Citation
Effects of sacrocaudal spinal cord lesions and transplants of fetal tissue on cutaneous reflexes of the tail

Material Information

Title:
Effects of sacrocaudal spinal cord lesions and transplants of fetal tissue on cutaneous reflexes of the tail
Creator:
Friedman, Robert Mark, 1961-
Publication Date:
Language:
English
Physical Description:
xii, 180 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Abnormal reflexes ( jstor )
Animal tails ( jstor )
Babinski reflex ( jstor )
Lesions ( jstor )
Linear regression ( jstor )
Physical trauma ( jstor )
Reflexes ( jstor )
Spinal cord ( jstor )
Tissue grafting ( jstor )
Transplantation ( jstor )
Cats ( mesh )
Department of Neuroscience thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Neuroscience -- UF ( mesh )
Electric Stimulation ( mesh )
Fetal Tissue Transplantation ( mesh )
Physical Stimulation ( mesh )
Reflex ( mesh )
Research ( mesh )
Spinal Cord -- transplantation ( mesh )
Spinal Cord Injuries -- surgery ( mesh )
Spinal Cord Injuries -- veterinary ( mesh )
Tail ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1995.
Bibliography:
Bibliography: leaves 169-179.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert Mark Friedman.

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:
028266499 ( ALEPH )
50179908 ( OCLC )
ALQ7399 ( NOTIS )

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Full Text









EFFECTS OF SACROCAUDAL SPINAL CORD LESIONS AND TRANSPLANTS
OF FETAL TISSUE ON CUTANEOUS REFLEXES OF THE TAIL














By

ROBERT MARK FRIEDMAN


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


1995




EFFECTS OF SACROCAUDAL SPINAL CORD LESIONS AND TRANSPLANTS
OF FETAL TISSUE ON CUTANEOUS REFLEXES OF THE TAIL
By
ROBERT MARK FRIEDMAN
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
1995


to absent friends


ACKNOWLEDGEMENTS
A number of individuals made significant contributions to the successful
completion of this research. First and foremost, I would like to recognize the
patience of my mentor Dr. Charles Vierck, Jr. Even though there may be more
productive student-mentor approaches, I feel that his hands off style of minimal
cajoling has made me a scientist better prepared to achieve my future research
goals. For not always getting on my goat, I sincerely recognize the contributions
of Dr. Brian Cooper to my early graduate research endeavors. Dr. Louis Ritz
receives a special acknowledgement, for I appreciate the opportunity to study tail
behaviors under his tutelage. I especially want to thank Dr. Paul Reier who made
numerous and invaluable contributions to this research. I also appreciate the time
and support of my other committee members, past and present: Drs. Paul Brown,
Christiana Leonard, John Munson, and Alan Spector.
Thanks go to Carolyn Baum for her assistance in working with the cats
involved in this research. Special thanks go to Jean Kaufman, for her help in other
aspects of my research is greatly appreciated.
I would also like to recognize the excellent care of the felines provided by
Ms. Barbara O'Steen, Kim Foli and Dan Theele, DVM. I especially would like to
thank the technical support of Anwaral Azam. Without his help in troubleshooting
electrical and computer problems, this research would have never gotten off the
ground.
in


The support of past and present fellow graduate students should not go
unmentioned. Gregory Schrimscher, Jianxin Bao, Audrey Kalehua, Diana
Glendinning, David Yeomans, Doug Swanson and Laura Errante made the
struggles of graduate school bearable. I especially appreciate the support of James
Makous for engaging discussions, advice and reflections on the scientific method,
life as research scientist, and the somatosensory system.
Love and special thanks go to my family. I would like to acknowledge the
understanding and support of my parents, Carolyn and Lester Friedman, who
wanted their son to grow up to be a doctor but will have to settle for a son with a
doctorate. I especially want to recognize the love and support of my sister Renee
Friedman who took longer to receive her Ph.D. than I, but to my chagrin still
finished tier's first. Alas, the unspoken love and contributions of Laura Friedman
to my life are immeasurable.
Financial support for this research was provided by the Center for
Neurobiological Sciences (NIMH grant MH15737), NIH grant NS27511 to Dr. Paul
J. Reier, the Mark F. Overstreet Fund for Spinal Cord Injury Research and the State
of Florida Impaired Drivers and Speeders Trust Fund.
IV


TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES ix
ABSTRACT xi
CHAPTERS
1 INTRODUCTION AND BACKGROUND 1
Consequences of Human Spinal Cord Injury 1
Treatments for Spinal Cord Injury 4
Behavioral Evaluation of Spinal Cord Injury 6
Animal Model Systems to Study Spinal Cord Injury 7
Overview of Dissertation 11
2 CUTANEOUS REFLEXES OF THE TAIL OF CATS BEFORE AND
AFTER SACROCAUDAL SPINAL LESIONS 13
Introduction 13
Methods 15
Results 25
Discussion 106
3 EFFECTS OF FETAL SPINAL TISSUE TRANSPLANTS ON
CUTANEOUS REFLEXES OF THE CATTAIL 115
Introduction 115
Methods 117
Results 121
Discussion 154
4 OVERALL DISCUSSION 164
REFERENCES 169
BIOGRAPHICAL SKETCH 180
v


LIST OF TABLES
Table 2-1
Summary of the multiple linear regression analysis of
reflex force of normal animals and after a
chronic sacrocaudal transection
. 39
Table 2-2
Summary of the multiple linear regression analysis
of peak reflex amplitude of normal animals
and after a chronic sacrocaudal transection
. 43
Table 2-3
Summary of the multiple linear regression analysis
of latency at peak reflex amplitude of normal animals
and after a chronic sacrocaudal transection
. 46
Table 2-4
Summary of the multiple linear regression analysis
of rise time of normal animals and after
a chronic sacrocaudal transection
. 50
Table 2-5
Summary of the multiple linear regression
analysis of half maximal reflex duration of normal
animals and after a chronic sacrocaudal transection ....
. 54
Table 2-6
Summary of the multiple linear regression analysis
of quarter maximal reflex duration of normal
animals and after a chronic sacrocaudal transection ....
. 58
Table 2-7
Summary of the multiple linear regression analysis
of reflex latency of normal animals and after a
chronic sacrocaudal transection
. 62
Table 2-8
Spearman rank order correlation coefficients between
measured characteristics of the reflex waveform
. 64
Table 2-9
Summary of the multiple linear regression analysis
of changes in reflex force over time after sacrocaudal
transection
. 68
Table 2-10
Summary of the multiple linear regression analysis
of changes in peak reflex amplitude over time
after sacrocaudal transection
. 71
Table 2-11
Summary of the multiple linear regression analysis
of changes in latency at peak reflex amplitude over time
after sacrocaudal transection
. 73
Table 2-12
Summary of the multiple linear regression analysis
of changes over time in the duration of decay to
0.5 maximal amplitude after sacrocaudal transection ....
. 76
VI


Table 2-13
Summary of the multiple linear regression analysis
of changes over time in the duration to decay to
0.25 maximal amplitude after sacrocaudal transection ....
. 77
Table 2-14
Summary of the multiple linear regression analysis
of changes in reflex latency after sacrocaudal transection .
. 81
Table 2-15
Summary of the multiple linear regression analysis
of changes in reflex rise time after sacrocaudal transection. .
. 83
Table 2-16
Comparison of 5 reflex characteristics for one animal
after a left hemisection and a group of 7 animals
pre- and post-transection
105
Table 2-17
Summary of the consequences of a transection of
the sacrocaudal spinal cord on the response
characteristics of the electrocutaneous reflex
108
Table 3-1
Anatomical evaluation of transplant survivability
and integration
122
Table 3-2
Summary of the multiple linear regression analysis
of reflex force of animals following transection or
transection plus transplantation
134
Table 3-3
Summary of the multiple linear regression analysis
of peak reflex amplitude of animals following transection
or transection plus transplantation
138
Table 3-4
Summary of the multiple linear regression analysis
of latency at peak reflex amplitude of animals following
transection or transection plus transplantation
142
Table 3-5
Summary of the multiple linear regression
analysis of reflex rise time of animals following
transection or transection plus transplantation
146
Table 3-6
Summary of the multiple linear regression analysis
of half maximal reflex duration of animals following
transection or transection plus transplantation
149
Table 3-7
Summary of the multiple linear regression analysis
of quarter maximal duration of animals following
transection or transection plus transplantation
153
Table 3-8
Summary of the multiple linear regression analysis of
reflex latency of animals following transection
or transection plus transplantation
157
v¡¡


Table 3-9
Summary of the consequences of transection plus
transplantation in comparison to the transection-only
lesion on the response characteristics of the
electrocutaneous reflex
159


LIST OF FIGURES
Figure 2-1 The testing apparati 18
Figure 2-2 Histological reconstructions of the sacrocaudal hemisections 27
Figure 2-3 Representative traces of tail reflexes to mechanical
stimulation 30
Figure 2-4 Frequency of responses to mechanical stimuli 32
Figure 2-5 Representative traces of tail reflexes to single
DC pulses of electrocutaneous stimulation 35
Figure 2-6 The relationship of reflex force to stimulus intensity 38
Figure 2-7 The relationship of peak reflex amplitude to stimulus
intensity 42
Figure 2-8 The relationship of latency at peak reflex amplitude to
stimulus intensity 45
Figure 2-9 The relationship of reflex rise time to stimulus intensity .... 49
Figure 2-10 The relationship of half maximal reflex duration to
stimulus intensity 53
Figure 2-11 The relationship of quarter maximal reflex duration
to stimulus intensity 57
Figure 2-12 The relationship of reflex latency to stimulus intensity 61
Figure 2-13 The time course of changes in reflex force 67
Figure 2-14 The time course of changes in peak reflex amplitude
and latency at peak reflex amplitude 70
Figure 2-15 The time course of changes in reflex duration 75
Figure 2-16 The time course of changes in reflex latency and rise time. . 80
Figure 2-17 Frequency of wind-down of the reflex with repetitive
stimulation 85
Figure 2-18 Frequency of wind-up with repetitive stimulation 87
Figure 2-19 Representative responses showing wind-up of the tail reflex. 90
Figure 2-20 The magnitude of changes in the reflex response
with repetitive stimulation 92
Figure 2-21 Reflex responses of the tail to trains of
electrocutaneous stimulation 94
IX


Figure 2-22 Reflex force in animals with variants of the sacrocaudal
lesion 96
Figure 2-23 Reflex characteristics following dorsal hemisection at S3 ... 99
Figure 2-24 Reflex characteristics following sacrocaudal transection
with an ischemic episode 101
Figure 2-25 Reflex characteristics following caudal spinal transection
at Ca3 103
Figure 3-1 Survival of transplant tissue 124
Figure 3-2 Coronal section through transplant tissue 127
Figure 3-3 Anatomical integration of transplant tissue with
host spinal cord 129
Figure 3-4 Longitudinal section through a transplant 131
Figure 3-5 Relationship of reflex force to stimulus intensity for animals
following transection or transection plus transplantation ... 133
Figure 3-6 Relationship of peak reflex amplitude to stimulus intensity
for animals following transection or transection plus
transplantation 137
Figure 3-7 Relationship of latency at peak reflex amplitude to stimulus
intensity for animals following transection or transection
plus transplantation 141
Figure 3-8 Relationship of reflex rise time to stimulus intensity for
animals following transection or transection plus
transplantation 145
Figure 3-9 Relationship of half maximal reflex duration to stimulus
intensity for animals following transection or transection
plus transplantation 148
Figure 3-10 Relationship of quarter maximal reflex duration
to stimulus intensity for animals following transection or
transection plus transplantation 152
Figure 3-11 Relationship of reflex latency to stimulus intensity for
animals following transection or transection plus
transplantation 156
x


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
EFFECTS OF SACROCAUDAL SPINAL CORD LESIONS AND TRANSPLANTS
OF FETAL TISSUE ON CUTANEOUS REFLEXES OF THE TAIL
By
ROBERT MARK FRIEDMAN
May 1995
Chairperson: Charles J. Vierck, Jr.
Major Department: Neuroscience
Cutaneous reflexes were measured in the tail of the cat to determine whether
cutaneous hyperreflexia is present after sacrocaudal spinal lesions and whether
changes in cutaneous reflexes are similar to those observed in human patients after
spinal cord injuries. Reflexes to electrocutaneous and mechanical stimulation of
the tail were evaluated prior to and after spinal cord lesions.
After a sacrocaudal spinal transection, the overall magnitude of
electrocutaneous tail reflexes was greater than that observed normally. Major
changes in the characteristics of the response included increases in peak reflex
amplitude, longer latencies to reach peak reflex amplitude, and longer reflex
durations. A long latency reflex could be exhibited, and the magnitude of the reflex
could show wind-up in magnitude with repeated stimulation. The changes in
XI


electrocutaneous reflexes developed progressively over a period of 3 months. The
expression of tail reflexes to mechanical cutaneous stimulation was highly variable.
However, there was a decrease in mechanical reflex threshold after sacrocaudal
transection. These changes in cutaneous reflexes did not occur after incomplete
lesions of the sacrocaudal spinal cord.
This animal model was investigated for its potential to evaluate treatments of
spinal cord injury. Consequences for tail reflexes were determined for animals that
received a transplant of fetal spinal tissue placed into an acute sacrocaudal
transection cavity. Eleven animals received fetal tissue. In 8 animals the transplant
successfully survived and integrated with the host spinal cord. In behavioral
studies of animals with surviving transplants, peak amplitude of the
electrocutaneous reflex was less than observed for animals with a chronic
transection. The duration of the electrocutaneous reflex for animals with
transplants was less than observed for transected animals without transplants and
was similar to normal animals. These findings indicate that the presence of
transplant tissue can modulate the cutaneous hyperreflexia that otherwise develops
after spinal cord transection.
XII


