJLRQV RI WKH KRVWJUDIW LQWHUIDFH WKH LQMXUHG +7 ILEHUV ZHUH WKLFNHQHG DQG DSSHDUHG WR HQGHG EOLQGO\ DW WKH KRVWJUDIW LQWHUIDFH IRUPLQJ ODUJH WHUPLQDO EXOEV )LJ Gf ,Q VRPH RI WKHVH H[DPSOHV QHDUE\ VHFWLRQV VWDLQHG

PAGE 115

)LJXUH ,QJURZWK RI +7 ILEHUV IURP WKH URVWUDO DQG YHQWUDO KRVW JUDIW LQWHUIDFH Df 6PDOO FDOLEHU +7 D[RQV DUURZVf FURVV WKH LQWHUIDFH DUURZKHDGVf EHWZHHQ WKH KRVW YHQWUDO JUD\ PDWWHU Kf DQG WKH WUDQVSODQW Wf Ef $ VLQJOH +7 D[RQ DUURZf ZLWKLQ D )6& JUDIW Ff 0DQ\ RI WKH KRVW +7 D[RQV VWRS ZLWKLQ WKH LQWHUIDFH DUURZKHDGVf ZKLOH VLQJOH ILEHUV DUURZf H[WHQG LQWR WKH JUDIW Gf,Q VRPH UHJLRQV RI WKH KRVWJUDIW LQWHUIDFH +7 D[RQV VWRS DEUXSWO\ DQG IRUP ODUJH WHUPLQDO EXOE HQGLQJV 7KLV UHJLRQ DUURZKHDGVf FRUUHVSRQGV WR DQ DUHD RI LQWHQVH *)$3 VWDLQLQJ LQ D QHDUE\ VHFWLRQ 6FDOH LQ DG P

PAGE 117

ZLWK *)$3 UHYHDOHG GHQVH JOLDO VFDUULQJ LQ WKH UHJLRQ ZKHUH WKHVH EXOERXV HQGLQJV ZHUH IRXQG 7\URVLQH +\GUR[\ODVH 7+f LPPXQRUHDFWLYLWY $OO RI WKH VWDLQHG JUDIWV FRQWDLQHG VRPH FHOOV WKDW H[KLELWHG 7+ OLNH LPPXQRUHDFWLYLW\ VHH EHORZf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nf $[RQV ZHUH EHVW YLVXDOL]HG LQ ORQJLWXGLQDO VHFWLRQ ZKHUH WKH\ GHVFHQGHG LQ WKH ODWHUDO IXQLFXOXV DQG DGMDFHQW WR WKH FHQWUDO FDQDO ZLWK OLWWOH EUDQFKLQJ HYLGHQW ,Q RI WUDQVSODQWV R[\WRFLQ LPPXQRUHDFWLYH ILEHUV ZHUH REVHUYHG ZLWKLQ WKH SDUHQFK\PD RI WKH JUDIWV ,Q HDFK FDVH WKH VWDLQHG ILEHUV ZHUH IRXQG QHDU WKH URVWUDO ERUGHU RI WKH WUDQVSODQWV )LJ D f 7KHVH ILEHUV ZHUH RI YHU\ VPDOO FDOLEUH DQG LQGLYLGXDO D[RQV H[KLELWHG YDULFRVLWLHV DORQJ WKHLU HQWLUH OHQJWK /LWWOH D[RQDO FROODWHUDOL]DWLRQ ZDV REVHUYHG +RZHYHU WKH ILEHUV RIWHQ WUDYHOHG LQ D FLUFXLWRXV PDQQHU ZKLFK ZDV GLIIHUHQW WKDQ WKDW

PAGE 118

REVHUYHG LQ WKH QRUPDO VSLQDO FRUG 7KH GLVWDQFH RI LQJURZWK RI WKHVH D[RQV ZDV VLPLODU WR WKDW GHVFULEHG IRU +7 DQG 7+ ILEHUV GHVFULEHG DERYH 1R R[\WRFLQFRQWDLQLQJ ILEHUV ZHUH VHHQ IRXQG DW GLVWDQFHV JUHDWHU WKDQ PP IURP WKH URVWUDO RU ODWHUDO LQWHUIDFH RI WKH JUDIW :KHUH LQMXUHG 2[ VWDLQHG ILEHUV ZHUH DSSRVHG WR KHDYLO\ VFDUUHG UHJLRQV RI WKH KRVWJUDIW LQWHUIDFH WKH D[RQV ZHUH RIWHQ WKLFNHQHG DV FRPSDUHG WR WKHLU DSSHDUDQFH LQ QRUPDO VSLQDO FRUG RU ZLWKLQ WKH JUDIW WLVVXH 2FFDVLRQDOO\ WKHVH WKLFNHQHG ILEHUV DVVXPHG D FRQILJXUDWLRQ VXJJHVWLQJ D UHYHUVDO LQ GLUHFWLRQ DW WKLV SRLQW )LJ Ef &DOFLWRQLQ JHQHUHODWHG SHSWLGH &*53f LPPXQRUHDFWLYLWY 7KH YDVW PDMRULW\ RI &*53 FRQWDLQLQJ ILEHUV DULVH IURP VPDOO GLDPHWHU SULPDU\ DIIHUHQWV IURP '5* FHOOV *LEVRQ HW DO n 0F1HLOO HW DO nf :LWKLQ WKH KRVW VSLQDO FRUG WKHVH ILEHUV ZHUH IRXQG SULPDULO\ ZLWKLQ ODPLQDH ,, DQG ,,, ZLWK D[RQV GHVFHQGLQJ WR ODPLQDH 9 DQG ; ,Q DGGLWLRQ WR VWDLQHG ILEHUV D SRSXODWLRQ RI PRWRQHXURQV DOVR H[KLELWHG &*53 LPPXQRUHDFWLYLW\ ZLWK VWDLQLQJ EHLQJ UHVWULFWHG WR WKH SHULNDU\RQ :KHQ KHPLVHFWLRQ JUDIWV ZHUH FXW LQ WKH VDJLWWDO SODQH KRVW &*53 ILEHUV ZHUH FRPPRQO\ VHHQ DW WKUHH UHJLRQV RI KRVW JUDIW DSSRVLWLRQ f WKH LQMXUHG RU LQWDFW GRUVDO URRWV f WKH DVFHQGLQJ DQG GHVFHQGLQJ WUDFWV RI /LVVDXHU DQG GRUVDO FROXPQV DQG f WKH FHQWUDO FDQDO RU ODPLQD ; UHJLRQ &*53 OLNH ILEHUV ZHUH REVHUYHG ZLWKLQ WKH JUDIWV DORQJ HDFK RI

PAGE 119

)LJXUH ,QJURZWK RI 2[ DQG &*53 FRQWDLQLQJ ILEHUV Df )LEHUV H[KLELWLQJ 2[OLNH LPPXQRUHDFWLYLW\ HQWHU D )6& WUDQVSODQW Wf IURP WKH URVWUDO KRVWKf JUDIW LQWHUIDFH DUURZKHDGVf 7KH ODEHOHG D[RQV DUH IRXQG PRVWO\ LQ WKH GRUVDO SRUWLRQ RI WKH JUDIW DUURZVf Ef +LJKHU SRZHU PLFURJUDSK RI WKH LQWHUIDFH EHWZHHQ D WUDQVSODQW Wf DQG WKH KRVW VSLQDO FRUG Kf DW WKH URVWUDO KRVWJUDIW LQWHUIDFH VLQJOH DUURZKHDGVf 6PDOO 2[OLNH D[RQV DUH IRXQG ZLWKLQ WKH VFDU DUURZVf ,Q DGGLWLRQ RWKHU D[RQV DVVXPHG D WKLFNHQHG DSSHDUDQFH MXVW URVWUDO WR WKH LQWHUIDFH GRXEOH DUURZKHDGVf Ff &*53 FRQWDLQLQJ ILEHUV DUURZVf HQWHU D )6& WUDQVSODQW Wf WR LQQHUYDWH WKH FDXGDO DQG GRUVDO TXDGUDQW 7KH KRVW VSLQDO FRUG Kf FDXGDO WR WKH JUDIW H[KLELWV QRUPDO &*53 LPPXQRUHDFWLYLW\ RI ILEHUV LQ WKH GRUVDO KRUQ DQG SXQFWDWH ODEHOLQJ RI PRWRQHXURQV LQ WKH YHQWUDO KRUQ 9+f ,Q WKLV VHFWLRQ D UHPQDQW RI D QHDUE\ GRUVDO URRW '5f LV DSSRVHG WR WKH JUDIW Gf +LJKHU SRZHU PLFURJUDSK RI &*53 SRVLWLYH ILEHUV DW WKH LQWHUIDFH EHWZHHQ D SRUWLRQ RI WKH KRVW GRUVDO URRW Kf DQG D WUDQVSODQW Wf 6PDOO FDOLEHU ILEHUV DUH VHHQ ZLWKLQ WKH JUDIW DUURZVf ,Q DGGLWLRQ VRPH ILEHUV WUDYHO SDUDOOHO WR WKH LQWHUIDFH VLQJOH DUURZKHDGVf ZKLOH RWKHUV ZLWKLQ WKH GRUVDO URRW H[KLELW EXOERXV HQGLQJV GLVWDO WR WKH JUDIW GRXEOH DUURZKHDGVf 6FDOH LQ DF LP EG [P

PAGE 121

UHJLRQV 0RVW RI WKH LQQHUYDWLRQ E\ &*53OLNH ILEHUV ZDV VHHQ LQ WKH GRUVDO KDOI RI WKH WUDQVSODQWV DQG QHDU WKH FDXGDO LQWHUIDFH )LJ F f ZKLOH D IHZ ILEHUV DOVR HQWHUHG WKH JUDIWV IURP WKH URVWUDO ERUGHU 7KH DEVROXWH GLVWDQFH RI LQJURZWK ZDV GLIILFXOW WR GLVFHUQ DV ILEHUV HQWHUHG WKH JUDIWV IURP DOO GLUHFWLRQV 0RVW RI WKH D[RQV ZHUH ORFDWHG ZLWKLQ PP RI DQ LQWHUIDFH +RZHYHU XQOLNH WKH +7 DQG 2[FRQWDLQLQJ D[RQV VRPH &*53VWDLQHG ILEHUV ZHUH DOVR IRXQG LQ GHHSHU UHJLRQV YHQWUDO H[WHQW RI WKH WUDQVSODQWV VHH 7HVVOHU HW DO n +RXOH DQG 5HLHU nf 7KH LQWHUIDFH EHWZHHQ LQMXUHG GRUVDO URRWV DQG WKH GRUVDO ERUGHU RI WKH JUDIWV DOVR UHYHDOHG &*53 LPPXQRUHDFWLYH ILEHUV WKDW WHUPLQDWHG ZLWKRXW H[WHQGLQJ LQWR WKH JUDIWV $V VHHQ ZLWK RWKHU KRVW ILEHU W\SHV VRPH RI WKHVH D[RQV DOVR H[KLELWHG ODUJH EXOERXV HQGLQJV LQGLFDWLYH RI D IDLOXUH RI HORQJDWLRQ ZKLOH RWKHUV FRXUVHG SDUDOOHO WR WKH LQWHUIDFH )LJ Gf &RPSDULVRQ RI GLIIHUHQW ILEHU W\SHV 7KH SDWWHUQV RI D[RQDO LQJURZWK ZHUH FRPSDUHG LQ WKUHH UHFLSLHQWV E\ VXSHULPSRVLQJ ORZPDJQLILFDWLRQ WUDFLQJV RI DGMDFHQW VHFWLRQV )LJ f ,Q HDFK FDVH WKH ILEHUV HQWHUHG WKH JUDIWV IURP KRVW UHJLRQV FRQWDLQLQJ D GHQVH LQQHUYDWLRQ RI WKH UHVSHFWLYH ILEHU W\SH &*53FRQWDLQLQJ D[RQV XVXDOO\ RFFXSLHG WKH GRUVDO DQG FDXGDO TXDGUDQW RI WKH JUDIWV 7KHVH ILEHUV VKRZHG WKH PRVW UREXVW SHQHWUDWLRQ RI WKH JUDIWV E\ DTXDOLWDWLYH FRPSDULVRQ RI ILEHU GHQVLW\ DQG WKH JUDIW DUHD

PAGE 122

)LJXUH 'UDZLQJ WXEH WUDFLQJV RI VHULDO VDJLWWDO VHFWLRQV LOOXVWUDWLQJ WKH GLVWULEXWLRQ RI LPPXQRF\WRFKHPLFDOO\ VWDLQHG D[RQV LQ D )6& JUDIW DW PRQWKV SRVWJUDIWLQJ 6HFWLRQV H[WHQG IURP WKH ODWHUDO Df WR PHGLDO Lf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f 6FDOH PP

PAGE 123

H

PAGE 124

RFFXSLHG E\ ODEHOHG D[RQV ,Q FRQWUDVW 2[ DQG +7 FRQWDLQLQJ D[RQV ZHUH UHVWULFWHG WR WKH URVWUDO ERUGHUV RI WKH JUDIWV 6RPH GHJUHH RI RYHUODS ZDV DSSDUHQW LQ WKH GLVWULEXWLRQ WKHVH ODWWHU ILEHU W\SHV 3UHVHQFH RI VWDLQHG FHOOV ZLWKLQ )6& WUDQVSODQWV 7KH DQWLVHUD XVHG LQ WKLV VWXG\ ZHUH VHOHFWHG EHFDXVH LQWULQVLF VSLQDO FRUG FHOOV FRQWDLQLQJ WKHVH DQWLJHQV DUH UDUH RU DEVHQW DQG ILEHUV ZLWKLQ WKH JUDIWV PD\ EH SUHVXPHG WR DULVH IURP VXSUDVSLQDO RU SHULSKHUDO RULJLQ 5HLHU HW DO nD %UHJPDQ nf +RZHYHU LQ VRPH RI WKHVH JUDIWV LPPXQRUHDFWLYH QHXURQV ZHUH REVHUYHG IROORZLQJ LQFXEDWLRQ LQ DQWLVHUD DJDLQVW +7 DQG 7+ 7DEOH f ,Q DGGLWLRQ &*53 FRQWDLQLQJ FHOOV WKDW VWDLQHG ZLWK D GLVWLQFWO\ GLIIHUHQW SDWWHUQ WKDQ PRWRQHXURQV RI WKH QRUPDO VSLQDO FRUG ZHUH DOVR REVHUYHG ZLWKLQ VRPH RI WKH JUDIWV 6LPLODU FHOOV ZHUH QRW IRXQG LQ WKH DGMDFHQW KRVW VSLQDO FRUG 7KHVH VWDLQHG QHXURQV PD\ UHIOHFW WKH GLIIHUHQWLDWLRQ RI D VPDOO QXPEHU RI QRUPDO FHOOV IRXQG LQ FHUYLFDO DQG VDFUDO UHJLRQV RI WKH VSLQDO FRUG 1HZWRQ HW DO n 1HZWRQ DQG +DPLOO n 0RXFKHW HW DO nf $OWHUQDWLYHO\ WKHVH FHOOV PD\ UHSUHVHQW DQ DEQRUPDO H[SUHVVLRQ RI HQ]\PHV RU SHSWLGH GXH WR WKH UHODWLYH GHDIIHUHQWHG VWDWH RI WKH JUDIW WLVVXH VHH &KDSWHU 'LVFXVVLRQf 'LVFXVVLRQ $[RQDO SURMHFWLRQV IRUPHG EHWZHHQ WUDQVSODQWV RI IHWDO QHXUDO WLVVXH DQG WKH KRVW VSLQDO FRUG PD\ SURYLGH DQ

PAGE 125

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n :DONHU DQG 0F$OOLVWHU n )RQVHFD HW

PAGE 126

DO n f WKH D[RQDO WUDFLQJ UHVXOWV GHPRQVWUDWHG WKDW FHOOV ZLWKLQ )6& JUDIWV HVWDEOLVK FRPSOH[ DQG ZLGHVSUHDG QHWZRUNV RI LQWULQVLF ILEHU SURMHFWLRQV 7KH LQWUDJUDIW FLUFXLWU\ FRQVLVWHG RI D SUHGRPLQDQFH RI VKRUW GLVWDQFH PPf SURMHFWLRQV DV ZHOO DV D VLJQLILFDQW FRQWLQJHQW RI D[RQV ZKLFK H[WHQGHG WKH OHQJWK RI WKH WUDQVSODQWV *UDIW HIIHUHQW SURMHFWLRQV 7KH WUDQVSODQWHG QHXURQV ZHUH DOVR DEOH WR H[WHQG D[RQV LQWR WKH DGMDFHQW VHJPHQWV RI WKH KRVW VSLQDO FRUG 7KHVH HIIHUHQW ILEHUV XVXDOO\ HQWHUHG WKH KRVW VSLQDO FRUG E\ SHQHWUDWLQJ UHJLRQV RI WKH KRVWJUDIW LQWHUIDFH ZKHUH DQ LQWHUYHQLQJ JOLDO ERUGHU ZDV QRW DSSDUHQW VHH EHORZf DQG WHUPLQDWHG ZLWKLQ ERWK GRUVDO DQG YHQWUDO UHJLRQV RI WKH VSLQDO FRUG %RWK WKH UHWURJUDGH DQG DQWHURJUDGH WUDFLQJ SDUDGLJPV SURYLGHG HYLGHQFH IRU D[RQDO RXWJURZWK H[WHQGLQJ DW OHDVW PP IURP WKH KRVWJUDIW LQWHUIDFH WKXV FRQILUPLQJ SUHYLRXV UHVXOWV IURP D PRUH OLPLWHG QHXURDQDWRPLFDO VWXG\ 5HLHU HW DO nDf 6LPLODU GLVWDQFHV RI D[RQDO RXWJURZWK KDYH EHHQ UHSRUWHG IRU EUDLQVWHP VXVSHQVLRQ JUDIWV LQ UDWV ZLWK FKHPLFDO OHVLRQV RI WKH VSLQDO FRUG 1RUQHV HW DO nf +RVW SURMHFWLRQV LQWR WUDQVSODQWV /RFDO LQWUDVSLQDO QHXURQV 7KH SDWWHUQV RI DIIHUHQW LQJURZWK IURP ORFDO KRVW QHXURQV ZHUH PRUH FRPSOH[ ,QMHFWLRQV RI )* LQWR HLWKHU WKH SHULSKHU\ RU WKH FHQWHU RI WKH WUDQVSODQWV UHVXOWHG LQ ODEHOLQJ RI KRVW QHXURQV $V VHHQ LQ SUHOLPLQDU\ :*$+53

PAGE 127

WUDFLQJ VWXGLHV IROORZLQJ WUDQVSODQWDWLRQ LQWR HLWKHU DFXWH 5HLHU HW DO nDf RU FKURQLF +RXOH DQG 5HLHU nf VSLQDO OHVLRQV PRVW UHWURJUDGHO\ ODEHOHG FHOOV ZHUH ORFDWHG ZLWKLQ PP RI WKH KRVWJUDIW LQWHUIDFH 7KLV FHOOXODU GLVWULEXWLRQ LV VLPLODU WR WKDW VHHQ ZLWK SHULSKHUDO QHUYH 316f JUDIWV WR WKH DGXOW VSLQDO FRUG 'DYLG DQG $JXD\R n 5LFKDUGVRQ HW DO nf DV ZHOO DV RWKHU UHJLRQV RI WKH &16 %HQIH\ DQG $JXD\R f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n )LQVHQ DQG =LPPHU nf 3ULYDW HW DO nf KDYH UHSRUWHG WKH PLJUDWLRQ RI WUDQVSODQWHG QHXURQV LQ WKH DGXOW UDW VSLQDO FRUG ,Q WKDW VWXG\ VHURWRQHUJLF QHXURQV ZHUH LGHQWLILHG DV IDU DV PP IURP EUDLQVWHP VXVSHQVLRQ JUDIWV SODFHG EHORZ D FRPSOHWH VSLQDO WUDQVHFWLRQ +RZHYHU WKLV REVHUYDWLRQ PD\ EH UHODWHG WR WKH H[WHQW RI GHQHUYDWLRQ RI WKH KRVW VSLQDO FRUG DV QR

PAGE 128

PLJUDWLRQ RI VHURWRQLQFRQWDLQLQJ FHOOV ZDV REVHUYHG IROORZLQJ D VLPLODU LQMHFWLRQ LQWR WKH OXPEDU FRUG RI UDWV IROORZLQJ FKHPLFDO D[RWRP\ RI WKH KRVW VHURWRQLQFRQWDLQLQJ ILEHUV )RVWHU HW DO nf ,Q ERWK RI WKHVH FDVHV WKH JUDIWV ZHUH PDGH RQH ZHHN DIWHU WKH LQLWLDO OHVLRQ :KLOH WKH H[WHQW WR ZKLFK D VLPLODU PRELOLW\ RI IHWDO QHXURQV RFFXUV LQ WKH DFXWHO\ LQMXUHG DGXOW VSLQDO FRUG KDV QRW \HW EHHQ GHWHUPLQHG WKLV K\SRWKHVLV PD\ EH WHVWDEOH E\ FRPELQLQJ SUHODEHOLQJ RI WKH JUDIWHG QHXURQV /LQGVD\ HW DO nf ZLWK D GRXEOHODEHOLQJ VWUDWHJ\ LQ WKH UHFLSLHQW :LFWRULQ HW DO nf )ROORZLQJ ODUJHU )* LQMHFWLRQV UHWURJUDGHO\ ODEHOHG KRVW QHXURQV ZHUH DOVR IRXQG DV IDU DV PP IURP WKH LQWHUIDFH ,Q FRQWUDVW WR WKH LQJURZWK IURP QHXURQV ERUGHULQJ WKH WUDQVSODQW WKHVH PRUH GLVWDQW KRVW FHOOV ZHUH REVHUYHG RQO\ ZKHQ WKH LQWUDJUDIW WUDFHU LQMHFWLRQV ZHUH SRVLWLRQHG QH[W WR WKH LQWHUIDFH )LJ f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