CHAPTER 1
INTRODUCTION AND BACKGROUND
Improved treatment strategies for spinal cord injury represent a major goal
for the health care industry. Approximately 10,000 new cases of spinal cord injury
are reported each year in the United States. The causes of spinal cord injuries are
numerous; however, they primarily involve automobile accidents, acts of violence,
falls and sports related injuries. Typically, these individuals are men between the
ages of 19 and 26. On a cost basis, including lost wages, billions of dollars are
spent each year on medical intervention, rehabilitation and chronic health care of
individuals with spinal cord injuries (U.S. Congress, 1990).
Consequences of Human Spinal Cord Injury
Initially after a spinal cord injury, the patient goes through a period of spinal
shock when all motor functions of spinal segments below the lesion are depressed.
After spinal shock recedes the neurological effects of spinal cord injury can be
dichotomized in terms of either negative (i.e., loss of functions) or positive (i.e.,
abnormal behaviors) signs (Young and Shahani, 1986).
Negative signs that are present below the spinal level of injury can include
a complete loss of voluntary movement (e.g., paraplegia or quadriplegia), weakness
1


2
or paresis of a limb, and a loss of bowel and bladder control. In addition, cutaneous
sensibility for fine touch, proprioception, pain and temperature sensations can be
disrupted or lost. Problems associated with sensory loss are significant, since a
patient with a lowered pain response to a noxious stimulus (hypoalgesia) or a
complete absence of pain (analgesia), may unknowingly experience a higher
incidence and severity of accidental injury (e.g., from a bum, cut, or pressure sore).
Positive signs after a lesion to the spinal cord can involve the development
of postural abnormalities, exaggerated flexion and tendon reflexes, muscle spasms,
and painful sensations. Both hyperreflexia and pain can interfere with the spinal
cord injured patient's ability to function. The different manifestations of
hyperreflexia that can occur after a spinal cord lesion are known collectively as the
spastic syndrome (Young and Shahani, 1986).
Alterations in muscle tone can include a fixed postural abnormality of a limb
(a dystonia or contracture), rigidity, and changes in stretch reflexes. The
exaggeration of segmental stretch reflexes is considered a crucial or defining
feature of spasticity. The alteration of segmental stretch reflexes is thought to (1)
underlie changes in threshold and amplitude of the tendon jerk (myotactic reflex),
(2) trigger a radiation of a local myotactic reflex to the entire limb, (3) trigger rapid
oscillatory movements of a limb (clonus), and (4) include modifications of tonic
stretch reflexes that cause abnormalities in posture (dystonia) of a limb (Dimitrijevic
and Nathan, 1967). Spasticity has been characterized as a velocity sensitive
increase in resistance to passive stretch of a limb with an accompanied increase


3
in tendon reflexes and hypertonia (Landau, 1974). In human patients with cortical
lesions, quantitative behavioral studies have characterized spasticity as a shift in
threshold to elicit stretch reflexes without an increase in magnitude or gain of the
response (Lee et al., 1987). However, similar studies have not been performed to
characterize spasticity in patients with spinal cord injuries.
After spinal cord injury in humans, cutaneous hyperreflexia is evident for
forms of stimulation other than muscular stretch. Cutaneous stimulation normally
elicits flexion reflexes only when the level of stimulation is nociceptive. However,
in a spastic patient, an innocuous cutaneous stimulus on the leg can produce a
brisk, stereotyped, involuntary, flexor movement that can include triple flexion at the
hip, knee and ankle, and crossed extension (Dimitrijevic and Nathan, 1967; Roby-
Brami and Bussel, 1987). The flexion reflex is normally a limited response in one
muscle or limb, but in spastic patients the response spreads to other muscles or
other limbs (Dimitrijevic and Nathan, 1968). In addition, involuntary flexor
movements of a limb can occur in the absence of a known cutaneous stimulus.
Because flexor spasms can increase in frequency in the presence of cutaneous
stimuli, it would appear that central flexor reflex mechanisms are involved (Shahani
and Young, 1973).
Another positive sign after spinal cord injury is the possible occurrence of a
central pain syndrome, despite diminution or loss of cutaneous sensations after
damage to the long ascending spinal pathways. Ninety percent of spinal cord
injured patients report some unpleasant sensory experiences; fifty percent of these


4
patients experience some pain sensations, and twenty-five percent report disabling
pain (Davis and Martin, 1947; Botterell et al., 1953; Kaplan et al., 1962;
Nepomuceno et al., 1979). Pain sensations can involve (1) a phantom limb pain
where the limb is sensed to be in a cramped, twisted position; (2) a visceral pain
where the patient perceives a fullness of the abdomen, fecal urgency and/or
bladder disturbances; (3) a dysesthesia, where the patient feels an unpleasant
sensation that can include diffuse burning or tingling; and (4) a neuralgia, where the
patient feels a hot, sharp, knifelike pain localized to a specific dermatome (Merskey
et al., 1986). With respect to sensations elicited by cutaneous stimulation, pain
perception varies in patients following spinal cord injuries (Lindblom, 1985). A
patient can have a lowered threshold for painful sensations (allodynia), where a
painful sensation is elicited by a stimulus that normally does not provoke pain. In
addition, a patient can report that a noxious stimulus elicits an increased sensation
of pain (hyperalgesia).
Treatments for Spinal Cord Injury
Multiple approaches are employed in treatment of the acute and chronic
effects of spinal cord injuries. In an attempt to control spasticity and pain following
a spinal cord injury, pharmacological and surgical interventions are used with
varying degrees of success (Dimitrijevic and Nathan, 1967; Davidoff, 1985).
Physical therapy and specialized mechanical devices (e.g., wheel chairs, crutches,
prosthetic devices, specialized hand splints) are utilized to improve independence


5
and provide some form of mobility (Trieschmann, 1988). However, there are
currently no available treatments to compensate for the loss of cutaneous sensibility
or development of chronic pain after spinal cord injury.
A new strategy that might ameliorate some effects of spinal cord injury is the
grafting of embryonic neural tissue. An example of the success of embryonic neural
tissue transplantation is found with Parkinson's disease, where grafting of cells from
embryonic brainstem has been reported to have measurable therapeutic benefits
for some patients (Lindvall et al., 1990; Freed et al., 1992). With this approach, the
goal can be to replace lost neurons, connections or neural pathways, to rescue
neurons at risk of cell death, or to modulate neural activity in the host nervous
system. Anatomical evidence suggests that some of these mechanisms may be
associated with the benefits derived from embryonic grafts in patients with
Parkinson's disease.
With respect to the spinal cord, anatomical studies have provided evidence
for the ability of embryonic spinal tissue to integrate and form axonal projections
with the host (Jakeman and Reier, 1991; Reier et al., 1994). Behavioral studies in
cats and rats have shown that fetal spinal transplants can promote recovery in
locomotor behavior (Yakoleff et al, 1989; Kunkel-Bagden and Bregman, 1990;
Anderson et a I., 1991; Goldberger, 1991; Kunkel-Bagden et al., 1991; Stokes and
Reier, 1992). In other experiments in acutely spinalized animals, long-latency
components of flexion reflexes have been shown to be enhanced by previous
transplants of fetal brain stem tissue (Moorman et al., 1990). Similarly, sexual


6
reflexes can be enhanced by transplantation of brain stem tissue (Privat et al.,
1989).
These studies are encouraging in the sense that neural grafts can modulate
or restore certain locomotor behaviors and reflexes that are diminished or lost as
a consequence of spinal cord injury. Improved recovery of locomotion as a result
of fetal cell transplantation could represent, at least in part, an attenuation of
abnormal reflexes, but little or no information is available to evaluate this possibility.
Thus, it is important to determine the effects of fetal transplantation on the
development of spasticity after spinal cord injury.
Behavioral Evaluation of Spinal Cord Injury
Evaluation of new treatment strategies for spinal cord injuries will require
quantitative evaluation in animals of both volitional and reflex behaviors in order to
fully assess the functional utility of these methodologies (Goldberger et al., 1990).
In animals and humans, evaluation of behavioral consequences of spinal cord injury
has relied primarily on subjective rating scales (e.g., Ashworth, 1964; Gale et al.,
1985). This approach has been successful in characterizing consequences of
spinal cord injuries and treatment strategies (Kerasidis et al., 1987). These rating
methods can be effective in specifying qualitative changes, but quantitative
changes in a behavioral function over time remain uncharacterized, irrespective of
issues of test sensitivity and interexaminer reliability.


7
Several different quantitative behavioral assays have been applied to
describe consequences of spinal cord injuries on volitional behaviors. Some of
these behavioral assays include the inclined plane test (Rivlin and Tator, 1977),
foot-falls during grid walking (Bresnehan et al., 1987; Kunkel-Bagden and Bergman,
1990), and quantitative footprint analysis of locomotion (Kunkel-Bagden and
Bergman, 1990). In addition, quantitative behavioral assays have been applied to
forelimb reaching and grasping after spinal cord lesions (Schrimsher and Reier,
1992,1993). Most of these tests evaluate gross behavioral performance by noting
whether an animal achieved the goal of a task. An issue with these behavioral
measures is that an animal with a lesion may utilize an abnormal motor strategy to
achieve the goal. Only a few behavioral paradigms have used electromyographic
and kinematic analyses (Barbeau and Rossignol, 1987; Goldberger, 1988) in
attempts to describe, quantitatively, changes in volitional performance.
Animal Model Systems to Study Spinal Cord Injury
A number of different types of spinal cord lesion have been performed in
order to determine which pathways must be interrupted to produce spasticity and/or
a loss of specific volitional behaviors (Wiesendanger, 1985). However, in the
attempt to understand spasticity in animals, there have been few studies that
quantify the different components of the spastic syndrome in order to relate to the
human condition. In evaluating the consequences of specific spinal cord lesions


8
on the development of spasticity, most animal studies have focused on alterations
of stretch reflexes.
Spinal transection. In cats with chronic spinal transection, behavioral signs
of spasticity are readily and consistently apparent (Fulton and Sherrington, 1932;
McCouch, 1947; Kozak and Westerman, 1966; Afelt, 1970; Bailey et al., 1980), but
rarely have been quantified (Little, 1986). As determined by subjective methods of
neurological evaluations of cats with complete thoracic transection (Bailey et al.,
1980), the limbs are hypotonic for the first few days below the level of the lesion.
The patellar tendon reflex is slightly exaggerated, and the cutaneous flexor reflex
is mildly exaggerated, with the possible presence of cross extension. By the end
of the first week, an increase in tone during passive stretch of a limb is apparent,
and a clasp-knife reflex can be elicited. Tendon reflexes are exaggerated with the
possible presence of clonus. Cutaneous flexor reflexes are exaggerated, and
flexor-extensor spasms might be present. Reflex stepping and standing begin to
appear. By 14 days, muscle tone is only mildly increased, yet all spinal reflexes are
exaggerated. Several early phases of reflex exaggeration have been distinguished
by quantitative methods (Little, 1986), but after 30 days muscle tone is markedly
increased, and spinal reflexes are easily elicited and markedly exaggerated.
A similar time-course has been reported for spinal monkeys. However,
depending on the quality of post surgical care, the lower limbs can become
contracted, rigid, and atrophied (Sahs and Fulton, 1940; Liu et al., 1966). In
paraplegic humans the sequence of hyporeflexia followed by hyperreflexia is similar


9
to that observed in the cat and monkey, except that the time-course of these events
is expanded.
Hemisection. The clinical picture of animals with a hemisection is less clear.
Some studies in rats and cats have reported signs of spasticity (Teasdall et al.,
1958, lesions at T4-L1; Murray and Goldberger, 1974, lesions at T13; Carter et al.,
1991, lesions at L5); whereas, others have not (Hultborn and Malmsten 1983,
lesions at T12 and L2; Carter et al., 1991, lesions at T12). When spasticity is
present after incomplete spinal lesions, the time-course of events is similar to that
of an animal with a transection except that weight bearing occurs in two weeks and
locomotion occurs by three weeks. In addition, hyperreflexive responses can
resolve at later post-injury time periods.
Staggered lateral hemisections. The consequences of staggered lateral
hemisection on reflexes and locomotion also have been investigated (Kato et al.,
1985). Initially there is a depression of segmental reflexes at 1 to 2 days. From 3
to 7 days, segmental reflexes are exaggerated, and unusual reflexes are observed
with the presence of airstepping and alternating limb movements. However, from
7 to 53 days, when the animal begins to stand and walk, abnormal reflexes subside
and segmental reflexes become less exaggerated. In primates, staggered bilateral
hemisection results in a clinical picture similar to that observed after complete
spinal transection (Turner, 1891; Lassek and Anderson, 1961). This difference
among species after staggered lateral hemisections may reflect differences in the


10
organization of the descending systems that control the spinal cord (Holstege and
Kuypers, 1987; Chung and Coggeshall, 1988).
Contusion injury. In animals, a weight drop technique (Allen, 1911) has been
used to model the rapid and traumatic contusive or compressive sort of insult that
causes damage in most patients with spinal cord injury. After a compression or
contusion injury, a hemorrhagic necrosis begins at the center of the insult and is
followed by neural degeneration and the development, over a period of weeks, of
a central cavitation. Modifications of the original technique have improved upon the
reproducibility of the injury (Wrathall et al., 1985; Anderson et al., 1988; Beattie et
al., 1988, Stokes et al., 1992). The behavioral consequences of contusion depend
on the degree of the injury (Gale et al., 1985). With respect to spasticity, both
cutaneous hyperreflexia (Gale et al., 1985) and alterations of physiologically
recorded monosynaptic reflexes (Thompson et al., 1992), develop over a period of
weeks after a contusion injury of the rat spinal cord.
Lesion factors in spasticity. The factors that are critically important to the
development of signs of spasticity in an animal include size, spinal level, and time
after the lesion. Together, these factors determine which pathways are spared, the
extent of deafferentation of the spinal cord, and the excitability of neurons caudal
to the lesion.
Variability in the chronic manifestation of hyperreflexia is apparent in humans
(Mailis and Ashby, 1990). For example, spasticity can be differentiated on the
bases of whether the lesion is spinal or supraspinal (Burke et al., 1972;


11
Wiesendanger, 1985). Following cortical lesions, extensor reflexes are released
over flexors, whereas flexor reflexes become exaggerated after spinal cord lesions.
In addition, not all hemiplegias from cortical or capsular lesions involve a sequential
development from early hypotonia to chronic hypertonia. And, recovery of volitional
control usually results in a decrease in hypertonia in these patients.
Overview of Dissertation
Preliminary to this dissertation research, Rhoton et al. (1988) adapted
traditional methods of human neurological examination to investigate the
consequences of caudal spinal lesions on the tails of cats. Soon after a caudal
spinal transection, the tip of the tail was flaccid. Then, over a period of weeks, the
tail developed heightened cutaneous flexion reflexes and increased tone
(resistance to stretch of tail muscles). Rigidity of the tail appeared, as did clonus
like responses to cutaneous stimulation. Subsequently, quantitative measurements
confirmed the impression from neurological testing that tone was greater for tail
muscles of lesioned cats than for normal cats (Ritz et al., 1992).
The dissertation research developed quantitative behavioral methods for
testing cutaneous reflexes. These procedures were evaluated and utilized to study
functional consequences of spinal cord injury and potentially therapeutic
interventions. The key aspect of this research was utilization of sacrocaudal spinal
lesions to evaluate effects of spinal cord lesions on cutaneous reflexes of the tail.
The advantage of a sacrocaudal spinal lesion is that effects of spinal transection


12
can be studied in the absence of bowel and bladder dysfunctions or disruptions in
gait or hindlimb reflexes. The lesions are made caudal to the lumbar enlargement
and are one to two segments caudal to the parasympathetic region (S1 and S2) of
the spinal cord that controls the bowel and bladder (Thor et al., 1989).
Chapter 2 describes studies designed to acquire precise measurements of
cutaneous reflexes in order to determine the time-course and magnitude of any
changes in cutaneous reflexes of the tails of cats after sacrocaudal spinal lesions.
Cutaneous reflexes of the tail were evaluated in normals and animals with spinal
cord lesions. This research determined that lesions of the sacrocaudal cord (S3-
Ca1) provide an appropriate model for investigating effects of transection of the
spinal cord on segmental reflexes of the limbs.
In the studies described in Chapter 3, this animal model was utilized to
evaluate a potential treatment for some of the debilitating effects of spinal cord
injury. Fetal spinal tissue was transplanted into an acute spinal cord transection
injury. The effects of transection and transplantation on cutaneous reflexes were
compared with effects of transection without introduction of a transplant.
In concluding, Chapter 4 discusses possible mechanisms for the cutaneous
hyperreflexia that is typically observed after a spinal cord injury. Also, mechanisms
for influences of fetal spinal transplantation on the consequences of spinal cord
injury are discussed.


CHAPTER 2
CUTANEOUS REFLEXES OF THE TAIL OF CATS BEFORE
AND AFTER SACROCAUDAL SPINAL LESIONS
Introduction
The consequences of a spinal cord transection, following a period of
areflexia (spinal shock), include a gradual development of heightened reflexes
caudal to the site of the lesion (Riddock, 1917; Dimitrijevic and Nathan, 1967). Both
phasic and tonic stretch reflexes are affected and are clinically defined as
hyperreflexic by the appearance of exaggerated tendon reflexes and a heightened
resistance to passive stretch of a muscle, respectively (Landau, 1974; Lance,
1980). Additionally, a limb can exhibit hyperreflexive cutaneous reflexes (Riddoch,
1917; Kuhn and Macht, 1949; Rushworth, 1966; Dimitrijevic and Nathan, 1968;
Shahani and Young, 1971, 1973; Meinck et al., 1985; Roby-Brami and Bussel,
1987). Clinically, cutaneous hyperreflexia is observed when an innocuous stimulus
to the skin of the foot or leg induces an exaggerated triple flexion of the hip, knee
and ankle. The Babinski reflex (dorsiflexion of the great toe with a fanning of the
other toes) is a cutaneous flexion reflex of clinical importance that can be exhibited
after a lesion to the spinal cord (Kugelberg et al., 1960; Roby-Brami et al., 1989).
13


14
Hyperreflexive reflex responses to cutaneous stimulation or muscle stretch are two
components of the spastic syndrome (Young and Shahani, 1986).
In order to study neural mechanisms underlying the development of
spasticity after spinal cord injury, studies in animals have induced hyperreflexia with
complete (Fulton and Sherrington, 1932; Afelt, 1970; Burke et al., 1972; Nelson and
Mendell, 1979; Bailey et al., 1980; Munson et al., 1986; Ritz et al., 1992) and
incomplete lesions (Turner, 1891; Wagley, 1945; Teasdall et al., 1958; Lassek and
Anderson, 1961; Fujimori et al., 1966; Murray and Goldberger, 1974; Kato et al.,
1985; Little, 1986; Carter et al., 1991; Thompson et al., 1992). These studies
primarily have characterized alterations in stretch reflexes. Only a few animal
studies have examined chronic changes in cutaneous (or polysynaptic) reflexes
after spinal cord lesions (Ranson and Hinsey, 1930; McCouch, 1947; Kozak and
Westerman, 1966; Liu et al., 1966; Hultborn and Malmsten, 1983).
Previously, the consequences of a lesion in the cat sacrocaudal spinal cord
which only affects the tail were investigated to develop a minimally disruptive model
of spinal cord injury. Rhoton et al. (1988) observed that a hemisection or complete
transection in the cat sacrocaudal spinal cord produced clinical signs of hypertonia,
cutaneous hyperreflexia, and clonus-like movements. These classical signs of
spasticity occurred in the absence of bowel and bladder dysfunctions or disruptions
in gait and hindlimb reflexes. Subsequent quantitative measurement (Ritz et al.,
1992) confirmed the changes in muscle tone. After a complete transection of the
spinal cord, the tail becomes actively ventroflexed in a midline position, and there


15
is a loss of sensation and volitional control. These functional alterations are akin
to paraplegia with spasticity that occurs in human patients with spinal cord injuries
(Young and Shahani, 1986).
To further investigate alterations in tail reflexes after lesions in the cat
sacrocaudal spinal cord, quantitative measurements of cutaneous reflexes of the
tail to electrocutaneous and mechanical stimuli were performed in normal animals
and in animals with lesions of the sacrocaudal spinal cord. Specifically, this study
addressed whether cutaneous reflexes are hyperactive after spinal transection, as
observed caudal to spinal cord injuries of human patients.
Methods
Subjects. A total of 11 adult cats (10 female, 1 male) served as subjects.
For 8 animals, data were collected before and after lesions of the sacrocaudal
spinal cord. Three animals were part of a prior study (Ritz et al., 1992) and had
previously received either a complete sacrocaudal transection (n=2) or a left
hemisection (n=1). When the animals were not performing in the behavioral
experiments, they were housed at the University of Florida Animal Resources
Facilities.
Surgery. All surgeries were performed under sterile conditions and gas
anesthesia (Flalothane or Isoflourane). Some cats received 50 mg/kg of ketamine
as a preanesthetic. The animal was intubated and a venous catheter was inserted
for infusion of fluids. Dorsal laminectomies were performed at the L6 and L7


16
vertebrae. Cats received a complete transection or a subpial transection cavity (~2-
3 mm long) at the S3/Ca1 level. The lesions were made with precision forceps and
suction. Four to 6 dural sutures secured the spinal cord after the lesion, and when
a subpial transection was performed, 4 pial sutures were added. Visual inspection
at the time of surgery confirmed the completeness of the lesion.
Postoperative care included antibiotic treatments (Amoxicillin, 50 mgs) for
7 days. Canned food was added to the diet. If necessary, the bladders were
manually expressed, but in most cases normal bladder function returned by the
second postoperative day.
At the termination of the behavioral experiments, the cats were anesthetized
with a lethal dose of sodium pentobarbital (100mg/kg) and transcardially perfused
with isotonic saline solution (0.9% with sodium nitroprusside; 0.5g/L) and heparin
sulfate (1.5cc/L), followed by a solution of 4.0% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.6). Upon extraction of the spinal cord, the completeness
of the lesion was determined, and the level of the lesion was identified.
Testing apparatus. The testing apparatus provided comfortable restraint of
the body and tail of each cat (Figure 2-1). Either a cat sack or a restraint harness
was used to situate the body of the cat, and the tail was bracketed at the base. To
provide quantitative measures of cutaneous reflexes, the tip of the tail was tethered
to a copper-beryllium leaf (2.5 cm by 8 cm by 0.5 mm) with an attached strain
gauge. The strain gauge was incorporated into a Wheatstone bridge circuit, and
the signal was amplified 1000 times by a direct current (DC) amplifier. The tether


Figure 2-1. The testing apparati. The cats were positioned with a cat sack (top) or
a restraint harness (bottom). The tail was bracketed at the base. Cutaneous
reflexes of the tail were measured by tethering the tip of the tail to a copper-
beryllium leaf containing a strain gauge.


18
Strain
Gauge


19
tension was held at 1.5 Newtons (N), which held the tail straight in an axial position.
Muscle contractions that moved any portion of the tail away from the axial position
were detectable as an increase in force as measured by the strain gauge. The
measured force assessed the strain produced by a tail reflex. Since the tail was
tethered in parallel, the setup was incapable of measuring the torque of the tail
reflex, which depends on the length of the tail distal to the pivot point (acting as a
moment arm), the angle of the movement, and the reflex force exerted. Attempts
to directly measure torque would encounter an inherent problem in that the pivot
point of tail reflexes were not fixed, but instead depended on stimulus intensity,
since recruited motoneurons course along the length of the tail. The response
forces were collected at a 1 KHz sampling rate and stored digitally in a file, using
computer hardware and software (RC-Electronics).
The timing of all stimulation and event triggering were performed by the
Stoelting Protege System's Stimulus Laboratory Controller with Laboratory
Automation System software.
Training paradigm. Cats were adapted to the testing room and then to the
testing apparatus. For restraint in cat sacks, adaptation to the testing apparatus
included rewards of dry food (BONKERS Cat-Treats or POUNCE Treats for Cats)
as well as physical affection. Training cats restrained with the harness entailed
rewards of liquid food. Subsequently, the proximal tail was braced, and then the
distal tail was tethered. Next, the cats were accustomed to passively receive
mechanical and electrocutaneous stimulation.