PAGE 129

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nf :LWK WKLV ODWWHU SDUDGLJP PRUH UHFHQW XOWUDVWUXFWXUDO HYLGHQFH KDV VKRZQ WKDW VXFK SULPDU\ DIIHUHQW ILEHUV FDQ IRUP V\QDSVHV ZLWK QHXURQV ZLWKLQ WKH JUDIW ,WRK DQG 7HVVOHU nf 9DULDELOLW\ LQ /DEHOLQJ 3DWWHUQV ([DPLQDWLRQ RI *)$3 DQG 1LVVO VWDLQHG VHFWLRQV UHYHDOHG UHJLRQV RI LQWLPDWH IXVLRQ EHWZHHQ KRVW DQG JUDIW WLVVXHV LQ QHDUO\ DOO VSHFLPHQV ,PPXQRF\WRFKHPLFDOO\ VWDLQHG VHFWLRQV DOVR VXJJHVWHG VRPH GHJUHH RI KRVWJUDIW QHXULWLF LQWHJUDWLRQ LQ WKH DEVHQFH RI ORQJGLVWDQFH JURZWK ([FHSW IRU D IHZ RXWVWDQGLQJ H[DPSOHV KRZHYHU WKH TXDQWLW\ RI KRVWJUDIW LQWHUDFWLRQV UHYHDOHG ZLWK WKH QHXURDQDWRPLFDO WUDFLQJ PHWKRGV ZDV PRUH OLPLWHG WKDQ WKDW H[SHFWHG IURP SUHOLPLQDU\

PAGE 130

ILQGLQJV 5HLHU HW DO n nD +RXOH DQG 5HLHU nf 7KLV GLIIHUHQFH EHWZHHQ WKH REVHUYHG ODEHOLQJ SDWWHUQV DQG WKH DQWLFLSDWHG H[WHQW RI QHXURQDO LQWHJUDWLRQ KDV DOVR EHHQ HQFRXQWHUHG LQ RWKHU VWXGLHV /XQG DQG +DUYH\ n 3ULW]HO HW DO n :LFWRULQ HW DO nf DQG PD\ EH LQKHUHQW WR WHFKQLFDO OLPLWDWLRQV RI WKH ODEHOLQJ SURFHGXUHV 7KH XVH RI DQ\ D[RQDO WUDFLQJ PHWKRGV LV GHSHQGHQW XSRQ WKH SODFHPHQW RI LQMHFWLRQV UHODWLYH WR WKH GLVWULEXWLRQ RI WHUPLQDO ILHOGV RU QHXURQDO FHOO ERGLHV RI LQWHUHVW %HFDXVH WKHVH LQMHFWLRQV VHOHFW D VPDOO VDPSOH RI WRWDO SURMHFWLRQV LW LV OLNHO\ WKDW WKH DEVROXWH ILEHU DQG FHOO QXPEHUV UHSUHVHQW D VDPSOLQJ RI WKH WRWDO D[RQDO SURMHFWLRQV IRUPHG EHWZHHQ KRVW DQG WUDQVSODQW WLVVXHV VHH DOVR +DUYH\ DQG /XQG n 3ULW]HO HW DO nf $ VHFRQG FRQFHUQ LQYROYHV WKH LGHQWLILFDWLRQ RI D[RQDO SURMHFWLRQV DURXQG WKH LQWHUIDFH UHJLRQ 7R SUHYHQW WKH SRVVLELOLW\ RI IDOVH LGHQWLILFDWLRQ RI SURMHFWLRQV LW LV QHFHVVDU\ WR DGRSW D FRQVHUYDWLYH LQWHUSUHWDWLRQ RI WKH LQMHFWLRQ VLWH DQG WKH UHJLRQ RI DFWLYH WUDFHU XSWDNH :DUU n 0HVXOXP nf +RZHYHU VLPLODU VWXGLHV KDYH VKRZQ WKDW UHWURJUDGH WUDFHU LQMHFWLRQV WKDW DUH VXFFHVVIXOO\ FRQILQHG WR D WUDQVSODQW RIWHQ IDLO WR GHPRQVWUDWH KRVWJUDIW SURMHFWLRQV WKDW KDYH EHHQ VKRZQ XVLQJ FRPSOHPHQWDU\ DQWHURJUDGH WHFKQLTXHV FI /XQG DQG +DUYH\ n +DUYH\ DQG /XQG n DOVR 3ULW]HO HW DO n &ODUNH HW DO nD 0F$OOLVWHU HW DO nf 0HDQZKLOH ODUJHU LQMHFWLRQV ZKLFK

PAGE 131

PD\ ODEHO ILEHUV QHDUHU WKH LQWHUIDFH DUH RIWHQ HOLPLQDWHG GXH WR GLIIXVLRQ LQWR WKH VXUURXQGLQJ WLVVXH $ ILQDO WHFKQLFDO FRQVLGHUDWLRQ LV WKH SRVVLELOLW\ RI XQLTXH D[RQDO WUDQVSRUW FKDUDFWHULVWLFV RI WKH QHXURQV ZLWKLQ WKH KRVW DQG JUDIW WLVVXHV )RU LQVWDQFH WKH WUDQVSRUW RI VRPH RU DOO RI WKHVH D[RQDO WUDFLQJ PROHFXOHV PD\ EH DOWHUHG IROORZLQJ D[RWRP\ UHJHQHUDWLRQ RU GHDIIHUHQWDWLRQ )HULQJD HW DO n n %HUNHOH\ DQG 9LHUFN nf )LQDOO\ GLIIHUHQW WUDFHUV PD\ SURGXFH GLIIHUHQW UHVXOWV GXH WR YDULDWLRQV LQ VHQVLWLYLW\ )RU H[DPSOH ZH REVHUYHG D JUHDWHU GHJUHH RI UHWURJUDGH ODEHOLQJ IROORZLQJ LQMHFWLRQV RI )* WKDQ ZH GLG IROORZLQJ LQMHFWLRQV RI +53:*$+53 LQ QRUPDO DQG WUDQVSODQWHG UDWV VHH DOVR &DEDQD DQG 0DUWLQ nf 7KH SUHVHQFH RI WKHVH DGGLWLRQDO YDULDEOHV HPSKDVL]HV WKH QHHG WR FRPSDUH UHVXOWV RI VHYHUDO WHFKQLTXHV ,VVXHV 5HODWHG WR $[RQDO *URZWK 7KH QDWXUH DQG WKH H[WHQW RI SURMHFWLRQV ZKLFK DUH IRUPHG EHWZHHQ KRVW DQG JUDIW WLVVXHV GHSHQG XSRQ ERWK WKH LQWULQVLF JURZWK FDSDFLW\ RI WKH QHXURQV DQG WKH UROH SOD\HG E\ QRQn QHXURQDO FHOOV DQG H[WUDFHOOXODU FRPSRQHQWV LQ WKH VXUURXQGLQJ HQYLURQPHQW &RQVLGHULQJ WKH SRVVLELOLW\ WKDW PDQ\ KRVWJUDIW LQWHUDFWLRQV PD\ EH ORFDWHG DW LQWHUIDFH UHJLRQV WKH YDULDELOLW\ LQ D[RQDO SURMHFWLRQ SDWWHUQV LQ DGGLWLRQ WR D UHIOHFWLRQ RI WHFKQLFDO DVSHFWV PD\ EH D UHVXOW RI WKH WLPHFRXUVH RI JUDIW GHYHORSPHQW DQG WKH

PAGE 132

UHODWLRQVKLS EHWZHHQ JUDIW PDWXUDWLRQ DQG WKH VHTXHQFH RI HYHQWV FRQVHTXHQW WR VSLQDO FRUG LQMXU\ ,QWULQVLF HOHPHQWV UHODWHG WR D[RQDO HORQJDWLRQ 7KH SUHVHQW WUDFLQJ H[SHULPHQWV VXJJHVW WKDW WKH RXWJURZWK RI D[RQV IURP )6& JUDIWV LV JUHDWHU LQ WHUPV RI RYHUDOO GLVWDQFH WKDQ WKH FRUUHVSRQGLQJ LQJURZWK RI VXSUDVSLQDO DQG PRUH GLVWDQW LQWULQVLF KRVW VSLQDO FRUG QHXURQV $ VLPLODU UHODWLRQVKLS KDV DOVR EHHQ VXJJHVWHG LQ RWKHU WUDQVSODQWDWLRQ PRGHOV LQ WKH DGXOW &16 2EOLQJHU DQG 'DV n 0F/RRQ DQG /XQG n 5DLVPDQ DQG (EQHU n )UHXQG HW DO nf 'LIIHUHQFHV LQ WKH H[WHQW RI D[RQDO HORQJDWLRQ FDQ EH DWWULEXWHG LQ SDUW WR YDU\LQJ JURZWK FDSDFLWLHV WKDW DUH FKDUDFWHULVWLF RI DGXOW DQG GHYHORSLQJ QHXURQV )RU H[DPSOH WKH SURGXFWLRQ RI VSHFLILF JURZWK DVVRFLDWHG SURWHLQV WKDW FRUUHODWH FORVHO\ ZLWK D[RQDO HORQJDWLRQ LV GHYHORSPHQWDOO\ UHJXODWHG DQG GHFUHDVHV ZLWK WKH PDWXUDWLRQ RI &16 QHXURQV -DFREVRQ HW DO nf &RPSDULVRQV RI DIIHUHQW SURMHFWLRQV IROORZLQJ WUDQVSODQWDWLRQ LQWR KRVWV RI GLIIHUHQW DJHV KDYH VKRZQ XQLIRUPO\ WKDW D[RQDO HORQJDWLRQ IURP QHZERUQ KRVW EUDLQ LQWR WUDQVSODQWV LV PRUH H[WHQVLYH WKDQ LQJURZWK IURP PDWXUH EUDLQ /XQG DQG +DUYH\ n 0F/RRQ DQG /XQG nf RU VSLQDO FRUG %UHJPDQ HW DO nf 6LPLODU GLIIHUHQFHV PD\ XQGHUOLH WKH JUHDWHU GHJUHH RI LQJURZWK RI &*53FRQWDLQLQJ DIIHUHQWV WKDQ WKDW RI VXSUDVSLQDO DIIHUHQW ILEHUV )XUWKHUPRUH WKHUH LV VRPH HYLGHQFH WKDW GLIIHUHQW W\SHV RI PDWXUH &16 QHXURQV H[KLELW

PAGE 133

GLIIHULQJ FDSDFLWLHV IRU D[RQDO HORQJDWLRQ LQWR IHWDO QHXUDO WUDQVSODQWV 3ULW]HO HW DO n 1RWKLDV HW DO n :LFWRULQ HW DO n 'RXFHW HW DO nf $VWURJOLDO FHOOV DQG D[RQDO RXWJURZWK 1RQQHXURQDO FHOOV SUHVHQW LQ DGXOW VSLQDO FRUG PD\ DOVR FRQWULEXWH WR WKH SDWWHUQV RI D[RQDO SURMHFWLRQV $VWURJOLDO HOHPHQWV ZLWKLQ WKH PDWXUH &16 XQGHUJR D UHVSRQVH WR LQMXU\ LH JOLRVLVf ZKLFK LQFOXGHV WKH K\SHUWURSK\ RI WKHLU SURFHVVHV DQG DQ LQFUHDVH LQ WKH SURGXFWLRQ RI *)$3 UHY LQ 1DWKDQLHO DQG 1DWKDQLHO n (QJ nf 7KH IRUPDWLRQ RI D JOLDO VFDU EHWZHHQ KRVW DQG JUDIW WLVVXHV PD\ SUHYHQW WKH GHYHORSPHQW RI D[RQDO SURMHFWLRQV EHWZHHQ WKH WZR WLVVXHV 5DLVPDQ DQG (EQHU n $]PLWLD DQG :KLWDNHU n 'DV n .UXJHU HW DO n 5HLHU nf :KLOH QR TXDQWLWDWLYH UHODWLRQVKLS ZDV IRXQG EHWZHHQ WKH DPRXQW RI JOLDO VFDUULQJ DW WKH LQWHUIDFH DQG QXPEHUV RI UHWURJUDGHO\ ODEHOHG FHOOV WKH LQIOXHQFH RI WKH VFDU RQ D[RQDO RXWJURZWK ZDV DSSDUHQW IURP TXDOLWDWLYH REVHUYDWLRQV $OWKRXJK WKH WUDQVSODQW D[RQV ODEHOHG ZLWK 3+$/ GLG QRW DSSHDU WR WUDYHO WKURXJK LQWHUIDFH UHJLRQV RI GHQVH JOLRVLV WKH\ UDUHO\ IRUPHG FOXEOLNH HQGLQJV LQGLFDWLYH RI DERUWLYH D[RQDO HORQJDWLRQ 5DPRQ \ &DMDO n 6XQJ n /LX]]L DQG /DVHN nf ,Q FRQWUDVW VXFK HQGLQJV ZHUH RIWHQ IRXQG LQ LPPXQRF\WRFKHPLFDOO\ VWDLQHG VHFWLRQV RI KRVW ILEHUV DW WKH KRVWJUDIW LQWHUIDFH RU DIWHU 3+$/ LQMHFWLRQV LQWR WKH KRVW VSLQDO FRUG

PAGE 134

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nf 5HVXOWV RI RWKHU VWXGLHV KDYH VXJJHVWHG WKDW WKH GHJUHH RI JOLRVLV ZLWKLQ D WUDQVSODQW PD\ UHIOHFW WKH H[WHQW RI D[RQDO LQWHJUDWLRQ EHWZHHQ KRVW DQG JUDIW WLVVXHV %MRUNOXQG + DQG 'DKO n %MRUNOXQG+ DQG 2OVRQ nf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

PAGE 135

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f FHOOV LQ WKH KRVW EHJLQ WR H[WHQG ILEHUV FRQVLGHUDEOH PDWXUDWLRQ RI WKH JUDIWV PD\ EH XQGHUZD\ E\ WKH WLPH WKDW D[RQV KDYH UHDFKHG WKH WUDQVSODQWV 7KH HQODUJHPHQW RI WKH VROLG WUDQVSODQWV ZLWKLQ WKH OHVLRQ FDYLW\ PD\ DOVR FRQWULEXWH WR YDULDWLRQV LQ WKH IRUPDWLRQ RI SURMHFWLRQV DV KRVW ILEHUV LQ YHQWUDO UHJLRQV DUH DSSRVHG WR WKH JUDIW WLVVXH DW DQ HDUOLHU VWDJH LQ LWV GHYHORSPHQW WKDQ WKH PRUH GRUVDO KRVW FHOOV DQG D[RQV .UXJHU HW DO nf 7KHVH LQWHUDFWLRQV PD\ EH HYHQ PRUH FRPSOH[ LQ FDVHV ZKHUH VRPH D[RQDO GLHEDFN RI KRVW ILEHUV SUHFHGHV UHJURZWK 7KH UDSLG UDWH DW ZKLFK URGHQW WLVVXH PDWXUHV PD\ DOVR FRQWULEXWH WR WKH SUHGRPLQDQFH RI VKRUW GLVWDQFH LQJURZWK :KLOH PDQ\ FRPSRQHQWV ZLWKLQ WKH HPEU\RQLF &16 WLVVXH VKRXOG IDYRU D[RQDO HORQJDWLRQ $TXLQR HW DO n %HDVOH\ DQG 6WDOOFXS n /LHVL DQG 6LOYHU nf LW LV OLNHO\ WKDW WKHVH HOHPHQWV DUH QRW XQLIRUPO\ DYDLODEOH WR DOO KRVW D[RQV

PAGE 136

LQ WKH LQMXUHG DGXOW &16 DW WKH DSSURSULDWH WLPH /LNHZLVH WKH SURJUHVVLYH H[SUHVVLRQ RI QRQSHUPLVVLYH PROHFXOHV LQ WKH PDWXULQJ JUDIW PD\ SOD\ D UROH LQ WKH OLPLWHG LQJURZWK REVHUYHG HJ P\HOLQDVVRFLDWHG SURWHLQV &DURQL DQG 6FKZDE nf )LQDOO\ WKH WLPLQJ RI D[RQDO HORQJDWLRQ UHODWLYH WR WKHVH IDFWRUV LV IXUWKHU FRPSOLFDWHG E\ WKH SRVVLELOLW\ WKDW WKH H[WHQW RI D[RQDO HORQJDWLRQ PD\ EH OLPLWHG E\ WKH IRUPDWLRQ RI V\QDSVHV ZLWKLQ WKH JUDIW RU WKH GHQHUYDWHG KRVW HQYLURQPHQW %HUQVWHLQ DQG %HUQVWHLQ nOOf 7KH ODVW VWXG\ RI WKLV VHULHV ZLOO H[SORUH VRPH RI WKHVH WLPLQJ LVVXHV DV WKH\ UHODWH WR WKH LQWHUDFWLRQV EHWZHHQ D ORQJ P\HOLQDWHG ILEHU WUDFW DQG WKH ORFDO FLUFXLWU\ HVWDEOLVKHG DW WKH KRVW JUDIW LQWHUIDFH

PAGE 137

&+$37(5 ,17(5$&7,216 %(7:((1 ,1-85(' &257,&263,1$/ 75$&7 $;216 $1' )(7$/ 63,1$/ &25' 75$163/$176 ,1 7+( $'8/7 5$7 ,QWURGXFWLRQ 7UDQVSODQWV RI IHWDO VSLQDO FRUG )6&f WLVVXH ZKHQ JUDIWHG LQWR OHVLRQV LQ WKH DGXOW VSLQDO FRUG PD\ VHUYH WR UHSODFH WLVVXH WKDW LV GHVWUR\HG E\ LQMXU\ RU GLVHDVH 5HLHU HW DO n nD +RXOH DQG 5HLHU n :LQLDOVNL DQG 5HLHU n 1RWKLDV HW DO nf ,Q DGGLWLRQ DUHDV ZLWKLQ WKHVH WUDQVSODQWV H[KLELW VRPH IHDWXUHV RI QRUPDO VSLQDO JUD\ PDWWHU 7KXV VXFK JUDIWV PD\ EH XVHG WR UHSODFH LQWULQVLF VSLQDO QHXURQV -DNHPDQ HW DO n &KDSWHU f (YLGHQFH REWDLQHG IURP QHXURDQDWRPLFDO WUDFLQJ DQG LPPXQRF\WRFKHPLFDO VWXGLHV KDV VKRZQ WKDW D[RQDO SURMHFWLRQV GHYHORS ERWK ZLWKLQ )6& WUDQVSODQWV DQG EHWZHHQ WKH WUDQVSODQWV DQG WKH VXUURXQGLQJ KRVW VSLQDO FRUG &KDSWHU f )RU H[DPSOH DIIHUHQW LQJURZWK IURP GRUVDO URRW ILEHUV DOVR 7HVVOHU HW DO nf VHURWRQHUJLF ILEHUV IURP EUDLQVWHP QXFOHL 5HLHU HW DO nDf R[\WRFLQFRQWDLQLQJ ILEHUV IURP K\SRWKDODPXV DQG ORFDO LQJURZWK IURP VSLQDO FRUG 5HLHU HW DO nDf QHXURQV KDV EHHQ GHPRQVWUDWHG )LQDOO\ FHOOV GLVWULEXWHG WKURXJKRXW WKH JUDIWV KDYH EHHQ VKRZQ WR H[WHQG LQWR WKH VXUURXQGLQJ VSLQDO FRUG DQG WHUPLQDWH DURXQG

PAGE 138

KRVW QHXURQV 7KH D[RQDO SURMHFWLRQV WKDW DUH IRUPHG EHWZHHQ FHOOV RI WKH KRVW VSLQDO FRUG DQG WKH WUDQVSODQWHG WLVVXH PD\ SURYLGH DQ DQDWRPLFDO EDVLV IRU WKH IRUPDWLRQ RI D QHXURQDO UHOD\ DFURVV D VSLQDO OHVLRQ VLWH 5HLHU n 5HLHU HW DO n &KDSWHU f 7KH WUDQVPLVVLRQ RI DVFHQGLQJ DQG GHVFHQGLQJ LQIRUPDWLRQ DFURVV D VSLQDO FRUG OHVLRQ LV OLNHO\ WR UHTXLUH LQWHUDFWLRQV EHWZHHQ ORQJ P\HOLQDWHG ILEHU WUDFWV LQ WKH KRVW VSLQDO FRUG DQG WKH ORFDO FLUFXLWU\ HVWDEOLVKHG EHWZHHQ KRVW DQG JUDIW WLVVXH QHDU WKH LQWHUIDFH 7R GDWH WKH DQDWRPLFDO EDVLV IRU VXFK LQWHUDFWLRQV KDV QRW EHHQ H[SORUHG WKRURXJKO\ 7KH UDW FRUWLFRVSLQDO WUDFW &67f RIIHUV VHYHUDO DGYDQWDJHV DV D PRGHO V\VWHP IRU H[DPLQLQJ WKH LQWHUDFWLRQV EHWZHHQ ORQJ ILEHU WUDFWV DQG )6& WUDQVSODQWV ,Q WKH URGHQW WKH &67 IRUPV D FRPSDFW ILEHU V\VWHP ZKLFK LV HDVLO\ LGHQWLILHG LQ KLVWRORJLFDO SUHSDUDWLRQV )XUWKHUPRUH DQWHURJUDGH WUDFLQJ PHWKRGV FDQ EH UHDGLO\ DSSOLHG WR GHWHUPLQH WKH SURMHFWLRQ SDWWHUQV RI WKLV VSHFLILF SRSXODWLRQ RI GHVFHQGLQJ ILEHUV *ULEQDX HW DO n -RRVWHQ HW DO n &DVDOH HW DO nf 7KH UHVSRQVH RI &67 D[RQV WR GLIIHUHQW W\SHV RI OHVLRQV KDV EHHQ ZHOO GRFXPHQWHG :KHQ WKH VSLQDO FRUG LV LQMXUHG LQ QHZERUQ FDWV UDWV RU KDPVWHUV D[RQV RI WKH GHYHORSLQJ &67 FLUFXPYHQW WKH OHVLRQ DQG H[WHQG WKURXJK WKH UHPDLQLQJ LPPDWXUH VSLQDO FRUG WLVVXH E\ WUDYHUVLQJ DOWHUQDWLYH SDWKZD\V 7KH LPPDWXUH D[RQV FRQWLQXH EH\RQG WKH OHVLRQ VLWH