20
Testing regimen. When possible, the animals were adapted and tested prior
to receiving a lesion (n=8). Following a sacrocaudal lesion, to evaluate possible
changes in the reflex over time, these animals were tested repeatedly over a period
of at least 7 months and up to a year. Five post-lesion time periods were
specifically examined: 7-14 days, 21-35 days, 70-90 days, 140-165 days, and
greater than 210 days. Due to the methods of restraint and concern for the welfare
of the animal, testing was only rarely attempted during the first week after surgery.
Three other animals were tested only at 3 years post-lesion.
Mechanical stimulation and reflex testing. All mechanical stimulation was
presented on the dorsal surface of the tail at dermatomal level Ca3/Ca4,
approximately 5 cm from the tip of the tail. Mechanical stimulation was presented
with a solenoid and consisted of a 50 ms cutaneous tap elicited through a blunt
metallic probe (1 mm in dia.) or an edge (a Steel T Pin, Create-A-Craft). The probe
was either in continuous contact with the skin or first made contact with the skin
during the tap. In all testing sessions, each stimulus condition was repeated 8
times with an interstimulus interval of 8 s.
Cutaneous response measures and data analysis. Different characteristics
of the response to the mechanical stimuli were tabulated. Individual trials were
evaluated with respect to (1) occurrence of a response, (2) reflex latency, (3) peak
reflex amplitude, and (4) complexity of the response. To tabulate reflex latency,
reflexes shorter than 30 ms were scored in one bin, responses greater than 30 ms
but less than 250 ms were placed in another bin, and reflexes occurring later than


21
250 ms after stimulus onset but less than 750 ms later were scored in a third bin.
Initial responses that occured at latencies greater than 750 ms were considered as
responses unrelated to the mechanical stimulus. The duration of the reflex was
tabulated into 4 bins: (1) less than 500 ms, (2) greater than 500 ms but less than
1 s, (3) greater than 1 s but less than 2 s, and (4) greater than 2 s. The amplitude
of the reflex was also tabulated into 4 bins: (1) less than 0.15 N, (2) greater than
0.15 N but less than 0.25 N, (3) greater than 0.25 N but less than 0.5 N, and (4)
greater than 0.5 N. To characterize the complexity of the reflex response 3 bins
were created. Simple responses, reflexes with less than 3 peak envelopes were
placed in one bin. Responses with greater than 3 peak envelopes but less than
7 peak envelopes were placed in a second bin. And responses with greater than
6 peak envelopes were placed in a third bin.
For each animal 3 to 4 blocks of trials were grouped together and scored to
acquire frequency values for the reflex measures. The population of frequency
values from pre-lesion and post-lesion animals with chronic transection lesions
were statistically compared with the Mann-Whitney Rank Sum Test.
Electrocutaneous stimulation. Innocuous electrocutaneous stimulation
consisted of a 5 ms pulse of constant current (DC) delivered via two wells (5 mm
in dia.) of electrode paste (EC 2 Electrode Cream; Grass Instrument Co.) which
were separated by 1 cm. The wells were punched out of a 1/8" thick polycushion
pad (Smith Nephew Rolyan Inc.) and were secured on the tail with elastic tape
(Elastikon, Johnson and Johnson Medical, Inc.). The stimulation site was the


22
dorsal surface of the tail at dermatomal level Ca3, approximately 8 to 10 cm from
the tip of the tail. Electrocutaneous stimulation was presented by Grass Instrument
Company equipment: a stimulator (Model S8), a stimulus isolation unit (Model SIU
5B), and a constant current unit (Model CCU 1A). The amplitude of the
electrocutaneous stimulation was manually set. A current monitor (built in house)
was utilized to measure the amount of current delivered by each stimulus.
Electrocutaneous reflex testing. Tail reflexes were elicited by a single DC
pulse of electrocutaneous stimulation. To determine intensity-response
relationships, stimuli were presented in blocks of 6 to 8 trials at each of 6 different
stimulus intensities in each testing session. Intensities of 2, 3, 4, 6, 8, and 10.5
mAmps/mm2 (in mAmps, these values were 0.4, 0.6, 0.8, 1.2, 1.6, and 2.1) were
presented in an ascending order of blocks. The intertrial intervals were 7 s.
Occasionally, reflex responses were recorded at various intensities of a 10 pulse,
50 Hz train of electrocutaneous stimulation.
Electrocutaneous response measures and data analysis. The stability of
reflex responses to repeated stimulation was evaluated by comparing the force of
responses on the first trial in a block of trials at one intensity with responses on all
subsequent trials presented at that intensity. For this purpose the force of the reflex
was determined by summing the amplitudes of responses over the first 500 ms after
stimulus onset. For a block of trials to be characterized as showing either wind-up
or wind-down to repeated stimulation, two criteria had to be satisfied. First, the
average change in reflex force had to be greater than 20 percent of the reflex force


23
estimate of the first response. And second, either the average change in reflex
force had to be greater than one standard deviation away from the reflex force
estimate of the first trial, or a consistently increasing or decreasing trend in reflex
force must be present.
For all other further characterizations of the reflex, averaged waveforms were
constructed for each stimulus intensity and period of testing. Individual trials were
not included in the average if there was an unstable baseline due to a volitional
body movement. Starting 10 ms prior to stimulus onset and ending 110 ms later,
data were entered into a waveform data file at the 1 KHz sampling rate. For the
next 400 ms of the response, the file received data at 5 ms intervals. From 400
msec until the end of the data collection window (1.5 s, or 3.5 s), the file received
data at 20 ms intervals.
Different characteristics of the averaged reflex waveforms were evaluated.
Reflex latency was measured by extrapolating back to the X-intercept of a line
made through the 10 and 90 percent peak amplitude points on the ascending limb
of the reflex. Peak reflex amplitude and the latency at peak reflex amplitude were
measured directly. The rise time of the ascending limb of the reflex was estimated
by calculating the slope of a line through the 25 and 75 percent peak amplitude
points on the ascending limb of the reflex. Two measures of reflex duration were
the latency of the reflex amplitude to decay to 50 percent and to 25 percent of the
peak reflex amplitude. An estimate of the overall force of the reflex was determined
by summing the reflex amplitudes from the time of stimulus offset until the decay of


24
the reflex response reached 25 percent of the peak reflex amplitude. The different
characteristics of the averaged waveform were calculated by a computer program
written in the C programming language.
Statistical analysis was performed with SigmaStat computer software (Jandel
Scientific Software). A mixed repeated-measures design was utilized for
comparison of a given characteristic of the tail reflex for normal animals and
animals with chronic sacrocaudal transection. The presence or absence of the
sacrocaudal lesion was treated as the categorical between-subjects factor.
Stimulus intensity was treated as a continuous within-subjects factor. Each
characteristic of the reflex was treated as a continuous dependent variable.
Multiple linear regression analysis was used to evaluate the results (Pedhazur,
1982). To perform the multiple linear regression analysis, the independent and
dependent variables were transformed, when appropriate, to pass tests of normality
(Kolmogorov-Smirnov test) and homoscedasticity (constant variance) and to
linearize the relationship between stimulus intensity and a characteristic of the
reflex. Criterion scaling was used to code for the subjects.
Multiple linear regression analysis was used to evaluate changes in the
reflex over time after sacrocaudal lesions. In the repeated-measures design, the
pre-lesion and 5 post-lesion time periods were treated as a categorical within-
subjects factor. Effect coding was utilized to vector code the categorical variable
for the multiple linear regression analysis. Stimulus intensity was treated as a
continuous within-subjects factor. Each characteristic of the reflex was treated as


25
a continuous dependent variable. Transformations of the independent and
dependent variables were the same as those used for comparisons of pre-lesion
and late post-lesion reflex characteristics.
Results
For 7 of the animals tested, spinal transactions were successfully placed at
sacrocaudal levels between the caudal half of S2 and the caudal half Ca1. These
7 animals comprised a group for analysis of chronic changes in cutaneous reflexes
after sacrocaudal transection. For 5 of these animals, pre-lesion and acute and
chronic post-lesion data were collected to evaluate the time-course of the effects
of the lesions. The reflex responses from 4 additional animals were treated
separately. For one animal, the sacrocaudal transection was located at rostral Ca1,
but evidence of an ischemic episode was present in caudal segments, where cresyl
violet staining revealed an abundance of macrophages and few neurons. In
another animal, the sacrocaudal transection was mistakenly placed at the Ca3
segment. In another animal, an incomplete sacrocaudal lesion at S3 cut through
the dorsal aspect of the spinal cord, disrupting the dorsal columns, Lissauer's tract,
and the dorsal aspect of the dorsolateral white matter, bilaterally (Figure 2-2,
bottom). A near perfect left hemisection at S3 was histologically confirmed for the
fourth animal (Figure 2-2, top).


Figure 2-2. Histological reconstructions of the sacrocaudal hemisections. The top
drawing shows the lesion from the animal with the left hemisection. The bottom
drawing shows the lesion from the animal with the bilateral dorsal hemisection.


2?


28
Responses to Mechanical Stimuli in Normal and Chronic Transection Animals
Examples of tail responses to mechanical stimuli are shown in Figure 2-3.
When tail reflexes could be elicited, various types of responses were expressed
which depended on both the type of mechanical stimulation and the specific animal
tested. Due to the variability observed in the reflexes produced by mechanical
stimuli, different characteristics of the reflex response were tabulated. And,
because of the inability of a single type of mechanical stimulus to consistantly elicit
reflexes in all animals, 4 different types of mechanical stimulation were used.
Reflex threshold to the mechanical stimuli in normal animals (n=7) and
animals with a chronic transection (n=7) was evaluated by determining the reflex
response frequency (Figure 2-4). The trend observed for a decrease in mechanical
reflex threshold for 3 of the 4 mechanical stimuli, as measured by an observed
increase in response frequency, was significant for stimulation with the blunt probe
that remained in contact with the skin (Mann-Whitney Rank Sum test; T = 24.0, P
= 0.008). Over 80 percent of the reflexes elicited were made up of less than 3
envelope peaks, and no differences were observed in the complexity of the reflex
between pre-lesion and chronic post-lesion animals. In post-lesion animals
increases were observed when compared with pre-lesion animals in the frequency
of reflexes with latencies between 30 and 250 ms which paralleled a decline in the
frequency of longer latency responses in post-lesion animals (e.g., edge stimulus,
on skin; P = 0.026). Long latency responses to the blunt probe (off skin) declined
in post-lesion animals (median frequency 0.00) from the frequency observed (0.26)


Figure 2-3. Representative traces of tail reflexes to mechanical stimulation. From
a normal (top) and an animal with a chronic sacrocaudal transection (bottom), the
traces show various reflex responses of the tail to mechanical stimulation. For
clarity, the reflex traces are offset. The bottom trace (top diagram) shows the
stimulus artifact when the mechanical probe displaces a passive tail.


Force (0.65 N/div) Force (0.65 N/div)
30
0.0 ms
800 ms/div
4.0 s


Figure 2-4. Frequency of responses to mechanical stimuli. For 4 different mechanical stimuli, the bar graph shows the
median probability of occurrence of a reflex response for the pre-lesion (grey columns, n=7) and chronic post-lesion periods
(n=7). A decrease in reflex threshold is observed post-lesion, as shown by an increase in probability of responding to the
mechanical stimuli. The lines represent 75 percent and 25 percent confidence intervals.


Probability (Median)
Blunt Probe On Blunt Probe Off Edge Probe On Edge Probe Off
w
ro
Mechanical Stimulus


33
in pre-lesion animals (Mann-Whitney Rank Sum test; T = 54.5, P = 0.0086). This
finding confirmed that the responses with latencies longer than 250 ms were most
likely volitional tail movements in response to the mechanical stimuli. And although,
in comparison to pre-lesion animals, there were trends in post-lesion animals for an
increase in reflex duration and peak reflex amplitude in response to the mechanical
stimuli, none were found to be statistically significant (Mann-Whitney Rank Sum
test).
Responses to Electrocutaneous Stimuli in Normal and Chronic Transection Animals
Representative traces of tail reflexes to single electrocutaneous stimuli are
shown in Figure 2-5. Reflexes occurred at short latencies with rapid rise times that
dissipated with a gradual decay. For normal animals and animals with a chronic
sacrocaudal transection, the tail responded similarly, allowing direct statistical
comparisons between pre- and post-lesion measurements of different features of
the reflex. For the following comparisons, averages from 4 pre-lesion sessions
were compared with averages from 2 or 3 sessions at the end of the testing period
for each animal.
Stimulation at 2 mA/mm2 was near threshold for elicitation of the reflex, and
responses were variably absent. Therefore, this intensity was not utilized for the
analyses of different characteristics of the reflex across stimulus intensities.
Comparing response probabilities for stimulation at 2 mA/mm2 during the pre-lesion
and chronic post-lesion periods, significant differences in reflex threshold were not


Figure 2-5. Representative traces of tail reflexes to single DC pulses of
electrocutaneous stimulation. From a pre-lesion recording (top) and after a chronic
sacrocaudal transection (bottom), the top 3 traces show reflex responses of the tail
to electrocutaneous stimulation at different stimulus intensities. For clarity, the
reflex traces are offset. The bottom trace shows the timing of the electrocutaneous
stimulus.


Force (0.65 N/div) Force (0.65 N/div)
35
.0 ms
800 ms/div
4.0 s
0.0 ms
800 ms/div
4.0 s


36
indicated (Mann-Whitney Rank Sum Test (T=60.0, P=0.694). For pre-lesion testing
at 2 mA/mm2, the median response probability was 0.6 (n=8), and the post-lesion
response probability was 0.75 (n=7).
In an overall evaluation of the electrocutaneous reflex, the force of the reflex
was observed to be larger for animals with a chronic transection than for normal
animals. The force of the reflex increased for both groups with higher levels of
stimulus intensity (Figure 2-6). Multiple regression analysis (Table 2-1) was
performed after the inverse exponential relationship was linearized by applying an
inverse transformation to the independent variable and a natural log transformation
to the dependent variable. The multiple linear regression analysis, which contained
stimulus intensity as a repeated continuous variable and the presence or absence
of the sacrocaudal lesion as the between-subject variable accounted for over 90
percent of the variance and was significant (r2 = 0.906; F = 30.59, P < 0.01). The
possible interaction between stimulus intensity and the presence of a lesion was not
significant (F= 3.04, P > 0.05). The main findings of the analysis were that: (1)
reflex force significantly increased with increases in stimulus intensity (F = 70.79,
P < 0.01); and (2) over this stimulus intensity range, there was a post-lesion
increase in reflex force (F = 7.34, P < 0.05).
An increase in peak reflex amplitude in animals with a chronic transection in
comparison to normal animals was found to be contributing factor for the
differences observed in reflex force between the two groups. Peak reflex amplitude
was observed to increase for both groups with higher levels of stimulus intensity,


Figure 2-6. The relationship of reflex force to stimulus intensity. Total reflex force at 5 different stimulus intensities is shown
pre-lesion (n=8; left) and after a chronic (>5 months) transection (n=7; right). Total reflex force was determined by summing
the reflex amplitudes from the time of stimulus offset until the decay of the reflex response reached 25 percent of peak
amplitude. The wider smooth lines (inverse exponential functions) represent the average response for each testing period.
Multiple regression analysis found that: (1) for both periods, reflex force significantly increased with increases in stimulus
intensity (P < 0.01); (2) over the stimulus intensity range, there was an overall post-lesion increase in reflex force (P < 0.05);
and (3) pre- and post-lesion differences in the increase in reflex force with increases in stimulus intensity were not significant
(P > 0.05).


Reflex Force (N)
Intensity (mA/mm2)
Reflex Force (N)
Intensity (mA/mm2)
CO
OO


39
Table 2-1. Summary of the multiple linear regression analysis of reflex force of
normal animals and after a chronic sacrocaudal transection.
Reflex Force
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
1.33
0.234
Treatment
1
39.4
7.34
<0.05
0.279
Subject
13
69.8
20.44
<0.01
0.773
0.94
0.041
Intensity
1
18.6
70.79
<0.01
0.904
-6.05
0.638
Treatment X
Intensity
1
0.8
3.04
>0.05
0.906
Residual
51
13.4
Total
67
127.8
30.59
<0.01
0.906
Transformations: Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Failed; Kurtosis=13.3, Skewness=2.34.
Homoscedasticity Test: Passed.


40
in what appeared to be a saturating nonlinear function (Figure 2-7). Multiple
regression analysis (Table 2-2) was performed after the relationship was linearized
by taking the reciprocal of the independent variable. The multiple linear regression
analysis, which contained stimulus intensity as a repeated continuous variable, the
presence or absence of the sacrocaudal lesion as the between-subject treatment
variable, and an interaction term between stimulus intensity and the possession of
a sacrocaudal lesion, accounted for over 90 percent of the variance and was
significant (r2 = 0.924; F = 39.06, P < 0.01). The main findings of the analysis were
that: (1) peak reflex amplitude significantly increased with increases in stimulus
intensity (F = 82.7, P < 0.01); (2) over this stimulus intensity range, there was a
post-lesion increase in the peak reflex amplitude (F = 9.13, P < 0.01); and (3) for
animals with a lesion, peak amplitude increased at a greater rate with increases in
stimulus intensity (F = 13.13, p < 0.01). The point of intersection of the ordinal
interaction was estimated be at 1.8 mA/mm2. Between normal animals and animals
with a chronic transection, peak reflex amplitudes were significantly different at
intensities greater than 2.4 mA/mm2 (Johnson-Neyman Technique).
Along with the differences observed between the two groups in peak reflex
amplitude, the latency at peak reflex amplitude was observed to remain relatively
constant in normal animals, but for animals with a lesion, latency at peak reflex
amplitude increased with higher levels of stimulus intensity (Figure 2-8). Multiple
regression analysis (Table 2-3) was performed after the relationship was linearized
by applying an inverse transformation to the independent variable and log


Figure 2-7. The relationship of peak reflex amplitude to stimulus intensity. Peak reflex amplitudes at 5 different stimulus
intensities are shown pre-lesion (left) and after a chronic transection (right). The larger smooth lines represent the average
response at each testing period. Multiple regression analysis found that: (1) for both periods, peak amplitude significantly
increased with increases in stimulus intensity (P < 0.01); (2) over the range of stimulus intensities, there was a post-lesion
increase in peak amplitude (P < 0.01); and (3) post-lesion, peak amplitude increased at a greater rate with increases in
stimulus intensity (P < 0.01).


Intensity (mA/mm2) Intensity (mA/mm2)
Peak Amplitude (N)
poo ooooooo
o -j- ro co A bn bo Peak Amplitude (N)
oooooooooo
o Ko co ^ di cn -Ni bo co
ZU


43
Table 2-2. Summary of the multiple linear regression analysis of peak reflex
amplitude of normal animals and after a chronic sacrocaudal transection.
Peak Amplitude
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.186
0.025
Treatment
1
1.230
9.13
<0.01
0.323
Subject
13
1.751
23.90
<0.01
0.781
1.015
0.039
Intensity
1
0.466
82.70
<0.01
0.904
-0.965
0.094
Treatment X
Intensity
1
0.074
13.13
<0.01
0.924
0.382
0.094
Residual
52
0.293
Total
68
3.521
39.06
<0.01
0.924
Transformations: Reciprocal of Intensity.
Normality Test: Failed; Kurtosis=0.105, Skewness=
Homoscedasticity Test: Passed.
0.836.


Figure 2-8. The relationship of latency at peak reflex amplitude to stimulus intensity. Latency at peak reflex amplitude at
5 different stimulus intensities are shown pre-lesion (left) and after a chronic transection (right). The larger smooth lines
represent the average response of each group. Multiple regression analysis found that: (1) latency at peak reflex amplitude
significantly increased with increases in stimulus intensity post-lesion (P < 0.01); (2) over the stimulus intensity range, there
was a post-lesion increase in latency at peak reflex amplitude (P < 0.01); and (3) increases in latency at peak reflex amplitude
with increases in stimulus intensity were significantly different in the pre- and post-lesion periods (P< 0.01).


Intensity (mA/mm2) Intensity (mA/mm2)
Latency at Peak Amplitude (msec)
Latency at Peak Amplitude (msec)
to
O
O
O)
O
00
o
o
o
to
o
N
O
O)
O
IO
00 o
o o
200


46
Table 2-3. Summary of the multiple linear regression analysis of latency at peak
reflex amplitude of normal animals and after a chronic sacrocaudal transection.
Latency at peak reflex amplitude
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.609
0.312
Treatment
1
3.39
13.99
<0.01
0.323
Subject
13
3.15
5.30
<0.01
0.781
0.898
0.068
Intensity
1
0.39
8.54
<0.01
0.904
-0.963
0.273
Treatment X
Intensity
1
0.77
16.85
<0.01
0.924
1.258
0.272
Residual
51
2.33
Total
67
10.30
10.53
<0.01
0.924
Transformations: Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Passed.


47
transformation to the dependent variable. The multiple linear regression model,
which contained stimulus intensity as a repeated continuous variable, the presence
or absence of the sacrocaudal lesion as the between-subject treatment variable,
and an interaction term between stimulus intensity and the presence of a
sacrocaudal lesion, accounted for over 90 percent of the variance and was
significant (r2 = 0.92; F = 10.53, P < 0.01). The main findings of the analysis were
that: (1) latency at peak reflex amplitude significantly increased with increases in
stimulus intensity for animals with a lesion (F = 8.54, P < 0.01); (2) over this
intensity range, there was a post-lesion increase in latency at peak reflex amplitude,
compared to pre-lesion values (F = 13.99, P < 0.01); and (3) for animals with a
lesion, latency at peak reflex amplitude increased at a greater rate with increases
in stimulus intensity (F = 16.85, P < 0.01). The point of intersection of the ordinal
interaction was estimated to be at 2.65 mA/mm2. Between normal animals and
animals with a chronic transection, the latency at peak reflex amplitude values were
significantly different at intensities greater than 3.1 mA/mm2 (Johnson-Neyman
Technique).
The rise time of the reflex was measured in order to investigate a possible
contribution to observed increases in peak amplitude at higher stimulus intensities
and in animals with a sacrocaudal lesion. Reflex rise time was observed to
increase for both groups with higher levels of stimulus intensity in what appeared
to be a saturating nonlinear function (Figure 2-9). Multiple regression analysis
(Table 2-4) was performed after the relationship was linearized by taking the


Figure 2-9. The relationship of reflex rise time to stimulus intensity. Reflex rise times at 5 different stimulus intensities are
shown for pre-lesion (left) and after a chronic transection (right). Reflex rise time was measured by calculating the slope
between the 25 and 75 percent peak amplitudes on the ascending limb of the reflex. The larger smooth lines represent the
average response of each group. Multiple regression analysis found that: (1) for both testing periods, reflex rise time
significantly increased with increases in stimulus intensity (P < 0.01); (2) over the intensity range, there was not an overall
difference in the reflex rise time between the pre- and post-lesion periods (P > 0.05); but (3) reflex rise time increased with
increases in stimulus intensity at a greater rate pre-lesion than post-lesion (P < 0.05).


Intensity (mA/mm2) Intensity (mA/mm2)
Rise Time (dynes/sec)
p p p P p ;-*-*--*-* N> IVJ NJ IO
k> -N b> bo o k> 4*. b) bo o Kj 4*. b>
Rise Time (dynes/sec)
OOOOOL
bro4^a5book) ^CT)CDok)T.CT)


50
Table 2-4. Summary of the multiple linear regression analysis of reflex rise time
of normal animals and after a chronic sacrocaudal transection.
Rise Time
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.082
0.012
Treatment
1
0.1410
2.52
>0.05
0.140
Subject
13
0.7280
55.57
<0.01
0.861
0.991
0.030
Intensity
1
0.0833
82.66
<0.01
0.943
-0.402
0.040
Treatment X
Intensity
1
0.0048
4.76
<0.05
0.949
0.096
0.040
Residual
52
0.0524
Total
68
1.0095
59.36
<0.01
0.949
Transformations: Reciprocal of Intensity, log of the dependent variable plus one.
Normality Test: Passed.
Homoscedasticity Test: Passed.


51
inverse of the independent variable and applying a log transformation to the
dependent variable. The multiple linear regression analysis, which contained
stimulus intensity as a repeated continuous variable, and an interaction term
between stimulus intensity and the possession of a sacrocaudal lesion, accounted
for over 90 percent of the variance and was significant (r2 = 0.95; F = 59.36, P <
0.01). The main findings of the model were that: (1) reflex rise time significantly
increases with increases in stimulus intensity (F = 82.7, P < 0.01); and (2) for the
between-subject variable, significant differences were not obtained for the presence
or absence of a lesion (F= 2.52, P > 0.05); and (3) for normal animals reflex rise
time increased at a greater rate with increases in stimulus intensity than in animals
with a lesion (F = 4.76, P < 0.05). The point of intersection of the ordinal interaction
was estimated be at -3.4 mA/mm2. Between normal animals and animals with a
chronic transection, the rise time values were significantly different at intensities
greater than -0.4 mA/mm2 (Johnson-Neyman Technique).
Two different measures of reflex duration were obtained in order to
determine the contribution of reflex duration to the reflex force estimate. It took
longer for animals with a lesion to reach half-maximal reflex duration when
compared to normal animals. Half maximal reflex duration was observed to
increase for both groups with higher levels of stimulus intensity (Figure 2-10). The
nonlinear relationships between stimulus intensity and half maximal reflex duration
were fit with hyperbola functions. Multiple regression analysis (Table 2-5) was
performed after the hyperbola relationship was linearized by applying an inverse


Figure 2-10. The relationship of half maximal reflex duration to stimulus intensity. Half maximal durations at 5 different
stimulus intensities are shown pre-lesion (left) and for animals with a chronic transection (right). The larger smooth curved
lines (hyperbola functions) represent the average response for each testing period. Multiple regression analysis found that:
(1) for both groups, half maximal reflex duration significantly increased with increases in stimulus intensity (P < 0.05); (2) over
this stimulus intensity range, there was a post-lesion increase in half maximal reflex duration (P < 0.05); and (3) increases
in half maximal reflex duration with increases in stimulus intensity were not significantly different pre- and post-lesion (P >
0.05).


Duration 0.5 max (msec)
Intensity (mA/mm2)
Duration 0.5 max (msec)
Intensity (mA/mm2)
CD
CO


54
Table 2-5. Summary of the multiple linear regression analysis of half maximal
reflex duration of normal animals and after a chronic sacrocaudal transection.
Half Maximal Duration
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
-1.04E-3
3.41 E-4
Treatment
1
1.01E-4
8.21
<0.05
0.298
Subject
13
1.60E-5
12.94
<0.05
0.769
0.933
5.01E-2
Intensity
1
2.97E-5
31.23
<0.05
0.857
7.65E-3
1.21 E-3
Treatment
X Intensity
1
1.40E-6
1.47
>0.05
0.861
Residual
51
4.85E-5
Total
67
2.92E-4
19.20
<0.01
0.861
Transformations: Reciprocal of Intensity minus 1; reciprocal of the dependent variable.
Normality Test: Failed; Kurtosis=7.45, Skewness=2.16.
Homoscedasticity Test: Passed.