PAGE 139

WR UHLQQHUYDWH WKH DSSURSULDWH WDUJHW UHJLRQV %UHJPDQ DQG *ROGEHUJHU n %HUQVWHLQ DQG 6WHO]QHU n6 6FKUH\HU DQG -RQHV n .DOLO n 7ROEHUW DQG 'HU nf 7KLV GHYHORSPHQWDO SODVWLFLW\ GHFUHDVHV ZLWKLQ WKH ILUVW WKUHH ZHHNV SRVWQDWDO LQ WKH UDW DQG LV QRW REVHUYHG IROORZLQJ D VLPLODU OHVLRQ LQ DGXOW DQLPDOV ,QVWHDG LQMXUHG DGXOW &67 D[RQV RIWHQ H[KLELW D SURJUHVVLYH UHWURJUDGH GHJHQHUDWLRQ DZD\ IURP WKH VLWH RI WKH LQMXU\ .DOLO DQG 6FKQHLGHU n )HULQJD HW DO n )LVKPDQ DQG .HOO\ nDE 7DWRU HW DO n 3DOOLQL HW DO nf 7R GDWH WKHUH LV OLWWOH HYLGHQFH WR LQGLFDWH WKDW DGXOW &67 D[RQV DUH FDSDEOH RI UHJHQHUDWLRQ RU FROODWHUDO VSURXWLQJ IROORZLQJ D[RWRP\ LQ WKH VSLQDO FRUG .DOLO DQG 5HK n 5LFKDUGVRQ HW DO n .XDQJ DQG .DOLO nf ,I WKLV OLPLWDWLRQ LV D UHVXOW RI LQKLELWRU\ LQIOXHQFHV ZLWKLQ WKH DGXOW VSLQDO FRUG HQYLURQPHQW LW PD\ EH SRVVLEOH WR SURPRWH WKH HORQJDWLRQ RI WKHVH D[RQV E\ WUDQVSODQWLQJ IHWDO WLVVXH DW WKH VLWH RI D VSLQDO LQMXU\ )RU H[DPSOH D UHFHQW UHSRUW KDV VXJJHVWHG WKDW WKH SUHVHQFH RI )6& WUDQVSODQWV DW D OHVLRQ VLWH ZLOO SURORQJ WKH SHULRG RI GHYHORSPHQWDO SODVWLFLW\ RI FRUWLFRVSLQDO WUDFW ILEHUV LQ \RXQJ UDWV %UHJPDQ HW DO nf ,W LV XQNQRZQ ZKHWKHU VLPLODU WUDQVSODQWV FDQ SUHYHQW UHWURJUDGH GHJHQHUDWLRQ RU VXSSRUW D VSURXWLQJ UHVSRQVH RI &67 D[RQV DIWHU LQMXU\ LQ WKH DGXOW 7KHUHIRUH WKH REMHFWLYH RI WKH SUHVHQW VWXG\ ZDV WR HVWDEOLVK ZKHWKHU WUDQVSODQWV RI )6& WLVVXH ZRXOG LQIOXHQFH

PAGE 140

WKH UHDFWLRQ RI &67 ILEHUV WR LQMXU\ DQG HLWKHU DOORZ WKH LQWHJUDWLRQ RI WKLV GHVFHQGLQJ WUDFW ZLWK QHXURQV DW WKH KRVWJUDIW LQWHUIDFH RU SHUPLW VRPH ILEHUV WR H[WHQG LQWR WKH WUDQVSODQWV %HFDXVH VHYHUDO VWXGLHV KDYH VKRZQ WKDW WKH UHWUDFWLRQ RI LQMXUHG &67 D[RQV FDQ RFFXU DV VRRQ DV D IHZ GD\V DIWHU LQMXU\ .DR HW DO n )LVKPDQ DQG .HOO\ nEf DQ DGGLWLRQDO SDUDGLJP ZDV LQFOXGHG WR GHWHUPLQH LI D GHOD\ EHWZHHQ WKH WLPH RI LQMXU\ DQG SODFHPHQW RI JUDIW WLVVXH ZRXOG VWLOO SHUPLW LQWHJUDWLRQ RI LQMXUHG &67 ILEHUV DQG )6& WUDQVSODQWV 3RUWLRQV RI WKLV VWXG\ KDYH EHHQ SUHVHQWHG LQ SUHOLPLQDU\ IRUP -DNHPDQ DQG 5HLHU n n 5HLHU HW DO nf 0DWHULDOV DQG 0HWKRGV $QLPDOV DQG 6XUJLFDO 3URFHGXUHV $GXOW IHPDOH UDWV DSSUR[LPDWHO\ ZHHNV RI DJHf ZHUH XVHG WKURXJKRXW WKLV VWXG\ 7KH QRUPDO &67 ZDV H[DPLQHG LQ UDWV UDWV UHFHLYHG OHVLRQV RQO\ DQG UHFHLYHG OHVLRQV DQG WUDQVSODQWV RI IHWDO UDW VSLQDO FRUG )6&f WLVVXH )LJ f 7KH H[SHULPHQWDO UHVXOWV ZHUH REWDLQHG RQO\ IURP UDWV ZLWK VXFFHVVIXO ODEHOLQJ RI WKH &67 DQG FRPSOHWH ELODWHUDO OHVLRQV RI WKH GRUVDO FROXPQV Q f 7KH OHVLRQ DQG WUDQVSODQWDWLRQ SURFHGXUHV ZHUH VLPLODU WR WKRVH GHVFULEHG SUHYLRXVO\ VHH &KDSWHU f $IWHU WKH UDWV ZHUH DQHVWKHWL]HG ZLWK NHWDPLQH DQG [\OD]LQH WKH GRUVDO VXUIDFH RI WKH VSLQDO FRUG ZDV H[SRVHG E\ SHUIRUPLQJ D ODPLQHFWRP\ RI WKH & RU 7 YHUWHEUDH 7KHQ D OHVLRQ RI

PAGE 141

%LODWHUDO &67 /HVLRQV &ORVH OHVLRQ FDYLW\ (X )6& WUDQVSODQW G[QR :*$+53 G WUDQVSRUW /(6,21 21/< 1 f G ZN GHOD\ (X )6& WUDQVSODQW PRPR G[QR :*$+53 3+$/ :*$+53 3+$/ G G WUDQV WUDQV '(/$<(' *5$)76 1 f G G WUDQV WUDQV $&87( *5$)76 1 f ),*85( ([SHULPHQWDO GHVLJQ ([SHULPHQWDO UDWV UHFHLYHG OHVLRQ RQO\ GHOD\HG JUDIWV RU DFXWH JUDIWV RI )6& WLVVXH DV RXWOLQHG :*$+53 RU 3+$/ ZHUH DSSOLHG ELODWHUDOO\ WR WKH VHQVRULPRWRU FRUWH[ DW YDULRXV WLPHV DIWHU WKH OHVLRQ RU WUDQVSODQW G GD\f

PAGE 142

PP LQ OHQJWK ZDV FUHDWHG E\ DVSLUDWLRQ 7KH OHVLRQ SURYLGHG D FDYLW\ IRU WUDQVSODQWDWLRQ DQG VHUYHG WR VHYHU WKH GRUVDO FROXPQV ELODWHUDOO\ 6RPH UDWV UHFHLYHG RYHU KHPLVHFWLRQ OHVLRQV %HUQVWHLQ DQG 6WHO]QHU nf ZKLFK H[WHQGHG WR WKH YHQWUDO IORRU RI WKH VSLQDO FRUG ,Q RWKHUV PRUH VKDOORZ OHVLRQV ZHUH FUHDWHG E\ VHDOLQJ WKH GRUVDO YHLQ ZLWK DQ RSKWKDOPLF FDXWHU\ 1HXURPHGLFV ,QFf DW VLWHV VHSDUDWHG E\ PP DQG VXEVHTXHQWO\ UHPRYLQJ WKH LQWHUYHQLQJ WLVVXH IURP WKH GRUVDO FROXPQV DQG GRUVDO KRUQ )6& WLVVXH ZDV SODFHG LQWR WKH OHVLRQ VLWH DQG WKH VSLQDO FRUG PHQLQJHV DQG PXVFOH LQFLVLRQV ZHUH FORVHG LQ OD\HUV DV GHVFULEHG LQ SUHYLRXV VWXGLHV 2QH JURXS RI UDWV Q f UHFHLYHG WUDQVSODQWV DFFRUGLQJ WR D GHOD\ SDUDGLJP )LJ f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f $ FUDQLRWRP\ ZDV WKHQ SHUIRUPHG WR H[SRVH ERWK KLQGOLPE DQG IRUHOLPE UHJLRQV RI WKH VHQVRULPRWRU FRUWH[ PP $3 PP 0/ UHODWLYH WR %UHJPD :HONHU n

PAGE 143

:LVH DQG -RQHV n *LRDQQL DQG /D0DUFKH nff 2Q HDFK VLGH WKH RYHUO\LQJ GXUD ZDV UHPRYHG DQG VXSHUILFLDO EOHHGLQJ ZDV FRQWUROOHG ZLWK SLHFHV RI DEVRUEDEOH JHODWLQ VSRQJH VRDNHG LQ ERYLQH WKURPELQ $IWHU WKH LQMHFWLRQV ZHUH FRPSOHWHG WKH ERQH GHIHFWV ZHUH FRYHUHG ZLWK D VPDOO SLHFH RI 'XUDILOP DQG WKH RYHUO\LQJ VNLQ FORVHG ZLWK VXWXUHV RU VXUJLFDO ZRXQG FOLSV +53 DQG :*$+53 LQMHFWLRQV 5DWV ZHUH ODEHOHG DW YDULRXV LQWHUYDOV DIWHU HLWKHU OHVLRQ RQO\ RU OHVLRQ SOXV WUDQVSODQWDWLRQ 7DEOH f 0XOWLSOH f LQMHFWLRQV RI LO RI b :*$+53 ZHUH PDGH LQWR HDFK VHQVRULPRWRU FRUWH[ XVLQJ D QLWURJHQ EXUVW DSSDUDWXV SLFRVSULW]HUf ,Q VRPH DQLPDOV DQ DOWHUQDWLYH PHWKRG RI WUDFHU DSSOLFDWLRQ ZDV XVHG ZKHUHE\ WXQJVWHQ ZLUHV FRQWDLQLQJ D EROXV PP GLDPHWHUf RI GULHG +53 DQG :*$+53 VROXWLRQ ZHUH SODFHG EHORZ WKH VXUIDFH RI HDFK FRUWH[ DQG UHPRYHG DIWHU WKH VROXWLRQ KDG GLVVROYHG 7KH GHJUHH RI WUDFW ODEHOLQJ ZDV VLPLODU XVLQJ HLWKHU PHWKRG $IWHU DOORZLQJ KRXUV IRU D[RQDO WUDQVSRUW WKH UHFLSLHQWV FRQWDLQLQJ +53:*$+53 LQMHFWLRQV ZHUH GHHSO\ DQHVWKHWL]HG ZLWK VRGLXP SHQWREDUELWDO DQG SHUIXVHG WUDQVFDUGLDOO\ ZLWK PO KHSDULQL]HG b 1D&O IROORZHG E\ PO IL[DWLYH b SDUDIRUPDOGHK\GH b JOXWDUDOGHK\GH LQ 0 6RUHQVRQnV SKRVSKDWH EXIIHUf 7LVVXH EORFNV LQFOXGLQJ WKH WUDQVSODQW DQG PP RI KRVW VSLQDO FRUG URVWUDO DQG FDXGDO WR WKH JUDIW ZHUH UHPRYHG 9LEUDWRPH VHFWLRQV RI

PAGE 144

P WKLFNQHVV ZHUH FXW LQ WKH VDJLWWDO RU KRUL]RQWDO SODQH DQG WKH VHFWLRQV ZHUH UHDFWHG ZLWKLQ KRXUV RI VHFWLRQLQJ DFFRUGLQJ WR WKH WHWUDPHWK\OEHQ]LGLQH 70%f SURWRFRO RI GH 2OPRV HW DO nf 6HFWLRQV ZHUH WKHQ PRXQWHG RQWR JHODWLQ FRDWHG VOLGHV DQG FRXQWHUVWDLQHG ZLWK b 1HXWUDO 5HG WR UHYHDO WKH F\WRDUFKLWHFWXUH RI WKH KRVW DQG WUDQVSODQW WLVVXHV 7$%/( 180%(56 2) 5$76 $1' 7,0( 32,176 )25 &67 /$%(/ $)7(5 /(6,21 25 /(6,21 $1' 75$163/$17$7,21 :*$+53 3+$/ /HVLRQ RQO\ 7UDQVSODQWV $FXWHf 7UDQVSODQWV $FXWHf & 7 & 7 & G OZN ZN PR 7UDQVSODQWV 'HOD\HGf & 7 7UDQVSODQWV 'HOD\HGf & PR PR 7LPH EHWZHHQ OHVLRQ RU WUDQVSODQWDWLRQ DQG SHUIXVLRQ ,Q DOO FDVHV :*$+53 ODEHOLQJ ZDV GRQH KRXUV EHIRUH VDFULILFH 3+$/ ODEHOLQJ ZDV GRQH GD\V SULRU WR VDFULILFH 'LVWDQFHV RI UHWUDFWLRQ RI &67 DW ZHHNV DIWHU OHVLRQ RU OHVLRQ DQG WUDQVSODQWDWLRQ ZHUH XVHG IRU TXDQWLWDWLYH DQDO\VLV

PAGE 145

7KH H[WHQW RI &67 UHWUDFWLRQ RU GLHEDFN ZDV H[DPLQHG LQ +53:*$+53 UHFLSLHQWV DW ZHHNV DIWHU LQMXU\ 7DEOH f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f ZLWK EULJKWILHOG RSWLFV DV LQ )LJ f 7KH LQWHUIDFH ZDV LGHQWLILHG E\ D WUDQVLWLRQ EHWZHHQ WKH KLJKO\ RUJDQL]HG F\WRDUFKLWHFWXUH RI WKH KRVW VSLQDO FRUG DQG WKH OHVV RUJDQL]HG JUDIW WLVVXH 7KH VHFRQG OLQH ZDV WKHQ GUDZQ URVWUDO WR WKH ILUVW DW WKH FDXGDO HQG RI WKH KHDYLO\ ODEHOHG &67 UHIHUUHG WR DV WKH EXON RI WKH WUDFWf VHH )LJ f )RU HDFK VHFWLRQ WKH GLVWDQFH EHWZHHQ WKH WZR OLQHV ZDV PHDVXUHG DQG FRUUHFWHG IRU WKH HQODUJHPHQW RI WKH GUDZLQJ $Q DYHUDJH YDOXH ZDV WKHQ REWDLQHG IRU HDFK DQLPDO XVHG LQ WKH TXDQWLWDWLYH DQDO\VLV *URXS PHDQV ZHUH WHVWHG IRU GLIIHUHQFHV DW WKH S OHYHO XVLQJ D RQH ZD\ $129$ DQG LQGLYLGXDO FRPSDULVRQV ZHUH GHWHUPLQHG E\ 7XNH\nV KVG 6SHQFH HW DO nf

PAGE 146

3KDVHROXV YXOJDULV OHXFRDDDOXWLQLQ I3+$/f LQMHFWLRQV $QWHURJUDGHO\ILOOHG &67 D[RQV DQG WHUPLQDOV ZHUH LGHQWLILHG E\ LPPXQRF\WRFKHPLFDO GHWHFWLRQ RI 3+$/ 9HFWRU /DERUDWRULHV ,QFfr 7KH WUDFHU ZDV DSSOLHG WR WKH FRUWH[ RI UDWV PRQWK JUDIWV Q GHOD\HG JUDIW PRQWKV Q Of XVLQJ D PRGLILFDWLRQ RI WKH PHWKRGV RI *HUIHQ DQG 6DZFKHQNR f $ b VROXWLRQ RI 3+$/ ZDV SUHSDUHG LQ P0 SKRVSKDWH EXIIHU S+ f *ODVV PLFURSLSHWWHV ZHUH FOHDQHG DQG EURNHQ WR D WLS GLDPHWHU RI [P $ FUDQLRWRP\ ZDV WKHQ SHUIRUPHG ELODWHUDOO\ DV DERYHf DQG LRQWRSKRUHWLF LQMHFWLRQV ZHUH PDGH LQWR HDFK FRUWH[ $IWHU DOORZLQJ GD\V IRU WUDQVSRUW RI WKH 3+$/ WKH UHFLSLHQWV ZHUH SHUIXVHG DV DERYH ZLWK IL[DWLYH FRQWDLQLQJ b SDUDIRUPDOGHK\GH DQG b JOXWDUDOGHK\GH 7KH FRUG EORFNV LQFOXGLQJ WKH WUDQVSODQW DQG PP RI WKH VXUURXQGLQJ URVWUDO DQG FDXGDO VSLQDO FRUG ZHUH UHPRYHG DQG SRVWIL[HG RYHUQLJKW DW r& )UHHIORDWLQJ 9LEUDWRPH VHFWLRQV RI P WKLFNQHVV ZHUH SURFHVVHG IRU WKH LGHQWLILFDWLRQ RI 3+$/ FRQWDLQLQJ FHOOV DQG SURFHVVHV DV GHVFULEHG LQ &KDSWHU 7KH VHFWLRQV ZHUH ILUVW ZDVKHG LQ 0 3RWDVVLXP 3KRVSKDWH %XIIHUHG 6DOLQH .3%6f DQG LQFXEDWHG IRU KUV LQ D SUHEORFNLQJ EDWK FRQWDLQLQJ b QRUPDO UDEELW VHUXP DQG b 7ULWRQ ; 7ULWRQ ; ZDV H[FOXGHG RU UHGXFHG WR b IRU VHFWLRQV VHOHFWHG IRU HOHFWURQ PLFURVFRS\ EHORZf $OO WKH VHFWLRQV ZHUH WKHQ LQFXEDWHG LQ JRDW DQWL3+$/ 9HFWRU f GLOXWHG LQ .3%6 IRU KRXUV DW r& DQG WKHQ

PAGE 147

IRU DGGLWLRQDO KRXUV DW URRP WHPSHUDWXUH 7KH VHFWLRQV ZHUH UHZDVKHG DQG WKHQ SURFHVVHG ZLWK ELRWLQ\ODWHG UDEELW DQWLJRDW ,J* f DQG 9HFWRU $YLGLQ%LRWLQSHUR[LGDVH &RPSOH[ $%&f 7KH ILQDO SHUR[LGDVH FRQMXJDWH ZDV UHDFWHG ZLWK + LQ WKH SUHVHQFH RI b GLDPLQREHQ]LGLQH '$%f 1LFNHOHQKDQFHG VHFWLRQV ZHUH FRXQWHUVWDLQHG ZLWK b &UHV\O 9LROHW RU b 1HXWUDO 5HG SULRU WR FRYHUVOLSSLQJ (OHFWURQ 0LFURVFRS\ )ROORZLQJ LQFXEDWLRQ LQ '$% WKRVH VHFWLRQV VHOHFWHG IRU HOHFWURQ PLFURVFRS\ ZHUH LQFXEDWHG IRU KU LQ b RVPLXP WHWUR[LGH 7KH VHFWLRQV ZHUH WKHQ VWDLQHG HQ EORF LQ b XUDQ\O DFHWDWH LQ PDOHDWH EXIIHU GHK\GUDWHG LQ DOFRKRO DQG SURS\OHQH R[LGH LQILOWUDWHG ZLWK (0 %HG DQG PRXQWHG EHWZHHQ YLQ\O VOLGHV 7KH UHJLRQV RI LQWHUHVW ZHUH SKRWRJUDSKHG DQG WKHQ FXW DQG UHPRXQWHG RQ SODVWLF EORFNV 8OWUDWKLQ VHFWLRQV ZHUH FROOHFWHG RQ FRSSHU JULGV DQG H[DPLQHG ZLWK D =HLVV (0& HOHFWURQ PLFURVFRSH 5HVXOWV :*$+53 7UDFW 7UDFLQJ 1RUPDO &67 $QWHURJUDGHO\ ODEHOHG FRUWLFRVSLQDO ILEHUV LQ QRUPDO DGXOW UDWV ZHUH GLVWULEXWHG LQWR WKUHH SDWKZD\V )LJ Df 'RQDWHOOH n 6FKUH\HU DQG -RQHV n *ULEQDX HW DO n &DVDOH HW DO n %UHJPDQ HW DO nf 7KH EXON RI WKH ODEHOHG ILEHUV ZHUH FRQFHQWUDWHG LQ WKH YHQWUDO SRUWLRQ RI WKH GRUVDO IXQLFXOXV GRUVDO &67f ZKLOH D VPDOOHU SDWKZD\

PAGE 148

ZDV REVHUYHG LQ WKH GRUVRODWHUDO IXQLFXOXV 6FKUH\HU DQG -RQHV nf :LWKLQ WKH FHUYLFDO VSLQDO FRUG ODEHOHG ILEHUV ZHUH DOVR HQFRXQWHUHG LQ WKH PRVW PHGLDO UHJLRQ RI WKH YHQWUDO IXQLFXOXV 9DKOVLQJ DQG )HULQJD nf $[RQDO ODEHOHG WHUPLQDO ILHOGV ZHUH FRQFHQWUDWHG LQ WKH PHGLDO GRUVDO KRUQ DQG LQWHUPHGLDWH JUH\ UHJLRQV DSSUR[LPDWH ODPLQDH ,,, 9 6WHLQHU DQG 7XUQHU nf ZKLOH IHZHU ILEHUV ZHUH IRXQG ZLWKLQ WKH VXSHUILFLDO GRUVDO KRUQ DQG ODWHUDO LQWHUPHGLDWH JUH\ ODPLQDH ,, DQG 9,,f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f ,Q DQLPDOV ZLWK FRPSOHWH OHVLRQV RI WKH GRUVDO IXQLFXOXV QR ODEHOHG ILEHUV ZHUH HYHU REVHUYHG LQ WKH GRUVDO FROXPQV FDXGDO WR WKH VLWH RI WKH OHVLRQ RU WUDQVSODQWV 7KHUHIRUH UHIHUHQFHV WR WKH FDXGDO H[WHQW RI WKH LQMXUHG GRUVDO &67 UHIHU H[FOXVLYHO\ WR WKH SRUWLRQ RI WKH WUDFW URVWUDO WR WKH OHVLRQ VLWH )ROORZLQJ VKDOORZ PLGOLQH OHVLRQV D VPDOO QXPEHU RI &67 ILEHUV LQ WKH ODWHUDO DQG YHQWUDO ZKLWH PDWWHU

PAGE 149

)LJXUH 'DUNILHOG PLFURJUDSKV RI WKH SDWWHUQV RI +53:*$+53 ODEHOLQJ LQ QRUPDO DQLPDOV DQG URVWUDO WR WKH VLWH RI OHVLRQ RU JUDIWLQJ Df 1RUPDO ODEHOLQJ SDWWHUQ LQ FHUYLFDO VSLQDO FRUG IROORZLQJ ELODWHUDO FRUWLFDO LQMHFWLRQV RI +53 DQG :*$+53 7KH EXON RI WKH &67 LV IRXQG LQ WKH YHQWUDO PRVW SRUWLRQ RI WKH GRUVDO FROXPQV Gf 6PDOOHU FRPSRQHQWV DUH ORFDWHG LQ WKH YHQWUDO Yf DQG ODWHUDO FROXPQV f $[RQV LQQHUYDWH WKH PHGLDO GRUVDO KRUQ DQG LQWHUPHGLDWH JUD\ UHJLRQV RI WKH VSLQDO FRUG DUURZKHDGVf 7UDQVYHUVH LP VHFWLRQ Ef 9DULDELOLW\ LQ DQWHURJUDGH ODEHOLQJ DW PP URVWUDO WR D OHVLRQ VLWH 7KH &67 RQ WKH OHIW RI WKLV KRUL]RQWDO VHFWLRQ LV ODEHOHG PXFK OHVV LQWHQVHO\ WKDQ WKDW RQ WKH ULJKW 7KLV OLJKWHU ODEHOLQJ SDWWHUQ DOORZHG H[DPLQDWLRQ RI WKH LQGLYLGXDO D[RQV DQG WHUPLQDO EXOEV DUURZKHDGVf URVWUDO WR D OHVLRQ 6FDOH LQ D E QP

PAGE 151

ZHUH VHHQ RFFDVLRQDOO\ EHORZ WKH OHYHO RI WKH OHVLRQ 7KHVH ILEHUV ZHUH SUREDEO\ VSDUHG E\ WKH OHVLRQ 7KUHH GD\V WR WZR ZHHNV SRVWOHVLRQ $QWHURJUDGH WUDQVSRUW UHYHDOHG D KLVWRSDWKRORJ\ IROORZLQJ SDUWLDO VSLQDO OHVLRQV LQYROYLQJ WKH &67 WKDW ZHUH VLPLODU WR WKDW GHVFULEHG LQ WKH PRXVH DQG UDW IROORZLQJ FRPSOHWH WUDQVHFWLRQ RI WKH VSLQDO FRUG )LVKPDQ DQG .HOO\ nDE 3DOOLQL HW DO nf ,Q WKH ILUVW IHZ GD\V DIWHU DVSLUDWLYH LQMXU\ WKHUH ZHUH VLJQV RI P\HOLQ EUHDNGRZQ DQG UHWURJUDGH GHJHQHUDWLRQ ZLWKLQ WKH GRUVDO &67 )LJ Df 7KH FDXGDO H[WHQW RI WKLV WUDFW ZDV PDUNHG E\ DQ DFFXPXODWLRQ RI KHPDWRJHQRXV FHOOV DQG WKH WLVVXH LQ WKH UHJLRQ RI WKH WUDFW ZDV QHFURWLF LQ DSSHDUDQFH 6RPH UHWUDFWLRQ EXOEV ZHUH SUHVHQW DW WKH HQGV RI ODUJHU D[RQV SUR[LPDO WR WKH OHVLRQ ,Q DGGLWLRQ ODEHOHG D[RQV DW WKH OHVLRQ VLWH ZHUH WKLFNHQHG )LJ Ef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f 7KH &67 ZLWKLQ WKH GRUVDO IXQLFXOXV KDG XQGHUJRQH UHWURJUDGH GHJHQHUDWLRQ DQG

PAGE 152

WKH WUDFW WHUPLQDWHG DSSUR[LPDWHO\ PP IURP WKH LQMXU\ VLWH $W WKLV VLWH WKH WUDFW DVVXPHG D WDSHUHG FRQILJXUDWLRQ XQGHU WKH GHJHQHUDWLQJ GRUVDO FROXPQ ILEHUV ,Q DGGLWLRQ WR WKH REVHUYHG UHWUDFWLRQ RI WKH EXON RI WKH &67 VRPH KHWHURJHQHLW\ LQ D[RQDO GLHEDFN RI LQGLYLGXDO ILEHUV ZDV DOVR QRWHG $OWKRXJK VRPH RI WKHVH D[RQV UHPDLQHG GLUHFWO\ DW WKH ZRXQG HGJH RU ZLWKLQ WKH QHFURWLF UHJLRQ RI WLVVXH )LJ Ef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f 7KH UHJLRQ EHWZHHQ WKH FDXGDO H[WHQW RI WKH GRUVDO &67 DQG WKH HGJH RI WKH OHVLRQ ZDV RIWHQ RFFXSLHG E\ F\VWLF FDYLWDWLRQ DQG FRQWDLQHG QHFURWLF WLVVXH DQG GHJHQHUDWLQJ ILEHUV %\ WKLV VL[ ZHHN WLPH SRLQW WKH DYHUDJH GLVWDQFH EHWZHHQ WKH HQG RI WKH GRUVDO &67 DQG WKH HGJH RI WKH UHPDLQLQJ WLVVXH ZDV PP Q f 7KH GLVWDQFH RI UHWUDFWLRQ ZDV JUHDWHU ZKHQ WKH

PAGE 153

)LJXUH 6DJLWWDO VHFWLRQV WKURXJK D OHVLRQ VLWH DW GD\V RU ZHHNV IROORZLQJ OHVLRQ RQO\ RU OHVLRQ SOXV WUDQVSODQWDWLRQ Df $W GD\V DIWHU D OHVLRQ /f WKH LQMXUHG &67 DUURZKHDGf LV RFFXSLHG E\ H[WHQVLYH KHPRUUKDJLQJ $ IHZ WKLFNHQHG D[RQV DQG WHUPLQDO EXOEV FDQ EH VHHQ URVWUDO WR WKH OHVLRQ VLWH ULJKW KDQG VLGH RI SKRWRf 7KH GRUVDO FROXPQV DUH VHHQ DERYH WKH ODEHOHG &67 :LWKLQ WKH YHQWUDO ZKLWH PDWWHU ERWWRP RI SKRWRf KHPRUUKDJLQJ LV OHVV H[WHQVLYH DUURZf DQG LQWHUUXSWHG WKLFNHQHG D[RQV UHPDLQ FORVHU WR WKH OHVLRQ VLWH Ef ,QMXUHG &67 D[RQV DW GD\V DIWHU D OHVLRQ DUH WKLFNHQHG LQ DSSHDUDQFH DUURZKHDGVf DQG VXUURXQGHG E\ KHPRUUKDJLF WLVVXH Ff %\ ZHHNV DIWHU D OHVLRQ WKH &67 MXVW DERYH WKH FHQWUDO FDQDO Ff LV WDSHUHG 7KH OHVLRQ VLWH LV RFFXSLHG E\ QHFURWLF WLVVXH DQG D VPDOO DPRXQW RI KHPRUUKDJLD LV SUHVHQW DUURZf Gf 0RVW RI WKH LQMXUHG &67 D[RQV KDYH UHWUDFWHG IURP WKH OHVLRQ DW ZHHNV SRVWLQMXU\ ZKLOH D IHZ D[RQV DUURZKHDGVf UHPDLQ FORVHU WR WKH LQMXU\ VLWH Hf 6HFWLRQ WKURXJK WKH JUD\ PDWWHU RI D ZHHN WUDQVSODQW 7f 7KH WLVVXH ZDV QRW \HW IXOO\ DSSRVHG WR WKH KRVW VSLQDO FRUG Kf If %\ ZHHNV DIWHU LQMXU\ DQG WUDQVSODQWDWLRQ &67 D[RQV KDYH UHWUDFWHG IURP WKH LQMXU\ VLWH $ IHZ D[RQV UHPDLQ FORVHU WR WKH HGJH RI WKH WLVVXH DUURZKHDGVf 6FDOH LQ DFH QP EG DQG I P

PAGE 155

OHVLRQV ZHUH PDGH DW WKH 7 YHUWHEUDO OHYHO PHDQ PP Q f WKDQ ZKHQ OHVLRQV ZHUH PDGH DW WKH & OHYHO PHDQ PP Q f S f )LJ RSHQ EDUVf $V VHHQ DW HDUOLHU SRVWLQMXU\ WLPHV LQGLYLGXDO ODEHOHG WHUPLQDO EXOEV ZHUH DOVR REVHUYHG LQ WKHVH VSHFLPHQV 7KHVH UHWUDFWLRQ EXOEV ZHUH HYLGHQW ERWK DW WKH HQG RI WKH WUDFW DQG DV IDU DV PP URVWUDO WR WKH OHVLRQ 0DQ\ RI WKH LQGLYLGXDO ILEHUV ZHUH GLUHFWO\ DSSRVHG WR HGJH RU ODWHUDO PDUJLQV RI WKHVH F\VWV 7KUHH WR IRXU PRQWKV SRVWLQMXU\ 7KH SDWWHUQ RI &67 GHJHQHUDWLRQ DQG FDYLWDWLRQ ZDV VLPLODU LQ OHVLRQ UHFLSLHQWV VDFULILFHG DW ORQJHU VXUYLYDO WLPHV PRQWKVf 7KH FDXGDO H[WHQW RI WKH &67 KDG UHWUDFWHG DSSUR[LPDWHO\ PP ZKLOH DGGLWLRQDO HQODUJHG D[RQ WHUPLQDOV ZHUH IRXQG URVWUDO WR WKH OHVLRQ )LJ Hf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

PAGE 156

)LJXUH 5HWUDFWLRQ RI WKH &67 IROORZLQJ OHVLRQ RQO\ RU OHVLRQ SOXV WUDQVSODQWDWLRQ %LODWHUDO LQMHFWLRQV RI :*$ +53 LQWR WKH VHQVRULPRWRU FRUWH[ Df %\ ZHHNV DIWHU LQMXU\ WKH ODEHOHG D[RQV RI WKH &67 KDYH UHWUDFWHG IURP WKH HGJH RI WKH OHVLRQ /f +RUL]RQWDO VHFWLRQ WKURXJK WKH OHYHO RI WKH GRUVDO FROXPQV 7KH GLVWDQFHV IURP WKH FDXGDO PRVW H[WHQW RI WKH WUDFW WR WKH OHVLRQ HGJH ZHUH PHDVXUHG DV VKRZQ ZKLWH OLQHVf 7KH HGJH RI WKH OHVLRQ LV VHHQ LQ WKH YHU\ ERWWRP RI WKH ILJXUH DQG YHULILHG E\ EULJKWILHOG LOOXPLQDWLRQ 1RWH WKDW LQGLYLGXDO ILEHUV LQ WKH ODWHUDO ZKLWH PDWWHU UHPDLQ QHDU WKH HGJH RI WKH OHVLRQ VLWH DUURZKHDGVf Ef +RUL]RQWDO VHFWLRQV WKURXJK WKH ODEHOHG &67 DW ZNV IROORZLQJ OHVLRQ SOXV WUDQVSODQWDWLRQ 7f $ VPDOO F\VW LV IRXQG EHWZHHQ WKH ODEHOHG &67 DQG WKH URVWUDO SRUWLRQ RI WKH JUDIW 6RPH ODEHOHG D[RQV H[WHQG DORQJ WKH PDUJLQV RI WKH F\VW DUURZKHDGf 7KH URVWUDO KRVWJUDIW LQWHUIDFH GRWWHG OLQHf ZDV GHWHUPLQHG E\ H[DPLQDWLRQ RI WKH F\WRDUFKLWHFWXUH XQGHU EULJKWILHOG LOOXPLQDWLRQ Ff 6HFRQG H[DPSOH RI ODEHOHG &67 DW ZHHNV DIWHU OHVLRQ RQO\ &\VWLF UHJLRQV VHSDUDWH WKH LQMXUHG WUDFW IURP WKH HGJH RI WKH OHVLRQ /f Gf ([DPSOH RI LQMXUHG &67 GLUHFWO\ DSSRVHG WR D ZHHN WUDQVSODQW 7f ,Q WKLV H[DPSOH QR F\VWV DUH SUHVHQW DW WKH KRVWJUDIW LQWHUIDFH GRWWHG OLQHf DQG ODEHOHG ILEHUV DUH VHHQ DW WKH HGJH RI WKH JUDIW Hf ,QMXUHG &67 ILEHUV UHWUDFW IURP WKH HGJH RI WKH OHVLRQ /f DW PRQWKV DIWHU OHVLRQ RQO\ If 7KUHH PRQWKV DIWHU OHVLRQ SOXV WUDQVSODQWDWLRQ LQMXUHG &67 D[RQV UHPDLQ DW WKH KRVWJUDIW LQWHUIDFH 6FDOH D I XQ

PAGE 158

)LJXUH $YHUDJH GLVWDQFHV EHWZHHQ WKH FDXGDO H[WHQW RI WKH GRUVDO &67 DQG WKH OHVLRQ RU KRVWJUDIW ERUGHU LQ 7 DQG & UHFLSLHQWV 2YHUDOO DYHUDJH IRU OHVLRQ RQO\ HPSW\ EDUVf PP Q f 2YHUDOO DYHUDJH IRU OHVLRQ DQG WUDQVSODQWDWLRQ KDWFKHG EDUVf PP Q f 7KHVH WZR JURXSV ZHUH GLIIHUHQW E\ 6WXGHQWnV W WHVW S f $129$ LQGLFDWHG WKDW WKH PHDQV RI DOO IRXU JURXSV ZHUH GLIIHUHQW S f 'LIIHUHQFHV ZHUH IRXQG E\ 7XNH\nV KVG EHWZHHQ WKH OHVLRQ RQO\ DQG OHVLRQ SOXV WUDQVSODQWDWLRQ DW ERWK WKH 7 DQG & YHUWHEUDO OHYHOV S f 7UDQVSODQWV WKDW ZHUH SDUWLDOO\ DSSRVHG WR WKH GRUVDO &67 ZHUH LQFOXGHG LQ WKH WRWDOV +RZHYHU WKUHH UHFLSLHQWV ZLWK IDLOHG JUDIWV ZHUH QRW LQFOXGHG

PAGE 159

5HWUDFWLRQ RI G&67 :HHNV $IWHU /HVLRQ RU /HVLRQ 7UDQVSODQW /HVLRQ 2QO\ /HVLRQ 7UDQVSODQW

PAGE 160

LQMXUHG ILEHUV RI WKH GRUVDO IXQLFXOXV )LJ Hf 7KH ODEHOHG GRUVDO &67 WUDFW ZDV PDUNHG E\ DQ DFFXPXODWLRQ RI EORRG FHOOV DQG PDFURSKDJHOLNH SURILOHV DW WKH HDUOLHVW WLPH SRLQWV %\ WZR ZHHNV SRVWJUDIWLQJ PRVW RI WKH ODEHOHG ILEHUV LQ WKH GRUVDO &67 KDG UHWUDFWHG IURP WKH HGJH RI WKH OHVLRQ )LJ Ff DQG D[RQV HQGHG LQ UHWUDFWLRQ EXOEV URVWUDO WR WKH OHVLRQ )LJ If 6L[ ZHHNV SRVWJUDIWLQJ 7UDQVSODQWV H[DPLQHG VL[ ZHHNV DIWHU WUDQVSODQWDWLRQ ZHUH ZHOO GHYHORSHG DQG PRVW KDG ILOOHG WKH OHVLRQ FDYLW\ 7KH DSSHDUDQFH RI WKH F\WRDUFKL WHFWXUH DQG GHJUHH RI P\HOLQDWLRQ ZHUH VLPLODU WR WKDW GHVFULEHG SUHYLRXVO\ 5HLHU HW DO nDf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f 7KH GLVWDQFH EHWZHHQ WKH HQG RI WKH GRUVDO &67 DQG WKH KRVWJUDIW LQWHUIDFH DFURVV DOO WKH ZHHN WUDQVSODQW UHFLSLHQWV ZDV s PP Q f 7KH GLVWDQFH UHIOHFWHG D GHFUHDVH LQ

PAGE 161

WKH H[WHQW RI UHWURJUDGH GHJHQHUDWLRQ DW ERWK WKH & PHDQ PP Q f DQG 7 PHDQ PP Q f YHUWHEUDO OHYHOV DV FRPSDUHG WR DQLPDOV ZLWK OHVLRQV RQO\ )LJ KDWFKHG EDUVf 2I WKH UHFLSLHQWV XVHG LQ WKH TXDQWLWDWLYH REVHUYDWLRQV WKUHH FRQWDLQHG JUDIWV WKDW ZHUH DSSRVHG WR WKH LQMXUHG &67 LQ WKH UHJLRQV QHDU WKH FHQWUDO FDQDO EXW WKH\ ZHUH QRW DSSRVHG WR WKH PRVW GRUVDO SRUWLRQV RI WKH WUDFW 7KHVH UHFLSLHQWV GLG QRW GLIIHU LQ WKH DYHUDJH GLVWDQFH RI &67 UHWUDFWLRQ IURP WKH UHPDLQGHU RI WKH JUDIW UHFLSLHQWV GLVWDQFHV PPf ,Q FRQWUDVW WKUHH DGGLWLRQDO JUDIW UHFLSLHQWV H[KLELWHG JRRG &67 ODEHOLQJ EXW KDG QR VXUYLYLQJ JUDIWV 2I WKHVH IDLOHG WUDQVSODQW UHFLSLHQWV WZR H[KLELWHG &67 UHWUDFWLRQ IRU GLVWDQFHV VLPLODU RU JUHDWHU WKDQ WKRVH VHHQ LQ OHVLRQRQO\ UHFLSLHQWV GLVWDQFHV PPf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

PAGE 162

SRVWJUDIWLQJ ,Q WKHVH DQLPDOV ODEHOHG &67 D[RQV ZHUH GLUHFWO\ DSSRVHG WR WKH KRVWJUDIW LQWHUIDFH )LJ If 'HOD\HG JUDIWV :KHQ HPEU\RQLF VSLQDO FRUG WLVVXH ZDV SODFHG LQWR OHVLRQV WKDW ZHUH SUHSDUHG RQH ZHHN SUHYLRXVO\ GHOD\HG JUDIWVf WKH TXDOLWDWLYH SDWWHUQ RI D[RQDO ODEHOLQJ ZDV VLPLODU WR WKDW REVHUYHG ZKHQ JUDIWV ZHUH SODFHG LQWR WKH DFXWH OHVLRQV 6RPH RI WKH GRUVDO &67 ILEHUV KDG UHWUDFWHG IURP WKH OHVLRQ VLWH DQG VPDOO F\VWLF FDYLWLHV ZHUH VRPHWLPHV SUHVHQW EHWZHHQ WKH FDXGDO HQG RI WKH WUDFW DQG WKH JUDIW $V VHHQ LQ WKH DFXWH LPPHGLDWHf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P EHIRUH WKH\ WHUPLQDWHG RU OHIW WKH SODQH RI VHFWLRQ 7KH PDMRULW\ RI WKHVH LQJURZLQJ ILEHUV ZHUH IRXQG QHDU WKH HQG RI WKH GRUVDO &67 LQ WKH GRUVDO DQG URVWUDO HGJH RI WKH JUDIW )LJ DGf +RZHYHU LQ RQH FDVH ILEHUV HQWHUHG IURP WKH ODWHUDO HGJH RI WKH JUDIW )LJ Hf 1R D[RQDO SURILOHV ZHUH IRXQG LQ WKH FDXGDO UHJLRQV RI DQ\ RI WKHVH WUDQVSODQWV

PAGE 163

)LJXUH ,QMHFWLRQV RI :*$+53 LQWR VHQVRULPRWRU FRUWH[ $[RQDO SURILOHV VWDLQHG ZLWK 70% DSSHDU WR H[WHQG DFURVV WKH KRVWJUDIW LQWHUIDFH GRWWHG OLQHVf DQG LQWR ZN )6& WUDQVSODQWV 7f +RUL]RQWDO P VHFWLRQV DEf %ULJKWILHOG SKRWRPLFURJUDSK RI 1HXWUDO 5HG VHFWLRQV WKURXJK WKH URVWUDO KRVWJUDIW LQWHUIDFH RI WZR VSHFLPHQV ,Q HDFK FDVH WKH LQWHUIDFH LV HYLGHQW E\ WKH SUHVHQFH RI D JOLDO ERUGHU VPDOO GHQVHO\ SDFNHG FHOOVf DORQJ WKH OHIW VLGH $ GLIIHUHQFH LQ F\WRDUFKLWHFWXUDO RUJDQL]DWLRQ LQGLFDWHG WKH KRVWJUDIW LQWHUIDFH RQ WKH ULJKW VPDOO EODFN GRWWHG OLQHf &RUUHVSRQGLQJ EORRG YHVVHOV DQG EDFNJURXQG UHDFWLRQ SURGXFW ODQGPDUNV LQ DFf DQG EGf DUH LGHQWLILHG E\ rf Ff 7ZR D[RQV ZKLWH DUURZKHDGVf HQWHU WKH )6& WUDQVSODQW VKRZQ LQ Df Gf 0RUH D[RQV HQWHU DQRWKHU JUDIW IURP D ZHOO IXVHG UHJLRQ RI WKH YHQWUDO KRVWJUDIW LQWHUIDFH DV VKRZQ LQ Ef Hf $[RQV DOVR HQWHU D )6& WUDQVSODQW IURP WKH GRUVRODWHUDO IXQLFXOXV If 2FFDVLRQDOO\ ODEHOHG D[RQV WUDYHO SDUDOOHO WR WKH KRVW JUDIW LQWHUIDFH ZKLWH Y DUURZKHDGVf 6FDOH LQ D I P