55
transformation to both the independent variable and the dependent variable. The
multiple linear regression analysis, which contained stimulus intensity as a repeated
continuous variable and the presence or absence of the sacrocaudal lesion as the
between-subject variable, accounted for over 80 percent of the variance and was
significant (r2 = 0.86; F = 19.2, P < 0.01). The interaction between stimulus
intensity and the presence of a lesion was not significant (F = 1.47, P > 0.05). The
main findings of the analysis were that: (1) half maximal reflex duration significantly
increased with increases in stimulus intensity (F = 31.23, P < 0.05); and (2) over
this stimulus intensity range, there was a post-lesion increase in half maximal reflex
duration (F = 8.21, P < 0.05).
One quarter maximal reflex duration values were observed to be greater in
animals with lesions in comparison to normal animals (Figure 2-11). Multiple
regression analysis (Table 2-6) was performed after the relationship was linearized
by taking the reciprocal of the independent variable and performing a natural log
transformation of the dependent variable. The multiple linear regression analysis,
which contained stimulus intensity as a repeated continuous variable, the presence
or absence of the sacrocaudal lesion as the between-subject treatment variable,
and an interaction term between stimulus intensity and the possession of a
sacrocaudal lesion accounted for 90 percent of the variance and was significant (r2
= 0.899; F = 27.92, P < 0.01). The main findings of the analysis were that: (1)
quarter maximal reflex duration significantly increased with increases in stimulus
intensity (F = 3.54, P < 0.01); (2) over this stimulus intensity range, there was a


Figure 2-11. The relationship of quarter maximal reflex duration to stimulus intensity. Quarter maximal reflex durations at
5 different stimulus intensities are shown pre-lesion (left) and after a chronic transection (right). The larger smooth lines
(inverse exponential functions) represent the average response for each testing period. Multiple regression analysis found
that: (1) for both periods, quarter maximal reflex duration significantly increased with increases in stimulus intensity (P < 0.01);
(2) over this stimulus intensity range, there was a post-lesion increase in the quarter maximal reflex duration (P < 0.05); and
(3) post-lesion, quarter maximal duration increased with increases in stimulus intensity at a greater rate than pre-lesion (P<
0.01).


Duration 0.25 max (msec)
Duration 0.25 max (msec)
Intensity (mA/mm2)


58
Table 2-6. Summary of the multiple linear regression analysis of quarter
maximal reflex duration of normal animals and after a chronic sacrocaudal
transection.
Quarter Maximal Duration
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.595
0.280
Treatment
1
11.60
5.55
<0.05
0.242
Subject
13
27.19
21.70
<0.01
0.810
0.965
0.043
Intensity
1
3.54
36.72
<0.01
0.884
-1.601
0.234
Treatment X
Intensity
1
0.07
7.68
<0.01
0.899
0.723
0.233
Residual
50
4.82
Total
66
43.07
27.92
<0.01
0.899
Transformations: Reciprocal of Intensity minus one; natural log of the dependent
variable.
Normality Test: Passed.
Homoscedasticity Test: Passed.


59
post-lesion increase in quarter maximal reflex duration (F = 5.55, P < 0.05); and (3)
for animals with a lesion, quarter maximal reflex duration increased at a greater rate
with increases in stimulus intensity (F = 7.68, P < 0.01). The point of intersection
of the ordinal interaction was estimated be at 2 mA/mm2. Comparing normal
animals and animals with a chronic transection, the quarter maximal durations were
significantly different at intensities greater than 2.3 mA/mm2 (Johnson-Neyman
Technique).
Reflex latency was observed to be shorter in normal animals in comparison
to animals with a transection of the sacrocaudal spinal cord. The latency of reflex
onset was observed to decrease for both groups with higher levels of stimulus
intensity in what appeared to be a saturating nonlinear function (Figure 2-12). The
nonlinear relationships between stimulus intensity and reflex latency were fit with
inverse exponential functions. Multiple regression analysis (Table 2-7) was
performed after the inverse exponential relationship was linearized by applying a
natural log transformation to the dependent variable. The multiple linear regression
analysis, which contained stimulus intensity as a repeated continuous variable and
the presence or absence of the sacrocaudal lesion as the between-subject variable
accounted for over 80 percent of the variance and was significant (r2 = 0.834; F =
16.24, P < 0.01). The possible interaction between stimulus intensity and the
presence of a lesion was not significant (F= 0.53, P > 0.05). The main findings of
the analysis were that: (1) reflex latency significantly decreased with increases in


Figure 2-12. The relationship of reflex latency to stimulus intensity. Reflex latency estimates at 5 different stimulus intensities
are shown pre-lesion (left) and after a chronic transection (right). Reflex latency was determined by extrapolating back to
the x intercept of a line through the 10 and 90 percent peak amplitude time points. The larger smooth lines (inverse
exponential functions) represent the average response for each testing period. Multiple regression analysis found that: (1)
for both periods, reflex latency significantly decreased with increases in stimulus intensity (P < 0.01); and (2) over the
intensity range, there was a post-lesion increase in reflex latency (P < 0.05).


Latency (msec)
2345678 9 10 11
Intensity (mA/mm2)


62
Table 2-7. Summary of the multiple linear regression analysis of reflex latency
of normal animals and after a chronic sacrocaudal transection.
Reflex Latency
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
4.403
1.158
Treatment
1
202.5
12.66
<0.01
0.343
Subject
13
208.0
8.46
<0.05
0.697
0.985
0.060
Intensity
1
80.3
42.43
<0.01
0.834
-5.664
0.772
Treatment X
Intensity
1
1.0
0.53
>0.05
0.834
0.382
0.094
Residual
51
98.4
Total
67
589.2
16.24
<0.01
0.834
Transformations: Log of Intensity.
Normality Test: Passed.
Homoscedasticity Test: Passed.


63
stimulus intensity (F = 42.43, P < 0.01); and (2) over this stimulus intensity range,
there was a increase in post-lesion reflex latency (F = 12.66, P < 0.01).
Flow the different measured characteristics of the reflex waveform were
related was examined with Spearman rank order correlation coefficients as shown
in Table 2-8. In normal prelesion animals, measures of force, half maximal
duration, quarter maximal duration and peak amplitude were significantly
intercorrelated. Reflex rise time was significantly correlated with reflex force, peak
amplitude and latency at peak reflex amplitude, but not with the measures of
duration. Latency at peak reflex amplitude was significantly correlated with the
measures of duration, but not with peak amplitude or reflex force. Reflex latency
was not significantly correlated with any of the other measured characteristics. In
contrast, all of the reflex characteristics were significantly intercorrelated in chronic
post-lesion animals. The correlations among the reflex characteristics were
significantly greater in the chronic post-lesion animals (Mann-Whitney Rank Sum
Test; T = 306.0, P =< 0.0001).
The Time-course of Changes in Reflex Characteristics
The time-course of changes in the electrocutaneous tail reflex after a
sacrocaudal spinal transection was evaluated for 5 animals that were tested
preoperatively and at 5 different post-lesion time periods. Post-lesion, alterations
in the electrocutaneous reflex progressively developed over a period of months.


64
Table 2-8. Spearman rank order correlation coefficients between
measured characteristics of the reflex waveform.
Latency at
Peak
Amplitude
Duration
0.25 max
Duration
0.5 max
Rise
Time
Reflex
Latency
Reflex
Force
Normal
Peak
Amplitude
0.013*
0.937
0.451
0.008
0.297
0.082
0.840
<0.001
-0.255
0.133
0.840
<0.001
Latency at Peak
Amplitude
0.650
<0.001
0.610
<0.001
-0.448
0.006
-0.004
0.980
0.208
0.082
Duration
0.25 max
0.710
<0.001
0.038
0.829
-0.311
0.073
0.740
<0.001
Duration
0.5 max
-0.112
0.520
-0.211
0.223
0.620
<0.001
Rise
Time
-0.238
0.162
0.518
0.002
Reflex
Latency
-0.326
0.056
Postlesion
Peak Amplitude
0.750
<0.001
0.870
<0.001
0.810
<0.001
0.740
<0.001
-0.470
0.006
0.940
<0.001
Latency at Peak
Amplitude
0.730
<0.001
0.750
<0.001
0.540
0.002
-0.720
<0.001
0.730
<0.001
Duration
0.25 max
0.950
<0.001
0.640
<0.001
-0.650
<0.001
0.970
<0.001
Duration
0.5 max
0.570
<0.001
-0.630
<0.001
0.930
<0.001
Rise
Time
-0.394
0.023
0.660
<0.001
Reflex
Latency
-0.542
0.001
For each cell, the top value is the correlation coefficient while
the bottom number is the corresponding P value.


65
The force estimate of the reflex was observed to increase over time after the
lesion for individual animals (Figure 2-13). For the group of subjects, a multiple
linear regression analysis (Table 2-9) accounted for over 70 percent of the variance
and was significant (r2 = 0.747; F = 30.8, P < 0.0001). Interactions between
stimulus intensity and reflex force at different times after the lesion were not
significant (P > 0.05). The main findings of the multiple regression analysis were:
(1) reflex force significantly increased with increases in stimulus intensity (t = -9.18,
P < 0.0001), and (2) the grouping of reflex force values into different post-lesion
time periods accounted for a significant proportion of the variance (Table 2-9). A
post hoc Scheffe analysis found that pre-lesion reflex force values were not
significantly different from post-lesion values at 7-14 and 21-35 days (P > 0.05), but
the reflex force at those 3 time periods was significantly less than at 70-90 days,
140-165 days, and >210 days (P < 0.05).
Peak reflex amplitude was observed to increase over time after the lesion for
individual animals (Figure 2-14). For the group of subjects, the multiple linear
regression analysis (Table 2-10) accounted for over 70 percent of the variance and
was significant (r2 = 0.743; F = 30.6, P < 0.0001). Interactions between stimulus
intensity and peak amplitude at different times after the lesion were generally not
significant (P > 0.05; except for the period 70-90 days, P = 0.0217). The main
findings of the multiple regression analysis were: (1) peak reflex amplitude
significantly increased with increases in stimulus intensity (t = -10.142, P < 0.0001),
and (2) the grouping of peak reflex amplitudes into different post-lesion time periods


Figure 2-13. The time course of changes in reflex force. Reflex force, averaged
across intensities for each of 5 animals (symbols), is shown at 6 different time
periods. The columns represent the average reflex force determined across
animals. A post hoc Scheffe analysis found that pre-lesion values were not
significantly different from the 7-14 and 21-35 day values (P > 0.05), but reflex force
at those 3 time periods was significantly less than the mean force at 70-90 days,
140-165 days, and >210 days (P < 0.05).


Reflex Force (N)
67
Time period (days)


68
Table 2-9. Summary of the multiple linear regression analysis of changes in
reflex force over time after sacrocaudal transection.
Reflex Force
Source
DF
SS F
P
R2
Regression
15
167.6
Residual
122
56.7
Total
137
224.3 30.8
<0.0001
0.747
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
7.236
<0.0001
2.120
0.2930
Subject
12.825
<0.0001
0.703
0.0548
Intensity
-9.181
<0.0001
-6.069
0.6610
7-14 days
-3.857
0.0002
-0.487
0.1263
21-35 days
-2.444
0.0159
-0.314
0.1285
70-90 days
3.648
0.0004
0.498
0.1366
140-165 days
4.233
<0.0001
0.535
0.1263
210 days plus
4.811
<0.0001
0.608
0.1263
Intensity X
7-14 days
3.374
0.7088
0.546
1.4586
Intensity X
21-35 days
1.126
0.2622
1.647
1.4620
Intensity X
70-90 days
-1.727
0.0867
-2.695
1.5610
Intensity X
140-165 days
-1.211
0.2281
-1.747
1.4421
Intensity X
>210 days
-0.319
0.7501
-0.460
1.4421
Transformations:
Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Failed.


Figure 2-14. The time course of changes in peak reflex amplitude and latency at
peak reflex amplitude. Peak amplitudes (top) and latency at peak amplitude
(bottom) for each of 5 animals (symbols) are shown at 6 different time periods,
averaged across intensities. The columns represent the average peak amplitude
and latency at peak reflex amplitude determined across animals. A post hoc
Scheffe analysis found that pre-lesion peak amplitudes were significantly less than
post-lesion amplitudes at 21-35, 70-90, 140-165 and >210 days (P < 0.05). Post
hoc Scheffe analysis found that pre-lesion latencies at peak amplitude were
significantly less than post-lesion latencies at 70-90, 140-165 and >210 days (P <
0.05).


Latency at Peak Amplitude (msec) Peak Amplitude (N)
70
Time Period (days)
Time Period (days)


71
Table 2-10. Summary of the multiple linear regression analysis of changes in
peak reflex amplitude over time after sacrocaudal transection.
Peak Amplitude
Source
DF
SS F
P
R2
Regression
15
3.75
Residual
124
1.29
Total
139
5.04 30.6
<0.0001
0.743
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
6.220
<0.0001
0.2030
0.0326
Subject
13.413
<0.0001
0.9837
0.0733
Intensity
-10.142
<0.0001
-0.9987
0.0985
7-14 days
-1.946
0.0538
-0.0368
0.0189
21-35 days
0.093
0.9258
0.0018
0.0189
70-90 days
1.599
0.1123
0.0328
0.0205
140-165 days
2.962
0.0037
0.0560
0.0189
210 days plus
4.133
<0.0001
0.0781
0.0189
Intensity X
7-14 days
0.982
0.3281
0.2144
0.2184
Intensity X
21-35 days
-0.416
0.6780
-0.0909
0.2184
Intensity X
70-90 days
-2.323
0.0217
-0.5430
0.2337
Intensity X
140-165 days
0.012
0.9897
0.0028
0.2159
Intensity X
>210 days
-0.253
0.8004
-0.0547
0.2159
Transformations
: Reciprocal of Intensity.
Normality Test:
Passed.
Homoscedasticity Test: Failed.


72
accounted for a significant amount of the variance (Table 2-10). Scheffe analysis
found that pre-lesion values were significantly less than post-lesion peak amplitude
levels at 21-35 days, 70-90 days, 140-165 days, and >210 days (P < 0.05).
Latency at peak reflex amplitude was observed to increase over time after
the lesion for the individual animals (Figure 2-14). The multiple linear regression
analysis (Table 2-11) for latency at peak reflex amplitude accounted for over 50
percent of the variance and was significant (r2 = 0.537; F = 12.1, P < 0.0001).
Interactions between stimulus intensity and time after the lesion were generally not
significant (P > 0.05; except for the period of >210 days, P = 0.0067). The main
findings of the multiple linear regression analysis were: (1) latency at peak reflex
amplitude significantly increased with increases in stimulus intensity (t = -3.736, P
= 0.0003), and (2) the grouping of latency at peak reflex amplitude values into post
lesion time periods accounted for a significant amount of the variance (Table 2-11).
Post hoc analysis found that pre-lesion values were significantly less than the mean
peak reflex latencies at 70-90 days, 140-165 days, and >210 days (Scheffe, P <
0.05).
Changes over time for several measures of reflex duration are shown in
Figure 2-15. Multiple linear regression analyses were performed on the measures
of half maximal reflex duration (Table 2-12, R2 = 0.539, F = 12.3, P < 0.0001) and
quarter maximal reflex duration (Table 2-13, R2 = 0.599, F = 15.7, P < 0.0001). For
half maximal reflex duration, interactions between stimulus intensity and duration
for different times after the lesion were generally not significant (P > 0.05; except


73
Table 2-11. Summary of the multiple linear regression analysis of changes in
latency at peak reflex amplitude over time after sacrocaudal transection.
Latency at peak reflex amplitude
Source
DF
SS F
P
R2
Regression
15
6.17
Residual
122
5.31
Total
137
11.48 12.1
<0.0001
0.537
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
1.408
0.1615
0.9063
0.6435
Subject
5.668
<0.0001
0.8200
0.1447
Intensity
-3.736
0.0003
-0.7634
0.2043
7-14 days
-1.813
0.0723
-0.0701
0.0386
21-35 days
0.269
0.7887
0.0104
0.0386
70-90 days
1.913
0.0580
0.0795
0.0416
140-165 days
3.005
0.0032
0.1161
0.0386
210 days plus
4.299
<0.0001
0.1689
0.0393
Intensity X
7-14 days
1.666
0.0981
0.7452
0.4472
Intensity X
21-35 days
0.867
0.3875
0.3879
0.4472
Intensity X
70-90 days
-1.304
0.1947
-0.6240
0.4786
Intensity X
140-165 days
-1.380
0.1700
-0.6103
0.4422
Intensity X
>210 days
-2.757
0.0067
-1.2758
0.4628
Transformations: Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Failed.


Figure 2-15. The time course of changes in reflex duration. Half maximal (top) and
quarter maximal (bottom) durations for each of 5 animals (symbols) are shown at
6 time periods, averaged across intensities. The columns represent the average
half maximal and quarter maximal durations determined across animals. A post hoc
Scheffe analysis found that pre-lesion half maximal durations were significantly less
than the values at 70-90, 140-165 and >210 days (P < 0.05). Post hoc Scheffe
analysis found that pre-lesion quarter maximal durations were not significantly
different from the 7-14 and 21-35 day values (P > 0.05), but quarter maximal
durations at these 3 time periods were significantly less than at 70-90 days, 140-
165 days, and >210 days (P < 0.05).


Duration 0.25 max (msec) Duration 0.5 max (msec)
75
Time Period (days)
Time Period (days)


76
Table 2-12. Summary of the multiple linear regression analysis of changes over
time in the duration of decay to 0.5 maximal amplitude after sacrocaudal
transection.
Duration at Half Maximal
Source
DF
SS
F
P
R2
Regression
15
0.000320
Residual
123
0.000274
Total
138
0.000594
12.3
<0.0001
0.539
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
3.741
0.0003
0.0015
0.0004
Subject
5.906
<0.0001
0.4847
0.0821
Intensity
6.549
<0.0001
0.0057
0.0009
7-14 days
2.136
0.0346
0.0006
0.0003
21-35 days
0.523
0.6019
0.0001
0.0003
70-90 days
-1.833
0.0692
-0.0005
0.0003
140-165 days
-2.954
0.0037
-0.0080
0.0003
210 days plus
-3.999
0.0001
-0.0011
0.0003
Intensity X
7-14 days
-0.943
0.3475
-0.0018
0.0019
Intensity X
21-35 days
-1.102
0.2724
-0.0021
0.0019
Intensity X
70-90 days
2.276
0.0245
0.0047
0.0021
Intensity X
140-165 days
1.002
0.3180
0.0019
0.0019
Intensity X
>210 days
-0.160
0.8733
-0.0003
0.0019
Transformations: Reciprocal of Intensity-1; reciprocal of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Failed.


77
Table 2-13. Summary of the multiple linear regression analysis of changes over
time in the duration to decay to 0.25 maximal amplitude after sacrocaudal
transection.
Duration at Quarter Maximal
Source
DF
SS
F
P
Ft2
Regression
15
55.0
Residual
123
36.8
Total
138
91.8
15.7
<0.0001
0.599
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
4.069
<0.0001
2.100
0.5160
Subject
8.743
<0.0001
0.698
0.0798
Intensity
-5.033
<0.0001
-1.603
0.3185
7-14 days
-3.359
0.0010
-0.340
0.1013
21-35 days
-3.303
0.0012
-0.334
0.1013
70-90 days
4.191
<0.0001
0.457
0.1091
140-165 days
2.967
0.0036
0.300
0.1012
210 days plus
3.536
0.0006
0.358
0.1012
Intensity X
7-14 days
2.093
0.0384
1.470
0.7023
Intensity X
21-35 days
1.788
0.0762
1.256
0.7023
Intensity X
70-90 days
-1.917
0.0575
-1.446
0.7543
Intensity X
140-165 days
-2.384
0.0186
-1.661
0.6966
Intensity X
>210 days
-0.754
0.4520
-0.526
0.6966
Transformations: Reciprocal of Intensity-1; natural log of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Failed.


78
for the time period of 70-90 days, P = 0.0245). For quarter maximal reflex duration,
some of the interactions between stimulus intensity and duration at different times
after the lesion approached significance (Table 2-13). The main findings of the
multiple regression analyses were that: (1) reflex duration significantly increased
with increases in stimulus intensity (Table 2-12, t = 6.549, P < 0.0001; Table 2-13,
t=-5033, P < 0.0001), and (2) the grouping of reflex durations at most of the post
lesion time periods accounted for a significant amount of the variance (Tables 2-12,
2-13). For the increasing trend over time in half maximal reflex duration, a post hoc
Scheffe analysis found that pre-lesion values were significantly less than post
lesion values at 70-90 days, 140-165 days, and >210 days (P < 0.05). For quarter
maximal duration, a post hoc Scheffe analysis found that pre-lesion values were not
significantly different from post-lesion values at 7-14 and 21-35 days (P > 0.05).
The quarter maximal durations at these 3 time periods were significantly less than
the mean quarter maximal reflex durations at 70-90 days, 140-165 days, and >210
days (P < 0.05).
The changes over time in reflex latency and rise time are shown in Figure 2-
16. For reflex latency, the multiple linear regression analysis accounted for over
60 percent of the variance and was significant (Table 2-14; r2 = 0.634; F = 18.2, P
< 0.0001). Interactions between stimulus intensity and latency at different times
after the lesion were not significant. The main findings of the multiple regression
analysis were: (1) reflex latency significantly decreased with increases in stimulus
intensity (t = -8.504, P < 0.0001), and (2) the grouping of reflex latency values into


Figure 2-16. The time course of changes in reflex latency and rise time. Reflex
latency (top) and rise time (bottom) for 5 animals (symbols) are shown at 6 time
periods, averaged across intensities. The columns represent the average reflex
latencies and reflex rise times determined across animals. A post hoc Scheffe
analysis found that pre-lesion latencies were significantly less than at all post-lesion
time periods (P < 0.05). Pre-lesion rise times were not significantly different from
post-lesion values at any time period after the lesion.


Rise Time (dynes/sec) Latency (msec)
25
20
15
10
5 -
A

o
A
Prelesion 7-14 21-35 70-90 140-165
Time Period (days)
>210
Time Period (days)


81
Table 2-14. Summary of the multiple linear regression analysis of changes in
reflex latency after sacrocaudal transection.
Reflex Latency
Source
DF
SS
F
P
R2
Regression
15
817
Residual
123
472
Total
138
1289
18.2
<0.0001
0.634
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
3.104
0.0024
6.3303
2.040
Subject
8.560
<0.0001
0.9445
0.110
Intensity
-8.504
<0.0001
-7.1937
0.846
7-14 days
-0.104
0.9170
-0.0379
0.363
21-35 days
0.005
0.9958
0.0019
0.363
70-90 days
1.660
0.0994
0.6473
0.390
140-165 days
3.393
0.0009
1.2295
0.362
210 days plus
2.527
0.0128
0.9156
0.362
Intensity X
7-14 days
0.184
0.8544
0.3441
1.871
Intensity X
21-35 days
1.709
0.0900
3.1984
1.872
Intensity X
70-90 days
-1.380
0.1700
-2.7382
1.984
Intensity X
140-165 days
-0.486
0.1627
-0.8901
1.831
Intensity X
>210 days
0.084
0.9329
0.1545
1.831
Transformations
: Log of Intensity.
Normality Test:
Failed; Passed.
Homoscedasticity Test: Passed.


82
post-lesion time periods accounted for a significant amount of the variance (Table
2-14). A post hoc Scheffe analysis of latency found that pre-lesion values were
significantly less than that observed for all post-lesion time periods (P < 0.05).
Reflex rise time at the different time periods is shown in Figure 2-16. The
multiple linear regression analysis accounted for over 70% of the variance and was
significant (Table 2-15, R2 = 0.74, P < 0.0001). Except for a significant effect of
intensity (P = < 0.0001) and an interaction between intensity and rise time at >210
days (P = 0.0387), no other significant differences were obtained for changes in rise
time.
Stability of the Electrocutaneous Reflex with Repetitive Stimulation
When electrocutaneous stimulation was presented in blocks of trials at a
single intensity, there were instances of wind-down (a decrease) or wind-up (an
increase) of reflex force estimates, when comparing the response to the first
stimulus with subsequent responses. The frequency of wind-down varied in normal
animals, ranging from 10 percent to 36 percent, with a median of 13.9 percent
(Figure 2-17). Post-lesion, wind-down increased for some animals and decreased
for others. For the population of 5 animals there was a slight decrease in the
occurrence of wind-down over the 5 different time periods after the lesion.
In normal animals wind-up occurred less often than wind-down, varying from
0 percent to 14 percent, with a median of 8 percent across subjects (Figure 2-18).
Post-lesion, the frequency of wind-up increased for most animals. Also, as early


83
Table 2-15. Summary of the multiple linear regression analysis of changes in
reflex rise time after sacrocaudal transection.
Rise Time
Source
DF
SS F
P
R2
Regression
15
1.021
Residual
123
0.361
Total
138
1.382 29.7
<0.0001
0.739
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
4.632
<0.0001
0.0949
0.0205
Subject
15.896
<0.0001
0.9712
0.0611
Intensity
-8.828
<0.0001
-0.4633
0.0525
7-14 days
-0.377
0.7068
-0.0038
0.0100
21-35 days
-1.648
0.1019
-0.0165
0.0100
70-90 days
0.762
0.4473
0.0083
0.0109
140-165 days
0.446
0.6561
0.0045
0.0100
210 days plus
1.897
0.0601
0.0190
0.0100
Intensity X
7-14 days
-0.662
0.5091
-0.0767
0.1159
Intensity X
21-35 days
-1.549
0.1240
-0.1795
0.1159
Intensity X
70-90 days
-1.397
0.1648
-0.1733
0.1240
Intensity X
140-165 days
1.840
0.0681
0.2109
0.1146
Intensity X
>210 days
2.089
0.0387
0.2394
0.1146
Transformations: Reciprocal of Intensity; log of the dependent variable+1.
Normality Test: Passed.
Homoscedasticity Test: Passed.