PAGE 165

$GGLWLRQDO D[RQDO SURILOHV ZLWKLQ WKH KRVW JUD\ PDWWHU ZHUH GLUHFWO\ DSSRVHG WR JUDIW WLVVXH LQ PDQ\ RI WKHVH UHFLSLHQWV $ IHZ RI WKHVH ODEHOHG D[RQV FRXUVHG SDUDOOHO WR WKH KRVWJUDIW LQWHUIDFH ZKLOH RWKHUV DSSHDUHG WR WHUPLQDWH MXVW URVWUDO WR WKH JUDIW RU GLVDSSHDU IURP WKH SODQH RI VHFWLRQ )LJ If $QWHURJUDGH 7UDQVSRUW RI 3+$/ 7UDQVSODQWV SODFHG LQWR DFXWH OHVLRQ FDYLWLHV )RXU PRQWKV DIWHU JUDIWLQJ HDUOLHVW WLPH H[DPLQHGf DOO RI WKH WUDQVSODQWV KDG ILOOHG WKH OHVLRQ FDYLWLHV DQG HDFK ZDV DSSRVHG WR WKH KRVW WLVVXH DORQJ WKH URVWUDO DQG ODWHUDO ERUGHUV 7KH 3+$/ LQMHFWLRQ VLWHV UHVXOWHG LQ KHDY\ ODEHOLQJ RI FHOOV WKURXJKRXW FRUWLFDO OD\HUV )LJ Df /DEHOHG WHUPLQDOV ZHUH IRXQG ZLWKLQ WKH GRUVRODWHUDO VWULDWXP DQG WKDODPXV LQ DOO RI WKHVH DQLPDOV DQG &67 D[RQV ZHUH IRXQG ERWK ZLWKLQ WKH WUDFWV DQG JUD\ PDWWHU URVWUDO WR WKH WUDQVSODQWV 0DQ\ RI WKH ODEHOHG &67 ILEHUV WHUPLQDWHG LQ ODUJH UHWUDFWLRQ EXOEV DV IDU DV PP IURP WKH URVWUDO ERUGHU RI WKH JUDIW )LJ Ef 0RVW RI WKHVH HQGLQJV ZHUH RI D W\SLFDO RYRLG VKDSH UDQJLQJ IURP Pf LQ GLDPHWHU $GGLWLRQDO D[RQDO HQGLQJV ZHUH PRUH LUUHJXODU LQ VKDSH VLPLODU WR WKRVH GHVFULEHG IROORZLQJ LQMXU\ WR WKH GRUVDO FROXPQV RI UDWV DQG RWKHU VSHFLHV 5DPRQ \ &DMDO n /DQFH nf

PAGE 166

7KH LGHQWLILHG &67 D[RQV KDG H[WHQGHG LQWR WKUHH RI WKH WUDQVSODQWV %DVHG XSRQ WKH ORFDWLRQ RI WKHVH ILEHUV ZLWKLQ WKH JUDIWV WKH D[RQV DSSHDUHG WR KDYH SHQHWUDWHG WKH WUDQVSODQWV IURP WKH URVWUDO DQG ODWHUDO KRVWJUDIW ERUGHUV 7KH D[RQV H[WHQGHG LQWR WKH WUDQVSODQWV IRU GLVWDQFHV UDQJLQJ IURP PP EHIRUH DUERUL]LQJ )LJ F Gf /DEHOHG &67 D[RQV ZLWKLQ WKH JUDIWV FRXOG EH IROORZHG IRU VRPH GLVWDQFH LQ WKHVH P VHFWLRQV 'UDZLQJ WXEH WUDFLQJV RI WKH ODEHOHG ILEHUV DFURVV VHYHUDO SODQHV RI IRFXV LQGLFDWHG WKDW RQO\ D IHZ &67 D[RQV LQ WKH JUDIWV KDG EHHQ ODEHOHG ZLWK WKH 3+$/ LQMHFWLRQV 1HYHUWKHOHVV HDFK RI WKH LQJURZLQJ D[RQV H[KLELWHG H[WHQVLYH EUDQFKLQJ ZLWKLQ WKH JUDIW )LJ f $[RQ FROODWHUDOV FRXOG EH IROORZHG DORQJ ORQJLWXGLQDO WUDMHFWRULHV [P ZLWKLQ D VHFWLRQf VLPLODU WR WKRVH UHSRUWHG LQ WKH VXSHUILFLDO GRUVDO KRUQ RI WKH QRUPDO FHUYLFDO VSLQDO FRUG &DVDOH HW DO nf ,Q DOO H[DPSOHV HQODUJHPHQWV UHVHPEOLQJ WHUPLQDO ERXWRQV :RXWHUORRG DQG *URHQHZHJHQ nf ZHUH IRXQG DORQJ WKH OHQJWKV RI WKH D[RQV DV ZHOO DV DW WKHLU HQGLQJV ,Q DGGLWLRQ WR WKH LQJURZWK RI &67 ILEHUV LQ WKHVH WUDQVSODQWV VRPH ODEHOHG D[RQV ZHUH DOVR REVHUYHG DGMDFHQW WR WKH URVWUDO DQG ODWHUDO ERUGHUV RI DOO VL[ RI WKHVH JUDIWV $[RQV WKDW GLG QRW HQWHU WKH WUDQVSODQWV FRXUVHG SDUDOOHO WR WKH KRVWJUDIW LQWHUIDFH DQG H[KLELWHG QRUPDO D[RQDO YDULFRVLWLHV DORQJ WKHLU OHQJWK

PAGE 167

)LJXUH /DEHOHG &67 ILEHUV IROORZLQJ LRQWRSKRUHWLF LQMHFWLRQV RI 3+$/ Df ([DPSOH RI DQ LQMHFWLRQ VLWH ZLWKLQ WKH VHQVRULPRWRU FRUWH[ Ef /DEHOHG ILEHUV DQG WHUPLQDO EXOEV ZLWKLQ WKH GRUVDO &67 DSSUR[ PP URVWUDO WR WKH VLWH RI WUDQVSODQWDWLRQ Ff &UHV\O 9LROHWVWDLQHG VHFWLRQ WKURXJK D PRQWK WUDQVSODQW FRQWDLQLQJ 3+$/ ODEHOHG D[RQV DUURZKHDGVf Gf (QODUJHPHQW RI ODEHOHG &67 ILEHUV ZLWKLQ D PRQWK )6& WUDQVSODQW 6FDOH LQ D QP 6FDOH LQ EFG XUQ

PAGE 168

R

PAGE 169

)LJXUH &67 D[RQV ODEHOHG E\ 3+$/ LQMHFWLRQV LQWR WKH FRUWH[ DUH VHHQ WR HQWHU DQG EUDQFK ZLWKLQ )6& WUDQVSODQWV Df +RUL]RQWDO VHFWLRQ WKURXJK D PRQWK )6& WUDQVSODQW ZLWKLQ WKH PHGLDO UHJLRQ RI WKH KRVW VSLQDO FRUG 7KH GRUVDO &67 LV ZHOO DSSRVHG WR WKH URVWUDO KRVWJUDIW LQWHUIDFH Ef 'UDZLQJ WXEH WUDFLQJ RI D[RQVf ZLWKLQ WKH WUDQVSODQW WKH YHVVHO DORQJ WKH LQWHUIDFH DUURZf FRUUHVSRQGV WR WKH YHVVHO LQ D 6FDOH LQ DE P

PAGE 171

'HOD\HG WUDQVSODQW $Q DGGLWLRQDO UHFLSLHQW UHFHLYHG D WUDQVSODQW SODFHG LQWR D FDYLW\ WKDW ZDV FUHDWHG RQH ZHHN SULRU WR WKH WLPH RI JUDIWLQJ 7KLV UDW ZDV UHWDLQHG IRU PRQWKV DIWHU WUDQVSODQWDWLRQ WR GHWHUPLQH WKH ORQJWHUP SHUVLVWHQFH RI LQJURZLQJ ILEHUV /DEHOHG &67 ILEHUV ZDV REVHUYHG ZLWKLQ WKH WUDQVSODQWHG WLVVXH )LJ f $ JURXS RI ODEHOHG ILEHUV ZDV REVHUYHG H[WHQGLQJ DFURVV WKH LQWHUIDFH EHWZHHQ WKH GRUVDO &67 DQG WKH JUDIW 7KHVH ILEHUV H[WHQGHG D VKRUW GLVWDQFH LQWR WKH URVWUDO SRUWLRQ RI WKH JUDIW DQG VKRZHG PXFK EUDQFKLQJ QHDU WKH LQWHUIDFH )LJ Df /DEHOHG &67 D[RQV DOVR FURVVHG WKH KRVWJUDIW LQWHUIDFH IURP WKH ODWHUDO ERUGHU ZKHUH D VPDOO SRUWLRQ RI WKH GRUVRODWHUDO IXQLFXOXV ZDV DGMDFHQW WR WKH WUDQVSODQW )LJ Ef :KLOH WKHVH ODEHOHG D[RQV ZHUH FRQFHQWUDWHG QHDU WKH SHULSKHU\ RI WKH JUDIW VRPH ZHUH DOVR IRXQG GHHSHU ZLWKLQ WKH JUDIW 7KH EUDQFKLQJ SDWWHUQV RI ODEHOHG D[RQV ZHUH VLPLODU WR WKRVH IRXQG LQ WKH PRQWK WUDQVSODQWV 7KH D[RQV EUDQFKHG DQG VXUURXQGHG WKH WUDQVSODQW QHXURQV DQG YDULFRVLWLHV ZHUH IRXQG DGMDFHQW WR DQG EHWZHHQ WKH FRXQWHUVWDLQHG SHULNDU\D )LJ FGf (OHFWURQ PLFURVFRS\ 5HJLRQV RI WKH WUDQVSODQWV WKDW FRQWDLQHG ODEHOHG FRUWLFRVSLQDO ILEHUV ZHUH H[DPLQHG ZLWK WKH HOHFWURQ PLFURVFRSH )LJ f $[RQDO SURILOHV FRQWDLQLQJ 3+$/ ZHUH GLVWULEXWHG WKURXJKRXW WKHVH DUHDV RI WKH JUDIW QHXURSLO

PAGE 172

)LJXUH &67 D[RQV ZLWKLQ D )6& JUDIW SODFHG LQWR D ZHHN ROG OHVLRQ DQG ODEHOHG E\ 3+$/ LQMHFWLRQV LQWR WKH FRUWH[ DW PRQWKV DIWHU JUDIWLQJ Df ,QJURZWK RI ODEHOHG &67 ILEHUV DUURZVf DFURVV WKH URVWUDO KRVWJUDIW LQWHUIDFH DQG LQWR WKH WUDQVSODQW 7f Ef 'DUNILHOG SKRWRPLFURJUDSK RI D[RQV H[WHQGLQJ LQWR WKH JUDIW 7f IURP WKH ODWHUDO KRVW Kf ERUGHU Ff /DEHOHG D[RQV ZLWKLQ &UHV\O 9LROHW VWDLQHG VHFWLRQ RI WKH JUDIW Gf +LJKSRZHU SKRWRPLFURJUDSK LOOXVWUDWHV EUDQFKLQJ DQG WHUPLQDO IRUPDWLRQ ZLWKLQ WKH JUDIW QHXURSLO 6FDOH LQ D QP E P FG QP

PAGE 174

)LJXUH (OHFWURQ PLFURVFRS\ RI WUDQVSODQW UHJLRQV FRQWDLQLQJ ODEHOHG &67 D[RQV Df /RZSRZHU HOHFWURQ PLFURJUDSK RI 3+$/ ODEHOHG XQP\HOLQDWHG D[RQV DQG SRVVLEOH WHUPLQDOV ZLWK WKH WUDQVSODQW DUURZVf Ef /LJKW PLFURJUDSK RI D SODVWLFHPEHGGHG VHFWLRQ FRQWDLQLQJ 3+$/ ODEHOHG ILEHUV ZLWKLQ D GHOD\HG JUDIW DW PRQWKV SRVWWUDQVSODQWDWLRQ Ff $ 3+$/ FRQWDLQLQJ WHUPLQDO WKDW LV QHDU D JUDIW GHQGULWH Gf Gf 6\QDSWLF HQGLQJV EHWZHHQ DQ XQODEHOHG D[RQ Xf DQG 3+$ / ODEHOHG &67 D[RQ DUURZf DQG D JUDIW GHQGULWH Gf 3RVW V\QDSWLF HOHFWURQGHQVH UHJLRQV DUH HYLGHQW DUURZKHDGVf Hf $[RGHQGULWLF V\QDSVH EHWZHHQ D 3+$/ ODEHOHG WHUPLQDO DUURZf DQG D GHQGULWH Gf ZLWKLQ D WUDQVSODQW 1RWH WKH HOHFWURQGHQVH SRVWV\QDSWLF WKLFNHQLQJ DUURZKHDGVf 6FDOH LQ D O2 P E [P FGH cLP

PAGE 176

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

PAGE 177

DSSRVLWLRQ RI WKHVH D[RQV DQG WKH JUDIW WLVVXH 7KHVH D[RQV PD\ SURYLGH WKH EDVLV IRU LQGLUHFW SRO\V\QDSWLF LQWHUDFWLRQV EHWZHHQ &67 D[RQV DQG WUDQVSODQW QHXURQV $OWHUQDWLYHO\ WKH H[WHQVLRQ RI GHQGULWHV RI WKH JUDIW QHXURQV DFURVV WKH URVWUDO LQWHUIDFH 0DKDOLN HW DO n &ODUNH HW DO nEf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n /DQFH n *LOVRQ DQG 6WHQVDDV n .DOLO DQG 6FKQHLGHU nf 7KLV UHWUDFWLRQ RI LQMXUHG D[RQV LV DVVRFLDWHG ZLWK UHSHDWHG H[WUXVLRQ RI D[RSODVP IURP WKH

PAGE 178

GLODWHG WHUPLQDOV .DR DQG &KDQJ n .DR HW DO nOOf DQG LV XVXDOO\ FRQVLGHUHG WR FRUUHVSRQG WR D IDLOXUH RI UHJHQHUDWLRQ ,Q WKH URGHQW &67 WKH SURFHVV RI UHWURJUDGH D[RQDO GHJHQHUDWLRQ LV IXUWKHU FRPSOLFDWHG E\ WKH YDVFXODU UHVSRQVH WR VSLQDO WUDXPD 6SHFLILFDOO\ WKH SURJUHVVLYH SDWKRORJLFDO HYHQWV IROORZLQJ FRQWXVLYH LQVXOW WR WKH VSLQDO FRUG UHVXOW LQ WKH IRUPDWLRQ RI D FRQLFDO UHJLRQ RI WLVVXH QHFURVLV 7KLV UHDFWLRQ OHDGV WR WKH IRUPDWLRQ RI F\VWLF FDYLWLHV ZKLFK H[WHQG LQWR WKH EDVH RI WKH GRUVDO FROXPQV LQ ERWK WKH URVWUDO DQG FDXGDO GLUHFWLRQV HJ %DOHQWLQH DQG 3DULV nD 1REHO DQG :UDWKDOO nf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

PAGE 179

UDSLGO\ ZKLOH VPDOOHU D[RQV SHUVLVW DW WKH HGJH RI D OHVLRQ IRU ORQJHU WLPHV XS WR VRPH PRQWKV DIWHU LQMXU\f EHIRUH GHJHQHUDWLQJ WR WKH QHDUHVW D[RQ FROODWHUDO 5DPRQ \ &DMDO n 7RZHU 02 /DQFH n .DOLO DQG 6FKQHLGHU nf 7KH YDULDWLRQ LQ UHWUDFWLRQ UDWHV ZLWKLQ WKH &67 KDV DOVR EHHQ REVHUYHG E\ RWKHU DXWKRUV XVLQJ DQWHURJUDGH WUDFLQJ WHFKQLTXHV HJ .DR HW DO n )LVKPDQ DQG .HOO\ nEf DQG WKXV LW PD\ EH UHVSRQVLEOH IRU WKH WDSHUHG DSSHDUDQFH RI WKH &67 VHHQ DIWHU LQMXU\ ,Q WKH SUHVHQW VWXG\ WKH UHWUDFWLRQ GLVWDQFH RI WKH LQMXUHG &67 ZDV OHVV ZKHQ OHVLRQV ZHUH PDGH LQ FHUYLFDO VSLQDO FRUG &f WKDQ ZKHQ VLPLODU OHVLRQV ZHUH PDGH IRXU VHJPHQWV ORZHU 7f 2QH UHDVRQ IRU WKLV ILQGLQJ PD\ EH WKDW WKH &67 D[RQV UHWUDFWHG WR WKH ODVW VXVWDLQLQJ FROODWHUDO DQG WKHUHIRUH WKH GLVWDQFH UHIOHFWV D JUHDWHU GHQVLW\ RI D[RQDO FROODWHUDOV LQ WKH FHUYLFDO HQODUJHPHQW WKDQ LQ XSSHU WKRUDFLF FRUG 'RQDWHOOH nOOn *ULEQDX HW DO nf 7KH GLVWDQFHV RI &67 UHWUDFWLRQ DW VL[ ZHHNV DIWHU SDUWLDO OHVLRQV ZHUH OHVV WKDQ WKRVH UHSRUWHG E\ 3DOOLQL HW DO nf DW GD\V DIWHU FRPSOHWH WUDQVHFWLRQ QR WUDQVSODQWf DW WKH 7 YHUWHEUDO OHYHO %DVHG XSRQ WKH SUHYLRXV DUJXPHQW WKLV GLIIHUHQFH PD\ EH SDUWO\ DWWULEXWHG WR WKH GLIIHUHQW VSLQDO OHYHOV $OWHUQDWLYHO\ YDULDWLRQV LQ D[RQDO UHWUDFWLRQ PD\ EH GXH WR WKH QDWXUH RI WKH OHVLRQ SDUWLDO YV FRPSOHWH WUDQVHFWLRQf +RZHYHU LW LV LPSRUWDQW WR QRWH WKDW WKH PHWKRGV XVHG WR GHWHUPLQH WKH GLHEDFN

PAGE 180

GLVWDQFH ZHUH DOVR FRQVLGHUDEO\ GLIIHUHQW ,Q WKDW VWXG\ 70% VWDLQHG VHFWLRQV ZHLH H[DPLQHG LQ EULJKWILHOG VHH DOVR )LJ f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f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

PAGE 181

ZHUH SODFHG LQWR WKH FRQWXVHG VSLQDO FRUG :LQLDOVNL DQG 5HLHU nf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

PAGE 182

GHYHORSLQJ VSLQDO FRUG PD\ SURYLGH D EHQHILFLDO WHUUDLQ IRU WKH HORQJDWLRQ RI D[RQ FROODWHUDOV LQWR WKH JUDIW WLVVXH %UHJPDQ HW DO nf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nf LQ ZKLFK UHWURJUDGH GHJHQHUDWLRQ RI &67 ILEHUV ZDV H[DPLQHG IROORZLQJ WUDQVSODQWDWLRQ RI )6& WLVVXH LQWR FRPSOHWH WUDQVHFWLRQV LQ DGXOW UDWV ,Q WKDW VWXG\ WKHUH ZHUH QR GLIIHUHQFHV LQ WKH DYHUDJH UHWUDFWLRQ RI LQMXUHG ILEHUV LQ WKH SUHVHQFH DQG DEVHQFH RI JUDIWV 7KH GLVFUHSDQF\ EHWZHHQ WKH SUHVHQW ILQGLQJV DQG WKDW UHSRUW PD\ UHIOHFW HLWKHU WKH PHWKRGV RI JXDQWLI\LQJ WKH GLVWDQFH RI D[RQDO GHJHQHUDWLRQ DERYHf RU WKH GLIIHUHQW OHYHOV RI WKH LQMXU\ & RU 7 YV 7f $OWHUQDWLYHO\ GLIIHUHQFHV LQ WKH

PAGE 183

H[WHQW RI WKH LQLWLDO OHVLRQ DQG SDWKRORJ\ SDUWLDO OHVLRQ YV FRPSOHWH WUDQVHFWLRQf DQG VXEVHTXHQW JUDIW VXUYLYDO DQG LQWHJUDWLRQ PD\ KDYH UHVXOWHG LQ WKH GLIIHUHQW RXWFRPHV &67 $[RQV 5HJHQHUDWH RU 6SURXW LQWR )6& 7UDQVSODQWV 7KH IDWH RI LQMXUHG &67 QHXURQV DQG WKHLU FDSDFLW\ IRU UHJHQHUDWLRQ KDV EHHQ FRQWURYHUVLDO 2QH K\SRWKHVLV WKDW UHFHLYHG VXSSRUW IRU PDQ\ \HDUV LV WKDW WKH LQMXUHG &67 QHXURQV GLHG DIWHU VSLQDO FRUG OHVLRQ 7KLV EHOLHI ZDV EDVHG XSRQ D GHFUHDVH LQ DQWHURJUDGH WUDQVSRUW RI WULWLDWHG SUROLQH )HULQJD HW DO nDf DQG DQ DSSDUHQW UHGXFWLRQ LQ WKH QXPEHU RI ODUJH %HW] FHOOV LQ WKH FRUWH[ +ROPHV DQG 0D\ n )HULQJD HW DO nEf ,Q DGGLWLRQ IHZHU &67 QHXURQV ZHUH UHWURJUDGHO\ ODEHOHG ZLWK +53 DSSOLHG SUR[LPDO WR D VSLQDO WUDQVHFWLRQ )HULQJD HW DO n )HULQJD DQG 9DKOVLQJ nf 7KH FRQWUDVWLQJ YLHZ KDV EHHQ WKDW WKH LQMXUHG QHXURQV VXUYLYH EXW VKULQN LQ VL]H .DOLO DQG 6FKQHLGHU n )HULQJD HW DO nE %DUURQ HW DO nf 7KH GHFUHDVH LQ DQWHURJUDGH DQG UHWURJUDGH ODEHOLQJ PD\ UHSUHVHQW DQ DOWHUDWLRQ LQ WKH D[RQDO WUDQVSRUW SURFHVVHV RI WKHVH QHXURQV IROORZLQJ LQMXU\ *RVKJDULDQ HW DO n 3UXLWW HW DO nf 7KHUH LV OLWWOH HYLGHQFH WR GDWH WKDW DGXOW &67 D[RQV DUH FDSDEOH RI UHJHQHUDWLRQ DIWHU LQMXU\ /DQFH nf 7KH LQDELOLW\ RI FHQWUDO D[RQV WR UHJHQHUDWH KDV IUHTXHQWO\ EHHQ DWWULEXWHG WR WKH SURKLELWLYH HQYLURQPHQW RI WKH DGXOW QHUYRXV V\VWHP +RZHYHU VRPH UHSRUWV KDYH VXJJHVWHG WKDW

PAGE 184

DGGLWLRQDO LQWULQVLF OLPLWDWLRQV PD\ EH XQLTXH WR WKH ORQJ P\HOLQDWHG ILEHU WUDFWV RI WKH DGXOW VXFK DV WKH &67 )RU H[DPSOH 5LFKDUGVRQ HW DO nf ZHUH DEOH WR GHPRQVWUDWH VSURXWLQJ RI VHYHUDO W\SHV RI VSLQDO FRUG DQG EUDLQVWHP QHXURQV LQWR SHULSKHUDO QHUYH JUDIWV EXW WKH\ ZHUH XQDEOH WR LGHQWLI\ UHJHQHUDWLQJ &67 D[RQV 2QH UHDVRQ IRU WKH ODFN RI FRUWLFRVSLQDO LQJURZWK LQWR WKHVH SHULSKHUDO QHUYHV PD\ KDYH EHHQ WKH GLVWDQFH IURP WKH SHULNDU\D WR WKH VLWH RI LPSODQWDWLRQ UHYLHZHG LQ $JXD\R nf $OWHUQD WLYHO\ WKH UHWURJUDGH +53 WUDFLQJ WHFKQLTXHV XVHG LQ WKHVH SHULSKHUDO QHUYH VWXGLHV PD\ KDYH EHHQ OLPLWHG LQ WKHLU VHQVLWLYLW\ WR GHWHFW VPDOO QXPEHUV RI HORQJDWLQJ &67 QHXURQV RU WR LGHQWLI\ D[RQV ZLWK DEQRUPDO UHWURJUDGH WUDQVSRUW FDSDFLWLHV 2WKHU UHVHDUFKHUV KDYH FODLPHG WKDW WKH WHUPLQDOV RI LQMXUHG &67 D[RQV GLIIHU IURP WKRVH RI LQMXUHG GRUVDO FROXPQ ILEHUV )LVKPDQ DQG .HOO\ nDEf 7KHVH DXWKRUV UHSRUWHG WKDW LQMXUHG &67 D[RQV DOO IRUPHG GLVWLQFW RYRLG WHUPLQDO UHWUDFWLRQ EXOEV ZKLOH LQMXUHG SULPDU\ DIIHUHQWV ZLWKLQ WKH GRUVDO FROXPQV H[KLELWHG HQGLQJV VXJJHVWLYH RI JURZWK FRQHV )URP WKHVH REVHUYDWLRQV WKH\ KDYH VXJJHVWHG WKDW WKH PRUSKRORJLFDO GLIIHUHQFH PD\ UHIOHFW D GLIIHUHQFH LQ WKH DELOLW\ WR UHJHQHUDWH DIWHU LQMXU\ 7KH SUHVHQW UHVXOWV LOOXVWUDWH WKDW DGXOW &67 D[RQV DUH FDSDEOH RI UHJHQHUDWLRQ RU FROODWHUDO VSURXWLQJ LQWR WUDQVSODQWV RI IHWDO VSLQDO FRUG WLVVXH 7KH GLVWDQFH RI LQJURZWK LV VLPLODU WR SUHYLRXV REVHUYDWLRQV IRU RWKHU

PAGE 185

GHVFHQGLQJ DQG ORFDO ILEHU V\VWHPV 5HLHU HW DO nD &KDSWHU f 7KH H[WHQW RI EUDQFKLQJ DQG WKH FDSDFLW\ RI LQMXUHG &67 D[RQV WR IRUP V\QDSVHV XSRQ JUDIW QHXURQV VXJJHVW WKDW WKH LQJURZWK RI HYHQ D IHZ &67 ILEHUV ZLWKLQ WKH JUDIWV PD\ EH VXIILFLHQW WR DFWLYDWH D QXPEHU RI QHXURQV ZLWKLQ WKH WUDQVSODQWV ,W LV LPSRUWDQW WR QRWH KRZHYHU WKDW VRPH DEQRUPDO FKDUDFWHULVWLFV RI WKH FRUWLFRVSLQDO D[RQV ZHUH IRXQG ZLWKLQ WKH JUDIWV )RU H[DPSOH WKH ODUJH WHUPLQDO SOH[XVHV )LJ Gf DQG WKH WRUWXRXV SDWKZD\ RI VRPH D[RQV )LJ Gf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n 1LHWR6DPSHGUR HW DO nf ,Q DGGLWLRQ LW KDV EHHQ VKRZQ LQ WKH SHULSKHUDO QHUYRXV V\VWHP WKDW D SULRU FRQGLWLRQLQJ OHVLRQ FDQ VHUYH WR HQKDQFH WKH UDWH RI D[RQDO UHJHQHUDWLRQ 0F4XDUULH nf ,W LV SRVVLEOH WKDW VLPLODU IDFWRUV PD\ LQIOXHQFH LQWHUDFWLRQV EHWZHHQ KRVW DQG JUDIW QHXURQV LQ WKH JUD\ PDWWHU RI WKH

PAGE 186

VSLQDO FRUG ,W ZDV XQNQRZQ ZKHWKHU WKH GLHEDFN RI WKH ORQJ P\HOLQDWHG ILEHU WUDFWV DIWHU LQMXU\ ZRXOG H[HUW DQ RSSRVLWH HIIHFW ZLWK D GHOD\ DQG WKXV SUHYHQW WKH LQJURZWK RI WKHVH WUDFWV LQWR )6& WUDQVSODQWV ,Q WKH SUHVHQW VWXG\ D GHOD\ RI IRXU GD\V WR RQH ZHHN EHWZHHQ FUHDWLQJ D OHVLRQ DQG LPSODQWDWLRQ RI IHWDO VSLQDO FRUG WLVVXH GLG QRW SUHYHQW WKH DSSRVLWLRQ RI &67 D[RQV WR WKH JUDIW RU WKH LQJURZWK RI &67 ILEHUV
PAGE 187

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

PAGE 188

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

PAGE 189

7KHVH FHOOV DUH GLVWULEXWHG ZLWKLQ ERWK P\HOLQDWHG DQG XQP\HOLQDWHG UHJLRQV RI QHXURSLO 6PDOO EXQGOHV RI P\HOLQDWHG ILEHUV DUH DOVR IRXQG ZLWKLQ WKH JUDIW WLVVXHV +RZHYHU WKHVH D[RQV DUH QRW RUJDQL]HG LQWR ORQJ WUDFWV H[WHQGLQJ WKH OHQJWK RI WKH WUDQVSODQWV $[RQDO WUDFLQJ DQG LPPXQRF\WRFKHPLFDO ILQGLQJV GHPRQVWUDWH WKH SUHVHQFH RI VKRUWGLVWDQFH SURMHFWLRQV EHWZHHQ KRVW DQG JUDIW WLVVXHV $IIHUHQW LQSXW LV LQGLFDWHG E\ WKH LQJURZWK RI KRVW D[RQV DULVLQJ IURP SULPDU\ DIIHUHQW ILEHUV ORFDO VSLQDO FRUG QHXURQV DQG FHOOV ZLWKLQ WKH KRVW FRUWH[ K\SRWKDODPXV DQG EUDLQVWHP *UDIW QHXURQV DOVR H[WHQG D[RQV LQWR WKH VXUURXQGLQJ KRVW VSLQDO FRUG ZKHUH WKH\ WHUPLQDWH DURXQG QHXURQV ZLWKLQ WKH GRUVDO DQG YHQWUDO JUD\ PDWWHU )LQDOO\ DQ H[WHQVLYH SDWWHUQ RI LQWULQVLF SURMHFWLRQV LV REVHUYHG ZLWKLQ WKH JUDIW WLVVXH ,Q DGGLWLRQ WR LQWHUDFWLRQV HVWDEOLVKHG E\ WKRVH D[RQV WKDW H[WHQG DFURVV WKH KRVWJUDIW LQWHUIDFH WKHUH LV DQ H[WHQVLYH LQWHUGLJLWDWLRQ RI QHXULWLF SURFHVVHV ZLWKLQ UHJLRQV RI IXVLRQ EHWZHHQ WKH KRVW DQG JUDIW WLVVXHV 7RJHWKHU WKHVH DQDWRPLFDO ILQGLQJV VHUYH WR GHILQH WKH HOHPHQWV WKDW PD\ FRPSRVH D QHXUDO UHOD\ DFURVV )6& JUDIWV %\ FRPELQLQJ HYLGHQFH RI DIIHUHQW LQWULQVLF DQG HIIHUHQW SURMHFWLRQV LW LV SRVVLEOH WR DVVHPEOH D YDULHW\ RI K\SRWKHWLFDO UHOD\ FLUFXLW SDWWHUQV WR VXEVHUYH WKH WUDQVPLVVLRQ RI DVFHQGLQJ GHVFHQGLQJ DQG VHJPHQWDO LQIRUPDWLRQ DFURVV D )6& JUDIW )LJ f

PAGE 190

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nDf 7KH DQDWRPLFDO RUJDQL]DWLRQ SUHVHQW HYHQ ZLWKLQ WKH LQWHUQHXURQDO SRSXODWLRQV RI WKH VSLQDO FRUG VXJJHVWV D JUHDW GHDO RI VSHFLILFLW\ LQ WHUPV RI D[RQDO SURMHFWLRQ SDWWHUQV DQG V\QDSWLF FRQQHFWLRQV 0ROHQDDU n *REHO HW DO n %UDV HW DO nf 7KXV WKH IRUPDWLRQ RI D IXQFWLRQDO UHOD\ DFURVV D )6& JUDIW PD\ UHTXLUH WKH UHSOLFDWLRQ RI VRPH GHJUHH RI VSHFLILFLW\ LQ WKH SDWWHUQV RI D[RQDO SURMHFWLRQV EHWZHHQ KRVW DQG JUDIW WLVVXHV 7KH DGXOW KLSSRFDPSXV KDV VHUYHG DV D PRGHO V\VWHP WR H[DPLQH WKH VSHFLILFLW\ RI D[RQDO SURMHFWLRQV EHWZHHQ KRVW DQG JUDIW WLVVXHV %MRUNOXQG DQG 6WHQHYL n 5DLVPDQ DQG (EQHU nf 7KHVH VWXGLHV KDYH VKRZQ WKDW WUDQVSODQWHG QHXURQV ZLOO UHLQQHUYDWH RQO\ WKH DSSURSULDWH WDUJHW UHJLRQV RI D GHQHUYDWHG KLSSRFDPSXV 7KH LQJURZWK RI KRVW ILEHUV

PAGE 191

)LJXUH 'LDJUDP RI K\SRWKHWLFDO UHOD\ FLUFXLWU\ HVWDEOLVKHG DFURVV D )6& WUDQVSODQW (YHU\ QHXUDO FLUFXLW LV FRPSRVHG RI Df LQSXW QHXURQV Ef LQWULQVLF QHXURQV DQG Ff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

PAGE 192

'HVFHQGLQJ 5HOD\ 3URSULRVSLQDO DQG 6HJPHQWDO 5HOD\

PAGE 193

LV DOVR GHSHQGHQW XSRQ D PDWFK EHWZHHQ WKH ILEHU W\SH DQG WKH QRUPDO WDUJHW UHJLRQ ,Q WKLV PRGHO D KLJK GHJUHH RI V\QDSWLF VSHFLILFLW\ DSSHDUV WR EH UHVWRUHG IROORZLQJ WUDQVSODQWDWLRQ 6LPLODU VSHFLILFLW\ KDV EHHQ GHPRQVWUDWHG IROORZLQJ WUDQVSODQWDWLRQ RI EUDLQVWHP VXVSHQVLRQ JUDIWV LQWR WKH WUDQVHFWHG DGXOW VSLQDO FRUG 3ULYDW n /RKLXV +LUVKILHOG DQG 5HLHU XQSXEOLVKHG REVHUYDWLRQVf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

PAGE 194

VXJJHVW WKDW VRPH HOHPHQWV ZLWKLQ WKH KRVW VSLQDO FRUG HQYLURQPHQW PD\ LQIOXHQFH WKH GLUHFWLRQ RI D[RQDO HORQJDWLRQ /LWWOH LQIRUPDWLRQ KDV EHHQ REWDLQHG UHJDUGLQJ WKH VSHFLILFLW\ RI DIIHUHQW JURZWK LQWR WKH WUDQVSODQWV 7KH GLIIHUHQWLDWLRQ RI GLVWLQFW 6*OLNH UHJLRQV ZLWKLQ WKH JUDIWV KRZHYHU PD\ SURYLGH D VXLWDEOH PRGHO IRU WHVWLQJ VRPH DVSHFWV RI WKLV D[RQDO VSHFLILFLW\ &KDSWHU f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nf 7UDQVSODQWV RI IHWDO VWULDWDO WLVVXH ZKHQ SODFHG LQWR VWULDWDO OHVLRQV LQ DGXOW DQLPDOV ZLOO UHFHLYH KRVW DIIHUHQW LQSXW IURP DSSURSULDWH EUDLQ UHJLRQV

PAGE 195

1HXURQV ZLWKLQ WKH JUDIWV DOVR H[WHQG HIIHUHQW SURMHFWLRQV LQWR WKH QRUPDO WDUJHW DUHDV ZLWKLQ WKH EUDLQ &ODUNH HW DO n :LFWRULQ HW DO n nf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nf 3RVVLEOH 5ROH RI )6& *UDIWV LQ 6HJPHQWDO DQG /RQJ7UDFW )XQFWLRQV 7KH DQDWRPLFDO FKDUDFWHULVWLFV RI KRVW DQG JUDIW SURMHFWLRQV FDQ EH FRPSDUHG ZLWK ZKDW LV NQRZQ DERXW WKH RUJDQL]DWLRQ RI WKH QRUPDO VSLQDO FRUG 7KLV LQIRUPDWLRQ PD\ WKHQ EH XVHG WR SUHGLFW WKH IXQFWLRQDO FDSDFLW\ WKDW D )6& JUDIW UHOD\ FLUFXLW PD\ ILOO LQ WKH LQMXUHG VSLQDO FRUG )RU H[DPSOH WKH YDVW PDMRULW\ RI FRQQHFWLRQV ZLWKLQ WKH QRUPDO VSLQDO FRUG DUH FRPSRVHG RI VKRUW SURMHFWLRQV RI WKH VSLQDO LQWHUQHXURQV 6]HQWDJRWKDL nnDE 0ROHQDDU n
PAGE 196

SOD\ DQ LPSRUWDQW UROH LQ WKH LQWHJUDWLRQ RI VHJPHQWDO SURMHFWLRQV ZLWK WKH LQWHUVHJPHQWDO DQG ORQJWUDFW V\VWHPV HJ 6]HQWDJRWKDL n 6FKHLEHO DQG 6FKHLEHO n /XQGEHUJ n -DQNRZVND DQG 5REHUWV n -DQNRZVND HW DO nOE %UDV HW DO nf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f FRQFOXGHG WKDW WKH UHWLFXORVSLQDO DQG SURSULRVSLQDO ILEHUV IRUP D FRQWLQXRXV SRO\V\QDSWLF SDWKZD\ WKDW RULJLQDWHV LQ WKH EUDLQVWHP DQG H[WHQGV WKH OHQJWK RI WKH VSLQDO FRUG +H SURSRVHG WKDW PXOWLV\QDSWLF SDWKZD\V LQYROYLQJ ERWK VKRUW DQG ORQJ SURSULRVSLQDO D[RQV FDQ SOD\ D VLJQLILFDQW UROH LQ WKH WUDQVPLVVLRQ RI GHVFHQGLQJ LQIRUPDWLRQ ,Q VWXGLHV HPSOR\LQJ D VLPLODU DSSURDFK WR WKDW XVHG E\ /OR\G 6KLN nf VXJJHVWHG WKDW PXOWLV\QDSWLF SDWKZD\V PD\ PHGLDWH WKH WUDQVPLVVLRQ RI GHVFHQGLQJ ORFRPRWRU LQIRUPDWLRQ IURP WKH EUDLQVWHP WR ORZHU VSLQDO FRUG OHYHOV

PAGE 197

/RQJ SRO\V\QDSWLF SDWKZD\V FDQ DOVR FRQWULEXWH WR VRPH W\SHV RI IXQFWLRQDO UHFRYHU\ LQ WKH DEVHQFH RI ORQJ WUDFW ILEHUV $ QXPEHU RI UHSRUWV KDYH LQGLFDWHG WKDW UHVLGXDO VHQVRU\ DQG PRWRU IXQFWLRQV FDQ EH UHWDLQHG DIWHU PXOWLSOH VWDJJHUHG KHPLVHFWLRQV DUH SHUIRUPHG WR GLVUXSW WKH ORQJ ILEHU WUDFWV -DQH HW DO nf UHSRUWHG UHFRYHU\ RI ORFRPRWRU EHKDYLRU LQ FDWV IROORZLQJ VXFK FURVVHG KHPLVHFWLRQV 6LPLODU VWXGLHV KDYH GHPRQVWUDWHG WKH UHFRYHU\ RI HOHPHQWV RI SDLQ VHQVDWLRQ DIWHU VWDJJHUHG KHPLVHFWLRQV LQ UDWV DQG SLJV %UHD]LOH DQG .LWFKHOO n %DVEDXP nf 7KXV WKH GHYHORSPHQW RI D UHOD\ FLUFXLW DFURVV D )6& JUDIW PD\ LPSURYH VRPH GHJUHH RI IXQFWLRQ E\ UHSODFLQJ WKH ORQJ ILEHU WUDFWV DW WKH OHVLRQ VLWH ZLWK QHZO\ IRUPHG SRO\n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f RI ORQJ WUDFW ILEHUV DFURVV D OHVLRQ 7KH XVH RI SHULSKHUDO QHUYH 316f JUDIWV PD\ EH ZHOO

PAGE 198

VXLWHG WR WKLV ODWWHU SUREOHP ,QLWLDO VWXGLHV LQGLFDWHG WKDW &16 QHXURQV SDUWLFXODUO\ WKRVH ORFDWHG QHDU WKH RULJLQ RI D 316 JUDIW ZLOO H[WHQG D[RQV IRU ORQJ GLVWDQFHV ZLWKLQ WKH SHULSKHUDO HQYLURQPHQW 'DYLG DQG $JXD\R f 0RUH UHFHQW H[SHULPHQWV LQYROYLQJ WKH SODFHPHQW RI 316 JUDIWV ZLWKLQ WKH RSWLF QHUYH KDYH GHPRQVWUDWHG WKDW ODUJH QXPEHUV RI ORQJ WUDFW ILEHUV FDQ EH GLUHFWHG WR DQ DSSURSULDWH WDUJHW UHJLRQ ZLWKLQ WKH &16 $JXD\R n %UD\ HW DO nf 7KHVH D[RQV WKHQ H[WHQG IRU D VKRUW GLVWDQFH ZLWKLQ WKH &16 ZKHUH WKH\ IRUP IXQFWLRQDO V\QDSVHV XSRQ QHXURQV LQ WKH WDUJHW UHJLRQ .LHUVWHDG HW DO nf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nf 7KHUHIRUH LI HLWKHU )6& RU 316 JUDIWV DUH XVHG WR SURPRWH IXQFWLRQDO UHFRYHU\ DIWHU D OHVLRQ RI WKH ORQJ WUDFW ILEHUV LW LV OLNHO\ WKDW WKRVH VHQVRU\ IXQFWLRQV WKDW DUH GHSHQGHQW XSRQ

PAGE 199

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n ,WD\D DQG YDQ +RHVHQ n -DQNRZVND DQG 6NRRJ nf ,Q DGGLWLRQ PHWKRGV HPSOR\LQJ PXOWLSOHODEHOLQJ SURFHGXUHV PD\ EH XVHG WR LGHQWLI\ WKH V\QDSWLF UHODWLRQVKLSV EHWZHHQ DVFHQGLQJ GHVFHQGLQJ DQG ORFDO FLUFXLW QHXURQV DW ERWK WKH OLJKW DQG XOWUDVWUXFWXUDO OHYHOV +HLPHU DQG =DERUV]N\ nf :LWK WKH DQDWRPLFDO EDVHOLQH HVWDEOLVKHG E\ WKHVH VWXGLHV IXWXUH WHVWV RI HOHFWURSK\VLRORJLFDO DQG EHKDYLRUDO PHDVXUHV DUH LQGLFDWHG WR GHWHUPLQH ZKHWKHU WKH SURMHFWLRQV EHWZHHQ KRVW DQG JUDIW WLVVXHV UHSUHVHQW IXQFWLRQDO LQWHUDFWLRQV %DVHG RQ WKHVH REVHUYDWLRQV HOHFWUR SK\VLRORJLFDO VWXGLHV DUH FXUUHQWO\ XQGHUZD\ WR H[DPLQH DVSHFWV RI V\QDSWLF IXQFWLRQ EHWZHHQ KRVW DQG JUDIW WLVVXHV 5HVXOWV RI SUHOLPLQDU\ VLQJOH XQLW H[WUDFHOOXODU UHFRUGLQJ

PAGE 200

H[SHULPHQWV VXJJHVW WKDW QHXURQV ZLWKLQ WKHVH JUDIWV DUH V\QDSWLFDOO\ DFWLYDWHG E\ VWLPXODWLRQ RI GRUVDO URRW ILEHUV 7KRPSVRQ HW DO nf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

PAGE 201

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

PAGE 202

5()(5(1&(6 $EHUFURPELH 0 f (VWLPDWLRQ RI QXFOHDU SRSXODWLRQV IURP PLFURWRPH VHFWLRQV $QDW 5HF $JXD\R $f $[RQDO UHJHQHUDWLRQ IURP LQMXUHG QHXURQV LQ WKH DGXOW PDPPDOLDQ FHQWUDO QHUYRXV V\VWHP ,Q &: &RWPDQ HGf 6\QDSWLF 3ODVWLFLW\ 1HZ
PAGE 203

%DUURQ .' 03 'HQWLQJHU $3RSS DQG 5 0DQNHV f 1HXURQV RI OD\HU 9E RI UDW VHQVRULPRWRU FRUWH[ DWURSK\ EXW GR QRW GLH DIWHU WKRUDFLF FRUG WUDQVHFWLRQ -1HXURSDWK([S1HXURO %DVEDXP $ f &RQGXFWLRQ RI WKH HIIHFWV RI QR[LRXV VWLPXODWLRQ E\ VKRUWILEHU PXOWLV\QDSWLF V\VWHPV RI WKH VSLQDO FRUG LQ WKH UDW ([S1HXURO %HDVOH\ / DQG :% 6WDOOFXS f 7KH QHUYH JURZWK IDFWRULQGXFLEOH ODUJH H[WHUQDO 1,/(f JO\FRSURWHLQ DQG QHXUDO FHOO DGKHVLRQ PROHFXOH1&$0f KDYH GLVWLQFW SDWWHUQV RI H[SUHVVLRQ LQ WKH GHYHORSLQJ UDW FHQWUDO QHUYRXV V\VWHP -1HXURVFL %HDWWLH 06 %7 6WRNHV DQG -& %UHVQDKDQ f ([SHULPHQWDO VSLQDO FRUG LQMXU\ 6WUDWHJLHV IRU DFXWH DQG FKURQLF LQWHUYHQWLRQ EDVHG RQ DQDWRPLF SK\VLRORJLFDO DQG EHKDYLRUDO VWXGLHV ,Q '* 6WHLQ DQG %$ 6DJHO HGVf 3KDUPDFRORJLFDO $SSURDFKHV WR 7UHDWPHQW RI %UDLQ DQG 6SLQDO &RUG ,QLXUY 1HZ
PAGE 204

%MRUNOXQG $ %+RIIHU 05 3DOPHU $ 6HLJHU DQG / 2OVRQ f 6XUYLYDO DQG JURZWK RI QHXURQV ZLWK HQNHSKDOLQOLNH LPPXQRUHDFWLYLW\ LQ IHWDO EUDLQ DUHDV JUDIWHG WR WKH DQWHULRU FKDPEHU RI WKH H\H 1HXURVFL %MRUNOXQG $ /LQGYDOO 2 ,VDFVRQ 3 %UXQGLQ :LFWRULQ 5( 6WUHFNHU '&ODUNH DQG6% 'XQQHWW f 0HFKDQLVPV RI DFWLRQ RI LQWUDFHUHEUDO QHXUDO LPSODQWV VWXGLHV RQ QLJUDO DQG VWULDWDO JUDIWV WR WKH OHVLRQHG VWULDWXP 7,16 %MRUNOXQG $ DQG 8 6WHQHYL f ,QWUDFHUHEUDO QHXUDO LPSODQWV 1HXURQDO UHSODFHPHQW DQG UHFRQVWUXFWLRQ RI GDPDJHG FLUFXLWULHV $QQ 5HY 1HXURVFL %MRUNOXQG + DQG 'DKO f *OLDO GLVWXUEDQFHV LQ LVRODWHG QHRFRUWH[ (YLGHQFH IURP LPPXQRKLVWRFKHPLVWU\ RI LQWUDRFXODU JUDIWV 'HY1HXURVFL %MRUNOXQG + 'DKO +DJOLG / 5RVHQJUHQ DQG / 2OVRQ f $VWURF\WLF GHYHORSPHQW LQ IHWDO SDULHWDO FRUWH[ JUDIWHG WR FHUHEUDO DQG FHUHEHOODU FRUWH[ RI LPPDWXUH UDWV 'HY %UDLQ 5HV %MRUNOXQG + DQG / 2OVRQ f $VWURF\WLF GHYHORSPHQW LQ LQWUDRFXODU DQG LQWUDFUDQLDO FRUWH[ FHUHEUL JUDIWV ,Q 'HYHORSLQJ DQG 5HJHQHUDWLQJ 9HUWHEUDWH 1HUYRXV 6\VWHPV 1HZ
PAGE 205

IURP JURXS ,, PXVFOH DIIHUHQWV LQ WKH FDW VSLQDO FRUG -&RPS1HXURO %UHD]LOH -( DQG 5/ .LWFKHOO f $ VWXG\ RI ILEHU V\VWHPV ZLWKLQ WKH VSLQDO FRUG RI WKH GRPHVWLF SLJ WKDW VXEVHUYH SDLQ -&RPS1HXURO %UHJPDQ %6 f 6SLQDO FRUG WUDQVSODQWV SHUPLW WKH JURZWK RI VHURWRQHUJLF D[RQV DFURVV WKH VLWH RI QHRQDWDO VSLQDO FRUG WUDQVHFWLRQ 'HY%UDLQ 5HV %UHJPDQ %6 DQG 0( *ROGEHUJHU f ,QIDQW OHVLRQ HIIHFW ,,, $QDWRPLFDO FRUUHODWHV RI VSDULQJ DQG UHFRYHU\ RI IXQFWLRQ DIWHU VSLQDO FRUG GDPDJH LQ QHZERUQ DQG DGXOW FDWV 'HY%UDLQ 5HV %UHJPDQ %6 ( .XQNHO%DJGHQ 0 0DF$WHH DQG $ 2n1HLOO f ([WHQVLRQ RI WKH FULWLFDO SHULRG IRU GHYHORSPHQWDO SODVWLFLW\ RI WKH FRUWLFRVSLQDO SDWKZD\ -&RPS1HXURO %UHJPDQ %6 DQG 35HLHU f 1HXUDO WLVVXH WUDQVSODQWV UHVFXH D[RWRPL]HG UXEURVSLQDO FHOOV IURP UHWURJUDGH GHDWK -&RPS1HXURO %XFKDQDQ -7 DQG +2 1RUQHV f 7UDQVSODQWV RI HPEU\RQLF EUDLQVWHP FRQWDLQLQJ WKH ORFXV FRHUXOHXV LQWR VSLQDO FRUG HQKDQFH WKH KLQGOLPE IOH[LRQ UHIOH[ LQ DGXOW UDWV %UDLQ 5HV %XOOLWW ( DQG $5 /LJKW f ,QWUDVSLQDO FRXUVH RI GHVFHQGLQJ VHURWRQHUJLF SDWKZD\V LQQHUYDWLQJ WKH URGHQW GRUVDO KRUQ DQG ODPLQD ; -&RPS1HXURO %X]VDNL DQG )+ *DJH f 0HFKDQLVPV RI DFWLRQ RI QHXUDO JUDIWV LQ WKH OLPELF V\VWHP &DQ 1HXURVFL &DEDQD 7 DQG *) 0DUWLQ f 'HYHORSPHQWDO VHTXHQFH LQ WKH RULJLQ RI GHVFHQGLQJ VSLQDO SDWKZD\V 6WXGLHV XVLQJ UHWURJUDGH WUDQVSRUW WHFKQLTXHV LQ WKH 1RUWK $PHULFDQ RSSRVXP 'LGHOSKLV YLUJLQLDQDf 'HY%UDLQ 5HV &DURQL 3 DQG 0( 6FKZDE f 7ZR PHPEUDQH SURWHLQ IUDFWLRQV IURP UDW FHQWUDO P\HOLQ ZLWK LQKLELWRU\ SURSHUWLHV IRU QHXULWH JURZWK DQG ILEUREODVW VSUHDGLQJ -&HOO %LRO

PAGE 206

&DVDOH ($5 /LJKW DQG $ 5XVWLRQL f 'LUHFW SURMHFWLRQ RI WKH FRUWLFRVSLQDO WUDFW WR WKH VXSHUILFLDO ODPLQDH RI WKH VSLQDO FRUG LQ WKH UDW &RPS 1HXURO &HUYHUR ) DQG $ ,JJR f 7KH VXEVWDQWLD JHODWLQRVD RI WKH VSLQDO FRUG $ FULWLFDO UHYLHZ %UDLQ &KXQJ *$ .HYHWWHU :' :LOOLV DQG 5 &RJJHVKDOO f $Q HVWLPDWH RI WKH UDWLR RI SURSULRVSLQDO WR ORQJ WUDFW QHXURQV LQW KH VDFUDO VSLQDO FRUG RI WKH UDW 1HXURVFL/HWW &ODUNH '6% 'XQQHWW 2 ,VDFVRQ '-6 6LULQDWKVLQJKML DQG $ %MRUNOXQG Df 6WULDWDO JUDIWV LQ UDWV ZLWK XQLODWHUDO QHRVWULDWDO OHVLRQV 8OWUDVWUXFWXUDO HYLGHQFH RI DIIHUHQW V\QDSWLF LQSXWV IURP WKH KRVW QLJURVWULWDO SDWKZD\ 1HXURVFL &ODUNH '3 %UXQGLQ 5( 6WUHFNHU 2* 1LOVVRQ $ %MRUNOXQG DQG /LQGYDOO Ef +XPDQ IHWDO GRSDPLQH QHXURQV JUDIWHG LQ D UDW PRGHO RI SDUNLQVRQnV GLVHDVH XOWUDVWUXFWXUDO HYLGHQFH IRU V\QDSVH IRUPDWLRQ XVLQJ W\URVLQH K\GUR[\ODVH LPPXQRF\WRFKHPLVWU\ ([S%UDLQ 5HV &OHPHQWH &' f 5HJHQHUDWLRQ LQ WKH YHUWHEUDWH FHQWUDO QHUYRXV V\VWHP ,QW5HY1HXURELRO &RLPEUD $ %3 6RGUH%RUJHV DQG 00 0DJDOKDHV f 7KH VXEVWDQWLD JHODWLQRVD 5RODQGL RI WKH UDW )LQH VWUXFWXUH F\WRFKHPLVWU\ DFLG SKRVSKDWDVHf DQG FKDQJHV DIWHU GRUVDO URRW VHFWLRQ -1HXURFYW &ROH -' f 7KH SDWKRSK\VLRORJ\ RI WKH DXWRQRPLF QHUYRXV V\VWHP LQ VSLQDO FRUG LQMXU\ ,Q /6 OLOLV HGf 6SLQDO &RUG '\VIXQFWLRQ $VVHVVPHQW 2[IRUG 2[IRUG 8QLYHUVLW\ 3UHVV SS &RPPLVVLRQJ -: f )HWDO ORFXV FRHUXOHXV WUDQVSODQWHG LQWR WKH WUDQVHFWHG VSLQDO FRUG RI WKH DGXOW UDW 6RPH REVHUYDWLRQV DQG LPSOLFDWLRQV 1HXURVFL &RWPDQ &: DQG 0 1LHWR6DPSHGUR f 3URJUHVV LQ IDFLOLWDWLQJ WKH UHFRYHU\ RI IXQFWLRQ DIWHU FHQWUDO QHUYRXV V\VWHP WUDXPD $QQ1<$FDG6FL 'DV *' f 1HXUDO WUDQVSODQWDWLRQ LQ WKH VSLQDO FRUG RI DGXOW UDWV &RQGLWLRQV VXUYLYDO F\WRORJ\ DQG FRQQHFWLYLW\ RI WKH WUDQVSODQWV -1HXURO6FL

PAGE 207

'DV *' f 1HXUDO WUDQVSODQWDWLRQ LQ VSLQDO FRUG XQGHU GLIIHUHQW FRQGLWLRQV RI OHVLRQV DQG WKHLU IXQFWLRQDO VLJQLILFDQFH ,Q *' 'DV DQG 5% :DOODFH HGVf 1HXUDO 7UDQVSODQWDWLRQ DQG 5HJHQHUDWLRQ 1HZ
PAGE 208

'XQQHWW 6% 2 ,VDFVRQ '-6 6LULQDWKVLQJKML '&ODUNH DQG $ %MRUNOXQG f 6WULDWDO JUDIWV LQ UDWV ZLWK XQLODWHUDO QHRVWULDWDO OHVLRQV ,,, 5HFRYHU\ IURP GRSDPLQHGHSHQGHQW PRWRU DV\PPHWU\ DQG GHILFLWV LQ VNLOOHG SDZ UHDFKLQJ 1HXURVFL 'XQQHWW 6% 7RQLROR $ )LQH &0 5\DQ $ %MRUNOXQG DQG 6' ,YHUVHQ f 7UDQVSODQWDWLRQ RI HPEU\RQLF YHQWUDO IRUHEUDLQ QHXURQV WR WKH QHRFRUWH[ RI UDWV ZLWK OHVLRQV RI QXFOHXV EDVDOLV PDJQRFHOOXODULV ,, 6HQVRULPRWRU DQG OHDUQLQJ LPSDLUPHQWV 1HXURVFL (EQHU )) 56 (U]XUXPOX DQG 60 /HH f 3HULSKHUDO QHUYH GDPDJH IDFLOLWDWHV IXQFWLRQDO LQQHUYDWLRQ RI EUDLQ JUDIWV LQ DGXOW VHQVRU\ FRUWH[ 3URF 1DWO $FDG 6FL 86$ (QJ /) f *OLDO ILEULOODU\ DFLGLF SURWHLQ $ UHYLHZ RI VWUXFWXUH IXQFWLRQ DQG FOLQLFDO DSSOLFDWLRQ ,Q 1HXURQDO DQG *OLDO 3URWHLQV 6WUXFWXUH )XQFWLRQ DQG &OLQLFDO $SSOLFDWLRQ 1HZ
PAGE 209

)HULQJD (5 +/ 9DKOVLQJ DQG %( 6PLWK f 5HWURJUDGH WUDQVSRUW LQ FRUWLFRVSLQDO QHXURQV DIWHU VSLQDO FRUG WUDQVHFWLRQ 1HXURORJ\ )HULQJD (5 +/ 9DKOVLQJ DQG 5& 'DXVHU Ef 7KH RUWKRJUDGH IORZ RI WULWLDWHG SUROLQH LQ FRUWLFRVSLQDO QHXURQV DW YDULRXV DJHV DQG DIWHU VSLQDO FRUG LQMXU\ -1HXURO1HXURVXUD3VYFKLDW‘ )HULQJD (5 DQG +/ 9DKOVLQJ f /DEHOHG FRUWLFRVSLQDO QHXURQV RQH \HDU DIWHU VSLQDO FRUG WUDQVHFWLRQ 1HXURVFL/HWW )LQVHQ % DQG =LPPHU f 7LPP VWDLQLQJ RI KLSSRFDPSDO QHUYH FHOO ERGLHV LQ WKH .\RWR UDW $ FHOO PDUNHU LQ DOOR DQG [HQRJUDIWLQJ RI UDW DQG PRXVH EUDLQ WLVVXH UHYHDOLQJ QHXURQDO PLJUDWLRQ 'HY%UDLQ 5HV )LVKPDQ 36 DQG -3 .HOO\ Df ,GHQWLILHG FHQWUDO D[RQV GLIIHU LQ WKHLU UHVSRQVH WR VSLQDO FRUG WUDQVHFWLRQ %UDLQ 5HV )LVKPDQ 36 DQG -3 .HOO\ Ef 7KH IDWH RI VHYHUHG FRUWLFRVSLQDO D[RQV 1HXURORJ\ )RQVHFD 0 'H)HOLSH DQG $ )DLUHQ f /RFDO FRQQHFWLRQV LQ WUDQVSODQWHG DQG QRUPDO FHUHEUDO FRUWH[ RI UDWV ([S %UDLQ 5HV )RUVVEHUJ + DQG 6 *ULOOQHU f 7KH ORFRPRWLRQ RI WKH DFXWH VSLQDO FDW LQMHFWHG ZLWK FORQLGLQH LY %UDLQ 5HV )RVWHU *$ 0 6FKXOW]EHUJ )+ *DJH $ %MRUNOXQG 7 +RNIHOW + 1RUQHV $& &XHOOR $$ 9HUKRIVWDG DQG 79LVVHU f 7UDQVPLWWHU H[SUHVVLRQ DQG PRUSKRORJLFDO GHYHORSPHQW RI HPEU\RQLF PHGXOODU\ DQG PHVHQFHSKDOLF UDSKH QHXURQHV DIWHU WUDQVSODQWDWLRQ WR WKH DGXOW UDW FHQWUDO QHUYRXV V\VWHP *UDIWV WR WKH VSLQDO FRUG ([S%UDLQ 5HV )UHHG :DQG +( &DQQRQ6SRRU f &RUWLFDO OHVLRQV LQFUHDVH UHLQQHUYDWLRQ RI WKH GRUVDO VWULDWXP E\ VXEVWDQWLD QLJUD JUDIWV %UDLQ 5HV )UHXQG 7) -3 %RODP $ %MRUNOXQG 8 6WHQHYL 6% 'XQQHWW -) 3RZHOO DQG $ 6PLWK f (IIHUHQW V\QDSWLF FRQQHFWLRQV RI JUDIWHG GRSDPLQHUJLF QHXURQV UHLQQHUYDWLQJ WKH KRVW QHRVWULDWXP $ W\URVLQH K\GUR[\ODVH LPPXQRF\WRFKHPLFDO VWXG\ -1HXURVFL

PAGE 210

*DJH )+ DQG %X]VDNL f &16 JUDIWLQJ SRWHQWLDO PHFKDQLVPV RI DFWLRQ ,Q )6HLO HGf 1HXUDO 5HJHQHUDWLRQ DQG 7UDQVSODQWDWLRQ 1HZ
PAGE 211

*REHO 6 :0 )DOOV *%HQQHWW 0 $EGHOPRXPHQWH + +D\DVKL DQG ( +XPSKUH\ f $Q (0 DQDO\VLV RI WKH V\QDSWLF FRQQHFWLRQV RI KRUVHUDGLVK SHUR[LGDVHILOOHG VWDONHG FHOOV DQG LVOHW FHOOV LQ WKH VXEVWDQWLD JHODWLQRVD RI DGXOW FDW VSLQDO FRUG -&RPS1HXURO *ROGEHUJHU 0( DQG 0 0XUUD\ f 3DWWHUQV RI VSURXWLQJ DQG LPSOLFDWLRQV IRU UHFRYHU\ RI IXQFWLRQ ,Q 6* :D[PDQ HGf $GYDQFHV LQ 1HXURORJ\ 9RO )XQFWLRQDO 5HFRYHU\ LQ 1HXURORJLFDO 'LVHDVH 1HZ
PAGE 212

+DXQ ) DQG 7&XQQLQJKDP f 6SHFLILF QHXURWURSKLF LQWHUDFWLRQV EHWZHHQ FRUWLFDO DQG VXEFRUWLFDO YLVXDO VWUXFWXUHV LQ GHYHORSLQJ UDW ,Q YLWUR VWXGLHV -&RPS1HXURO +HLPHU / DQG / =DERUV]N\ f 1HXURDQDWRPLFDO 7UDFW7UDFLQJ 0HWKRGV 5HFHQW 3URJUHVV 1HZ
PAGE 213

-DFREVRQ 5' 9LUDJ DQG -+3 6NHQH f $ SURWHLQ DVVRFLDWHG ZLWK D[RQ JURZWK *$3 LV ZLGHO\ GLVWULEXWHG DQG GHYHORSPHQWDOO\ UHJXODWHG LQ UDW &16 -1HXURVFL -DHJHU &% DQG 5' /XQG f 7UDQVSODQWDWLRQ RI HPEU\RQLF RFFLSLWDO FRUWH[ WR WKH WHFWDO UHJLRQ RI QHZERUQ UDWV $ OLJKW PLFURVFRSLF VWXG\ RI WKH RUJDQL]DWLRQ DQG FRQQHFWLYLW\ RI WKH WUDQVSODQWV -&RPS1HXURO -DNHPDQ /% DQG 35HLHU f 7KH UHVSRQVH RI FRUWLFRVSLQDO WUDFW ILEHUV IROORZLQJ LQMXU\ DQG WUDQVSODQWDWLRQ LQ WKH DGXOW UDW VSLQDO FRUG 6RFA1HXURVFL$EVW -DNHPDQ /% DQG 35HLHU f $[RQDO SURMHFWLRQV EHWZHHQ IHWDO VSLQDO FRUG WUDQVSODQWV DQG WKH DGXOW UDW VSLQDO FRUG 6RF1HXURVFL$EVW -DNHPDQ /% DQG 35HLHU f 5HJHQHUDWLRQ RU VSURXWLQJ RI FRUWLFRVSLQDO WUDFW D[RQV LQWR IHWDO VSLQDO FRUG WUDQVSODQWV LQ WKH DGXOW UDW 6RF1HXURVFL$EVW -DNHPDQ /% 35HLHU %6 %UHJPDQ (% :DGH 0 'DLOH\ 5.DVWQHU %7 +LPHV DQG $ 7HVVOHU f 'LIIHUHQWLDWLRQ RI VXEVWDQWLD JHODWLQRVDOLNH UHJLRQV LQ LQWUDVSLQDO DQG LQWUDFHUHEUDO WUDQVSODQWV RI HPEU\RQLF VSLQDO FRUG WLVVXH LQ WKH UDW ([S1HXURO -DQH -$ -3 (YDQV DQG /( )LVKHU f $Q LQYHVWLJDWLRQ FRQFHUQLQJ WKH UHVWLWXWLRQ RI PRWRU IXQFWLRQ IROORZLQJ LQMXU\ WR WKH VSLQDO FRUG 1HXURVXUJ -DQNRZVND ( $ /XQGEHUJ :5REHUWV DQG 6WXDUW f $ ORQJ SURSULRVSLQDO V\VWHP ZLWK GLUHFW HIIHFW RQ PRWRQHXURQHV DQG RQ LQWHUQHXURQHV LQ WKH FDW OXPERVDFUDO FRUG ([S%UDLQ 5HV -DQNRZVND ( DQG :5REHUWV f $Q HOHFWUR SK\VLRORJLFDO GHPRQVWUDWLRQ RI WKH D[RQDO SURMHFWLRQV RI VLQJOH VSLQDO LQWHUQHXURQHV LQ WKH FDW -3K\VLRO -DQNRZVND ( DQG % 6NRRJ f /DEHOOLQJ RI PLGOXPEDU QHXURQHV SURMHFWLQJ WR FDW KLQGOLPE PRWRQHXURQHV E\ WUDQVQHXURQDO WUDQVSRUW RI D KRUVHUDGLVK SHUR[LGDVH FRQMXJDWH 1HXURVFL/HWW

PAGE 214

-RKQVRQ 0, DQG 53 %XQJH f 3ODVWLFLW\ LQ QHXURWUDQVPLWWHU H[SUHVVLRQ DQG WKH XVH RI QHXURQDO UHOD\V LQ VSLQDO FRUG UHSDLU ,Q && .DR 53 %XQJH DQG 35HLHU HGVf 6SLQDO &RUG 5HFRQVWUXFWLRQ 1HZ
PAGE 215

.URPHU /) $ %MRUNOXQG DQG 8 6WHQHYL f ,QWUDFHSKDOLF HPEU\RQLF QHXUDO LPSODQWV LQ WKH DGXOW UDW EUDLQ *URZWK DQG PDWXUH RUJDQL]DWLRQ RI EUDLQVWHP FHUHEHOODU DQG KLSSRFDPSDO LPSODQWV &RPS 1HXURO .UXJHU 6 6LHYHUV & +DQVHQ 0 6DGOHU DQG 0 %HUU\ f 7KUHH PRUSKRORJLFDOO\ GLVWLQFW W\SHV RI LQWHUIDFH GHYHORS EHWZHHQ DGXOW KRVW DQG IHWDO EUDLQ WUDQVSODQWV LPSOLFDWLRQV IRU VFDU IRUPDWLRQ LQ WKH DGXOW FHQWUDO QHUYRXV V\VWHP -&RPS1HXURO .XDQJ 5= DQG .DOLO f 6SURXWLQJ RI FRUWLFRVSLQDO ILEHUV LQWR GHQHUYDWHG FRQWUDODWHUDO VSLQDO FRUG UHYHDOV VSHFLILFLW\ RI QHZ D[RQ DUERUV 6RF1HXURVFL$EVW .XQNHO%DJGHQ ( DQG %6 %UHJPDQ f 7UDQVSODQWV DOWHU WKH GHYHORSPHQW RI VHQVRULPRWRU IXQFWLRQ DIWHU QHRQDWDO VSLQDO FRUG GDPDJH ([S %UDLQ 5HVILQ SUHVVf /D0RWWH && f 2UJDQL]DWLRQ RI GRUVDO KRUQ QHXURWUDQVPLWWHU V\VWHPV ,Q 7
PAGE 216

/XQGEHUJ $ f &RQYHUJHQFH RI H[FLWDWRU\ DQG LQKLELWRU\ DFWLRQ RQ LQWHUQHXURQHV LQ WKH VSLQDO FRUG ,Q 0$% %UD]LHU HGf 7KH ,QWHUQHXURQ 8&/$ )RUXP 0HG VFL 1R /RV $QJHOHV 8QLY RI &DOLIRUQLD 3UHVV SS 0DKDOLN 77( )LQJHU 6WURPEHUJ DQG / 2OVRQ f 6XEVWDQWLD QLJUD WUDQVSODQWV LQWR GHQHUYDWHG VWULDWXP RI WKH UDW 8OWUDVWUXFWXUH RI JUDIW DQG KRVW LQWHUFRQQHFWLRQV &RPS 1HXURO 0F$OOLVWHU -3 65 &REHU (5 6FKDLEOH DQG 3' :DONHU f 0LQLPDO FRQQHFWLYLW\ EHWZHHQ VL[ PRQWK QHRVWULDWDO WUDQVSODQWV DQG WKH KRVW VXEVWDQWLD QLJUD %UDLQ 5HV 0F&RQQHOO 6. f 0LJUDWLRQ DQG GLIIHUHQWLDWLRQ RI FHUHEUDO FRUWLFDO QHXURQV DIWHU WUDQVSODQWDWLRQ LQWR WKH EUDLQV RI IHUUHWV 6FLHQFH 0F/RRQ 6& DQG 5' /XQG f 'HYHORSPHQW RI IHWDO UHWLQD WHFWXP DQG FRUWH[ WUDQVSODQWHG WR WKH VXSHULRU FROOLFXOXV RI DGXOW UDWV &RPS 1HXURO 0F1HLOO '/ 5( &RJJHVKDOO DQG 60 &DUOWRQ f $ OLJKW DQG HOHFWURQ PLFURVFRSLF VWXG\ RI FDOFLWRQLQ JHQHUHODWHG SHSWLGH LQ WKH VSLQDO FRUG RI WKH UDW ([S1HXURO 0F4XDUULH,* f 7KH HIIHFW RI D FRQGLWLRQLQJ OHVLRQ RQ WKH UHJHQHUDWLRQ RI PRWRU D[RQV %UDLQ 5HV 0HO]DFN 5 DQG 3' :DOO f 3DLQ PHFKDQLVPV D QHZ WKHRU\ 6FLHQFH 0HQHWUH\ GH 3RPPHU\ DQG ) 5RXGLHU f 3URSULRVSLQDO ILEHUV UHDFKLQJ WKH OXPEDU HQODUJHPHQW LQ WKH UDW 1HXURVFL/HWW 0HVXOXP 00 f 7UDFLQJ 1HXURQDO &RQQHFWLRQV ZLWK +RUVHUDGLVK 3HUR[LGDVH 1HZ
PAGE 217

0RODQGHU & 4 ;X & 5LYHUR0HOLDQ DQG *UDQW f &\WRDUFKLWHFWRQLF RUJDQL]DWLRQ RI WKH VSLQDO FRUG LQ WKH UDW ,, 7KH FHUYLFDO DQG XSSHU WKRUDFLF FRUG -&RPS1HXURO 0ROHQDDU f 7KH GLVWULEXWLRQ RI SURSULRVSLQDO QHXURQV SURMHFWLQJ WR GLIIHUHQW PRWRQHXURQDO FHOO JURXSV LQ WKH FDWnV EUDFKLDO FRUG %UDLQ 5HV 0RRUPDQ 6-5 :KDOHQ DQG +2 1RUQHV f 6WXGLHV RI KLQGOLPE UHIOH[HV DIIHFWHG E\ LPSODQWV RI IHWDO EUDLQVWHP WLVVXH LQ WKH VSLQDO FRUG RI WKH UDW 6RF1HXURVFL$EVW 0RXFKHW 3 0 0DQLHU 0 'LHWO & )HXHUVWHLQ $ %HURG 0 $UOXLVRQ / 'HQRUR\ DQG 7KLEDXOW f ,PPXQRKLVWR FKHPLFDO VWXG\ RI FDWHFKRODPLQHUJLF FHOO ERGLHV LQ WKH UDW VSLQDO FRUG %UDLQ 5HV%XOO 0XIVRQ (5 /DEEH DQG '* 6WHLQ f 0RUSKRORJLF IHDWXUHV RI HPEU\RQLF QHRFRUWH[ JUDIWV LQ DGXOW UDWV IROORZLQJ IURQWDO FRUWLFDO DEODWLRQ %UDLQ 5HV 1DIWFKL 1( 6$EUDKDPV 60 &UDLQ (5 3HWHUVRQ -0 +LOOHU DQG (6LPRQ f 3UHVHQFH RI OHXFLQHHQNHSKDOLQ LQ RUJDQRW\SLF H[SODQWV RI IHWDO PRXVH VSLQDO FRUG 3HSWLGHV 1DWKDQLHO (-+ DQG '5 1DWKDQLHO f 7KH UHDFWLYH DVWURF\WH $GY &HOO 1HXURELRO 1HZWRQ %: DQG 5: +DPLOO f 7KH PRUSKRORJ\ DQG GLVWULEXWLRQ RI UDW VHURWRQLQHUJLF LQWUDVSLQDO QHXURQV $Q LPPXQRKLVWRFKHPLFDO VWXG\ %UDLQ 5HV%XOO 1HZWRQ %: %( 0DOH\ DQG 5: +DPLOO f ,PPXQRKLVWRFKHPLFDO GHPRQVWUDWLRQ RI VHURWRQLQ QHXURQV LQ DXWRQRPLF UHJLRQV RI WKH UDW VSLQDO FRUG %UDLQ 5HV 1LHWR6DPSHGUR 0 -3 .HVVODN 5% *LEEV DQG &: &RWPDQ f (IIHFWV RI FRQGLWLRQLQJ OHVLRQV RQ WUDQVSODQW VXUYLYDO FRQQHFWLYLW\ DQG IXQFWLRQ $QQ 1< $FDG6FL 1REHO /DQG -5 :UDWKDOO f 7KH EORRGVSLQDO FRUG EDUULHU DIWHU LQMXU\ 3DWWHUQ RI YDVFXODU HYHQWV SUR[LPDO DQG GLVWDO WR D WUDQVHFWLRQ LQ WKH UDW %UDLQ 5HV

PAGE 218

1RUQHV + $ %MRUNOXQG DQG 8 6WHQHYL f 7HPSRUDO SDWWHUQ RI QHXURJHQHVLV LQ VSLQDO FRUG RI UDW $Q DXWRUDGLRJUDSKLF VWXG\ f§ 7LPH DQG VLWHV RI RULJLQ DQG PLJUDWLRQ DQG VHWWOLQJ SDWWHUQV RI QHXUREODVWV %UDLQ 5HV 1RUQHV + $ %MRUNOXQG DQG 8 6WHQHYL f 5HLQQHUYDWLRQ RI WKH GHQHUYDWHG DGXOW VSLQDO FRUG RI UDWV E\ LQWUDVSLQDO WUDQVSODQWV RI HPEU\RQLF EUDLQ VWHP QHXURQV &HOO 7LVVXH 5HV 1RUQHV + $ %MRUNOXQG DQG 8 6WHQHYL f 7UDQVSODQWDWLRQ VWUDWHJLHV LQ VSLQDO FRUG UHJHQHUDWLRQ ,Q -5 6ODGHN DQG '0 *DVK HGVf 1HXUDO 7UDQVSODQWV 'HYHORSPHQW DQG )XQFWLRQ 1HZ
PAGE 219

3ULW]HO 0 2 ,VDFVRQ 3 %UXQGLQ / :LNOXQG DQG $ %MRUNOXQG f $IIHUHQW DQG HIIHUHQW FRQQHFWLRQV RI VWULDWDO JUDIWV LPSODQWHG LQWR WKH LERWHQLF DFLG OHVLRQHG QHRVWULDWXP LQ DGXOW UDWV ([S %UDLQ 5HV 3ULYDW $ + 0DQVRXU $ 3DY\ 0 *HIIDUG DQG ) 6DQGLOORQ f 7UDQVSODQWDWLRQ RI GLVVRFLDWHG IRHWDO VHURWRQLQ QHXURQV LQWR WKH WUDQVHFWHG VSLQDO FRUG RI DGXOW UDWV 1HXURVFL/HWW 3ULYDW $ + 0DQVRXU 1 5DMDRIHWUD DQG 0 *HIIDUG f ,QWUDVSLQDO WUDQVSODQWV RI VHURWRQHUJLF QHXURQV LQ WKH DGXOW UDW %UDLQ 5HV %XOO 3UXLWW -1 (5 )HULQJD DQG 5/ 0F%ULGH f &RUWLFRVSLQDO D[RQV SHUVLVW LQ FHUYLFDO DQG KLJK WKRUDFLF UHJLRQV ZHHNV DIWHU D 7 VSLQDO FRUG WUDQVHFWLRQ 1HXURORJ\ 5DLVPDQ DQG )) (EQHU f 0RVV\ ILEUH SURMHFWLRQV LQWR DQG RXW RI KLSSRFDPSDO WUDQVSODQWV 1HXURVFL 5DNLF 3& f /RFDO &LUFXLW 1HXURQV &DPEULGJH0$ 0,7 3UHVV 5DOVWRQ +f 7KH ILQH VWUXFWXUH RI /DPLQDH ,, DQG ,,, RI WKH 0DFDTXH VSLQDO FRUG -&RPS1HXURO 5DPRQ \ &DMDO 6 f 'HJHQHUDWLRQ DQG 5HJHQHUDWLRQ RI WKH 1HUYRXV 6\VWHP /RQGRQ 7UDQVODWHG E\ 50 0D\ +DIQHU 3XEOLVKLQJ &RPSDQ\ 5DQVRKRII f 6XUJLFDO LQWHUYHQWLRQ DIWHU WUDXPDWLF LQMXU\ ,Q :) :LQGOH HGf 7KH 6SLQDO &RUG DQG ,WV 5HDFWLRQ WR 7UDXPDWLF ,QMXU\ 1HZ
PAGE 220

5HLHU 3%6 %UHJPDQ DQG -5 :XMHN f ,QWUDVSLQDO WUDQVSODQWV RI HPEU\RQLF VSLQDO FRUG WLVVXH LQ DGXOW DQG QHRQDWDO UDWV HYLGHQFH IRU WRSRJUDSKLFDO GLIIHUHQWLDWLRQ DQG D[RQDO LQWHUDFWLRQV ZLWK WKH KRVW &16 ,Q $ %MRUNOXQG DQG 8 6WHQHYL HGVf 1HXUDO *UDIWLQJ LQ WKH 0DPPDOLDQ &16 $PVWHUGDP (OVHYLHU 6FLHQFH 3XEOLVKHUV SS 5HLHU 3%6 %UHJPDQ DQG -5 :XMHN Df ,QWUDVSLQDO WUDQVSODQWDWLRQ RI HPEU\RQLF VSLQDO FRUG WLVVXH LQ QHRQDWDO DQG DGXOW UDWV -&RPS1HXURO 5HLHU 3%6 %UHJPDQ DQG -5 :XMHN Ef ,QWUDVSLQDO WUDQVSODQWDWLRQ RI IHWDO VSLQDO FRUG WLVVXH $Q DSSURDFK WRZDUG IXQFWLRQDO UHSDLU RI WKH LQMXUHG VSLQDO FRUG ,Q 0 *ROGEHUJHU $ *RULR DQG 0 0XUUD\ HGVf 3URFHHGLQJV RI WKH ,QWHUQDWLRQDO 6\PSRVLXP RQ 3ODVWLFLW\ DQG 'HYHORSPHQW RI WKH 0DPPDOLDQ 6SLQDO &RUG 3DGRYD /LYLDQD 3UHVV )LGLD 5HVHDUFK 6HULHV SS 5HLHU 3/) (QJ DQG /% -DNHPDQ f 5HDFWLYH DVWURF\WH DQG D[RQDO RXWJURZWK LQ WKH LQMXUHG &16 ,V JOLRVLV UHDOO\ DQ LPSHGLPHQW WR UHJHQHUDWLRQ" ,Q )6HLO HGf 1HXUDO 5HJHQHUDWLRQ DQG 7UDQVSODQWDWLRQ 1HZ
PAGE 221

5H[HG % f 7KH F\WRDUFKLWHFWRQLF RUJDQL]DWLRQ RI WKH VSLQDO FRUG LQ WKH FDW -&RPS1HXURO 5LFKDUGVRQ 30 80 0F*XLQHVV DQG $$JXD\R f 3HULSKHUDO QHUYH DXWRJUDIWV WR WKH UDW VSLQDO FRUG 6WXGLHV ZLWK D[RQDO WUDFLQJ PHWKRGV %UDLQ 5HV 5LFKDUGVRQ 30 90. ,VVD DQG $$JXD\R f 5HJHQHUDWLRQ RI ORQJ VSLQDO D[RQV LQ WKH UDW -1HXURFYW 5RELQVRQ *$ DQG 0( *ROGEHUJHU f 7KH GHYHORSPHQW DQG UHFRYHU\ RI PRWRU IXQFWLRQ LQ VSLQDO FDWV,, 3KDUPDFRORJLFDO HQKDQFHPHQW RI UHFRYHU\ ([S%UDLQ 5HV 6DQGOHU $1 DQG &+ 7DWRU f 5HYLHZ RI WKH HIIHFW RI VSLQDO FRUG WUDXPD RQ WKH YHVVHOV DQG EORRG IORZ LQ WKH VSLQDO FRUG 1HXURVXUD 6DXQGHUV 5' // 'XJDQ 3 'HPHGLXN ('0HDQV / $ +RUURFNV DQG '. $QGHUVRQ f (IIHFWV RI PHWK\OSUHGQLVRORQH DQG WKH FRPELQDWLRQ RI DOSKDWDFRSKHURO DQG VHOHQLXP RQ DUDFKLGRQLF DFLG PHWDEROLVP DQG OLSLG SHUR[LGDWLRQ LQ WUDXPDWL]HG VSLQDO FRUG WLVVXH 1HXURFKHP 6FKHLEHO 0( DQG $% 6FKHLEHO f $ VWUXFWXUDO DQDO\VLV RI VSLQDO LQWHUQHXURQV DQG UHQVKDZ FHOOV ,Q 0$% %UD]LHU HGf 7KH ,QWHUQHXURQ 8&/$ )RUXP 0HG 6FL 1R /RV $QJHOHV 8QLY RI &DOLIRUQLD 3UHVV SS 6FKPXHG /& DQG -+ )DOORQ f )OXRUR*ROG D QHZ IOXRUHVFHQW UHWURJUDGH D[RQDO WUDFHU ZLWK QXPHURXV XQLTXH SURSHUWLHV %UDLQ 5HV 6FKUH\HU 'DQG (* -RQHV f *URZWK DQG WDUJHW ILQGLQJ E\ D[RQV RI WKH FRUWLFRVSLQDO WUDFW LQ SUHQDWDO DQG SRVWQDWDO UDWV 1HXURVFL 6FKUH\HU 'DQG (* -RQHV f *URZLQJ FRUWLFRVSLQDO D[RQV E\SDVV QHRQDWDO UDW VSLQDO FRUG 1HXURVFL 6H\EROG 9 DQG 5 (OGH f ,PPXQRKLVWRFKHPLFDO VWXGLHV RI SHSWLGHUJLF QHXURQV LQ WKH GRUVDO KRUQ RI WKH VSLQDO FRUG -+LVWRFKHP&YWRFKHP 6H\EROG 9 DQG 5 (OGH f 1HXURWHQVLQ LPPXQRUHDFWLYLW\ LQ WKH VXSHUILFLDO ODPLQDH RI WKH GRUVDO KRUQ RI WKH UDW /LJKW PLFURVFRSLF VWXGLHV RI FHOO ERGLHV DQG SUR[LPDO GHQGULWHV -&RPS1HXURO

PAGE 222

6KHSDUG *0 f 1HXURELRORT\ 1HZ
PAGE 223

6WHQHYL 8 $ %MRUNOXQG DQG 1$ 6YHQJDDUG f 7UDQVSODQWDWLRQ RI FHQWUDO DQG SHULSKHUDO PRQRDPLQH QHXURQV WR WKH DGXOW UDW EUDLQ 7HFKQLTXHV DQG FRQGLWLRQV IRU VXUYLYDO %UDLQ 5HV 6WHUQEHUJHU /$ f ,PPXQRFYWRFKHPLVWUY 1HZ
PAGE 224

7ROEHUW '/ DQG 7 'HU f 5HGLUHFWHG JURZWK RI S\UDPLGDO WUDFW D[RQV IROORZLQJ QHRQDWDO S\UDPLGRWRP\ LQ FDWV -&RPS1HXURO 7RZHU 66 f 3\UDPLGDO OHVLRQ LQ WKH PRQNH\ %UDLQ 9DKOVLQJ +/ DQG (5 )HULQJD f $ YHQWUDO XQFURVVHG FRUWLFRVSLQDO WUDFW LQ WKH UDW ([S1HXURO 9LHUFN &5+ &RKHQ DQG %< &RRSHU f (IIHFWV RI VSLQDO OHVLRQV RQ WHPSRUDO UHVROXWLRQ RI FXWDQHRXV VHQVDWLRQV 6RPDWRVHQVRU\ 5HV 9LHUFN &-' *UHHQVSDQ / $ 5LW] DQG '&
PAGE 225

:LOOLV :' 5% /HRQDUG DQG '5 .HQVKDOR f 6SLQRWKDODPLF WUDFW QHXURQV LQ WKH VXEVWDQWLD JHODWLQRVD 6FLHQFH :LQGOH :) &' &OHPHQWH DQG :: &KDPEHUV f ,QKLELWLRQ RI IRUPDWLRQ RI D JOLDO EDUULHU DV D PHDQV RI SHUPLWWLQJ D SHULSKHUDO QHUYH WR JURZ LQWR WKH EUDLQ -&RPS1HXURO :LQGOH :) f 7KH 6SLQDO &RUG DQG ,WV 5HDFWLRQ WR 7UDXPDWLF ,QMXU\ 1HZ
PAGE 226

%,2*5$3+,&$/ 6.(7&+ /\Q %XUUHOO -DNHPDQ ZDV ERUQ LQ 6FKHQHFWDG\ 1HZ
PAGE 227

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fn AKGMOASVRSK\ % 0XQVRQ URIHVVRU RI 1HXURVFLHQFH FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSVBDQG D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWF &KDU 3URIHVV 1HXURVFLHQFH FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSA DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRIYFLU 3KLORV 5RJHU 0 5HHS $VVLVWDQW 3URIHVVRM RI 1HXURVFLHQFH

PAGE 228

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f§f %DUEDUD 6 %UHJPDQ I $VVRFLDWH 3URIHVVRU RI $QDWRP\ DQG &HOO %LRORJ\ *HRUJHWRZQ 8QLYHUVLW\ 7KLV GLVVHUWDWLRQ ZDV VXEPLWWHG WR WKH *UDGXDWH )DFXOW\ RI WKH &ROOHJH RI 0HGLFLQH DQG WR WKH *UDGXDWH 6FKRRO DQG ZDV DFFHSWHG DV SDUWLDO IXOILOOPHQW RI WKH UHTXLUHPHQWV IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 0D\ 'HDQ &ROOHJH RI 0HGLFLQH

PAGE 229

81,9(56,7< 2) )/25,'$