Figure 2-17. Frequency of wind-down of the reflex with repetitive stimulation. For
the 6 time periods, the graphs show the median probability of occurrence of a
diminished reflex response with repetitive stimulation at one stimulus intensity for
each animal (top) and for the group of 5 subjects (bottom). A decreased frequency
of wind-down was observed following sacrocaudal lesions. The lines represent 75
percent and 25 percent confidence intervals.


Wind-down Probability
85
0.70
0.60
Time Period (days)

Pre-lesion

7 to 14
no
21 to 35

70 to 90

140 to 165
s
>210
Time Period (days)


Figure 2-18. Frequency of wind-up with repetitive stimulation. For the 6 time
periods, the graphs show the median probability of occurrence of wind-up of the
reflex response with repetitive stimulation at a constant intensity for each subject
(top) and for the group (bottom). The increase in frequency of wind-up developed
progressively over time after sacrocaudal lesions. The lines represent 75 percent
and 25 percent confidence intervals.


Wind-up Probability
87
0.70
0.60
>.
S
ro
-O
o
1
CL
Q.
3
i
"O
c
£

Pre-lesion

7 to 14
21 to 35

70 to 90
B
140 to 165
Q
>210
Subject 1 Subject 2 Subject 3 Subject 4 Subject 5
Time Period (days)
Time Period (days)


Full Text
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EFFECTS OF SACROCAUDAL SPINAL CORD LESIONS AND TRANSPLANTS
OF FETAL TISSUE ON CUTANEOUS REFLEXES OF THE TAIL
By
ROBERT MARK FRIEDMAN
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
1995

to absent friends

ACKNOWLEDGEMENTS
A number of individuals made significant contributions to the successful
completion of this research. First and foremost, I would like to recognize the
patience of my mentor Dr. Charles Vierck, Jr. Even though there may be more
productive student-mentor approaches, I feel that his hands off style of minimal
cajoling has made me a scientist better prepared to achieve my future research
goals. For not always getting on my goat, I sincerely recognize the contributions
of Dr. Brian Cooper to my early graduate research endeavors. Dr. Louis Ritz
receives a special acknowledgement, for I appreciate the opportunity to study tail
behaviors under his tutelage. I especially want to thank Dr. Paul Reier who made
numerous and invaluable contributions to this research. I also appreciate the time
and support of my other committee members, past and present: Drs. Paul Brown,
Christiana Leonard, John Munson, and Alan Spector.
Thanks go to Carolyn Baum for her assistance in working with the cats
involved in this research. Special thanks go to Jean Kaufman, for her help in other
aspects of my research is greatly appreciated.
I would also like to recognize the excellent care of the felines provided by
Ms. Barbara O'Steen, Kim Foli and Dan Theele, DVM. I especially would like to
thank the technical support of Anwaral Azam. Without his help in troubleshooting
electrical and computer problems, this research would have never gotten off the
ground.
in

The support of past and present fellow graduate students should not go
unmentioned. Gregory Schrimscher, Jianxin Bao, Audrey Kalehua, Diana
Glendinning, David Yeomans, Doug Swanson and Laura Errante made the
struggles of graduate school bearable. I especially appreciate the support of James
Makous for engaging discussions, advice and reflections on the scientific method,
life as research scientist, and the somatosensory system.
Love and special thanks go to my family. I would like to acknowledge the
understanding and support of my parents, Carolyn and Lester Friedman, who
wanted their son to grow up to be a doctor but will have to settle for a son with a
doctorate. I especially want to recognize the love and support of my sister Renee
Friedman who took longer to receive her Ph.D. than I, but to my chagrin still
finished tier's first. Alas, the unspoken love and contributions of Laura Friedman
to my life are immeasurable.
Financial support for this research was provided by the Center for
Neurobiological Sciences (NIMH grant MH15737), NIH grant NS27511 to Dr. Paul
J. Reier, the Mark F. Overstreet Fund for Spinal Cord Injury Research and the State
of Florida Impaired Drivers and Speeders Trust Fund.
IV

TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES ix
ABSTRACT xi
CHAPTERS
1 INTRODUCTION AND BACKGROUND 1
Consequences of Human Spinal Cord Injury 1
Treatments for Spinal Cord Injury 4
Behavioral Evaluation of Spinal Cord Injury 6
Animal Model Systems to Study Spinal Cord Injury 7
Overview of Dissertation 11
2 CUTANEOUS REFLEXES OF THE TAIL OF CATS BEFORE AND
AFTER SACROCAUDAL SPINAL LESIONS 13
Introduction 13
Methods 15
Results 25
Discussion 106
3 EFFECTS OF FETAL SPINAL TISSUE TRANSPLANTS ON
CUTANEOUS REFLEXES OF THE CATTAIL 115
Introduction 115
Methods 117
Results 121
Discussion 154
4 OVERALL DISCUSSION 164
REFERENCES 169
BIOGRAPHICAL SKETCH 180
v

LIST OF TABLES
Table 2-1
Summary of the multiple linear regression analysis of
reflex force of normal animals and after a
chronic sacrocaudal transection
. . 39
Table 2-2
Summary of the multiple linear regression analysis
of peak reflex amplitude of normal animals
and after a chronic sacrocaudal transection
. . 43
Table 2-3
Summary of the multiple linear regression analysis
of latency at peak reflex amplitude of normal animals
and after a chronic sacrocaudal transection
. . 46
Table 2-4
Summary of the multiple linear regression analysis
of rise time of normal animals and after
a chronic sacrocaudal transection
. . 50
Table 2-5
Summary of the multiple linear regression
analysis of half maximal reflex duration of normal
animals and after a chronic sacrocaudal transection ....
. . 54
Table 2-6
Summary of the multiple linear regression analysis
of quarter maximal reflex duration of normal
animals and after a chronic sacrocaudal transection ....
. . 58
Table 2-7
Summary of the multiple linear regression analysis
of reflex latency of normal animals and after a
chronic sacrocaudal transection
. . 62
Table 2-8
Spearman rank order correlation coefficients between
measured characteristics of the reflex waveform
. . 64
Table 2-9
Summary of the multiple linear regression analysis
of changes in reflex force over time after sacrocaudal
transection
. . 68
Table 2-10
Summary of the multiple linear regression analysis
of changes in peak reflex amplitude over time
after sacrocaudal transection
. . 71
Table 2-11
Summary of the multiple linear regression analysis
of changes in latency at peak reflex amplitude over time
after sacrocaudal transection
. . 73
Table 2-12
Summary of the multiple linear regression analysis
of changes over time in the duration of decay to
0.5 maximal amplitude after sacrocaudal transection ....
. . 76
VI

Table 2-13
Summary of the multiple linear regression analysis
of changes over time in the duration to decay to
0.25 maximal amplitude after sacrocaudal transection ....
. 77
Table 2-14
Summary of the multiple linear regression analysis
of changes in reflex latency after sacrocaudal transection . .
. 81
Table 2-15
Summary of the multiple linear regression analysis
of changes in reflex rise time after sacrocaudal transection. .
. 83
Table 2-16
Comparison of 5 reflex characteristics for one animal
after a left hemisection and a group of 7 animals
pre- and post-transection
105
Table 2-17
Summary of the consequences of a transection of
the sacrocaudal spinal cord on the response
characteristics of the electrocutaneous reflex
108
Table 3-1
Anatomical evaluation of transplant survivability
and integration
122
Table 3-2
Summary of the multiple linear regression analysis
of reflex force of animals following transection or
transection plus transplantation
134
Table 3-3
Summary of the multiple linear regression analysis
of peak reflex amplitude of animals following transection
or transection plus transplantation
138
Table 3-4
Summary of the multiple linear regression analysis
of latency at peak reflex amplitude of animals following
transection or transection plus transplantation
142
Table 3-5
Summary of the multiple linear regression
analysis of reflex rise time of animals following
transection or transection plus transplantation
146
Table 3-6
Summary of the multiple linear regression analysis
of half maximal reflex duration of animals following
transection or transection plus transplantation
149
Table 3-7
Summary of the multiple linear regression analysis
of quarter maximal duration of animals following
transection or transection plus transplantation
153
Table 3-8
Summary of the multiple linear regression analysis of
reflex latency of animals following transection
or transection plus transplantation
157
v¡¡

Table 3-9
Summary of the consequences of transection plus
transplantation in comparison to the transection-only
lesion on the response characteristics of the
electrocutaneous reflex
159

LIST OF FIGURES
Figure 2-1 The testing apparati 18
Figure 2-2 Histological reconstructions of the sacrocaudal hemisections . 27
Figure 2-3 Representative traces of tail reflexes to mechanical
stimulation 30
Figure 2-4 Frequency of responses to mechanical stimuli 32
Figure 2-5 Representative traces of tail reflexes to single
DC pulses of electrocutaneous stimulation 35
Figure 2-6 The relationship of reflex force to stimulus intensity 38
Figure 2-7 The relationship of peak reflex amplitude to stimulus
intensity 42
Figure 2-8 The relationship of latency at peak reflex amplitude to
stimulus intensity 45
Figure 2-9 The relationship of reflex rise time to stimulus intensity .... 49
Figure 2-10 The relationship of half maximal reflex duration to
stimulus intensity 53
Figure 2-11 The relationship of quarter maximal reflex duration
to stimulus intensity 57
Figure 2-12 The relationship of reflex latency to stimulus intensity 61
Figure 2-13 The time course of changes in reflex force 67
Figure 2-14 The time course of changes in peak reflex amplitude
and latency at peak reflex amplitude 70
Figure 2-15 The time course of changes in reflex duration 75
Figure 2-16 The time course of changes in reflex latency and rise time. . . 80
Figure 2-17 Frequency of wind-down of the reflex with repetitive
stimulation 85
Figure 2-18 Frequency of wind-up with repetitive stimulation 87
Figure 2-19 Representative responses showing wind-up of the tail reflex. . 90
Figure 2-20 The magnitude of changes in the reflex response
with repetitive stimulation 92
Figure 2-21 Reflex responses of the tail to trains of
electrocutaneous stimulation 94
IX

Figure 2-22 Reflex force in animals with variants of the sacrocaudal
lesion 96
Figure 2-23 Reflex characteristics following dorsal hemisection at S3 ... 99
Figure 2-24 Reflex characteristics following sacrocaudal transection
with an ischemic episode 101
Figure 2-25 Reflex characteristics following caudal spinal transection
at Ca3 103
Figure 3-1 Survival of transplant tissue 124
Figure 3-2 Coronal section through transplant tissue 127
Figure 3-3 Anatomical integration of transplant tissue with
host spinal cord 129
Figure 3-4 Longitudinal section through a transplant 131
Figure 3-5 Relationship of reflex force to stimulus intensity for animals
following transection or transection plus transplantation ... 133
Figure 3-6 Relationship of peak reflex amplitude to stimulus intensity
for animals following transection or transection plus
transplantation 137
Figure 3-7 Relationship of latency at peak reflex amplitude to stimulus
intensity for animals following transection or transection
plus transplantation 141
Figure 3-8 Relationship of reflex rise time to stimulus intensity for
animals following transection or transection plus
transplantation 145
Figure 3-9 Relationship of half maximal reflex duration to stimulus
intensity for animals following transection or transection
plus transplantation 148
Figure 3-10 Relationship of quarter maximal reflex duration
to stimulus intensity for animals following transection or
transection plus transplantation 152
Figure 3-11 Relationship of reflex latency to stimulus intensity for
animals following transection or transection plus
transplantation 156
x

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
EFFECTS OF SACROCAUDAL SPINAL CORD LESIONS AND TRANSPLANTS
OF FETAL TISSUE ON CUTANEOUS REFLEXES OF THE TAIL
By
ROBERT MARK FRIEDMAN
May 1995
Chairperson: Charles J. Vierck, Jr.
Major Department: Neuroscience
Cutaneous reflexes were measured in the tail of the cat to determine whether
cutaneous hyperreflexia is present after sacrocaudal spinal lesions and whether
changes in cutaneous reflexes are similar to those observed in human patients after
spinal cord injuries. Reflexes to electrocutaneous and mechanical stimulation of
the tail were evaluated prior to and after spinal cord lesions.
After a sacrocaudal spinal transection, the overall magnitude of
electrocutaneous tail reflexes was greater than that observed normally. Major
changes in the characteristics of the response included increases in peak reflex
amplitude, longer latencies to reach peak reflex amplitude, and longer reflex
durations. A long latency reflex could be exhibited, and the magnitude of the reflex
could show wind-up in magnitude with repeated stimulation. The changes in
XI

electrocutaneous reflexes developed progressively over a period of 3 months. The
expression of tail reflexes to mechanical cutaneous stimulation was highly variable.
However, there was a decrease in mechanical reflex threshold after sacrocaudal
transection. These changes in cutaneous reflexes did not occur after incomplete
lesions of the sacrocaudal spinal cord.
This animal model was investigated for its potential to evaluate treatments of
spinal cord injury. Consequences for tail reflexes were determined for animals that
received a transplant of fetal spinal tissue placed into an acute sacrocaudal
transection cavity. Eleven animals received fetal tissue. In 8 animals the transplant
successfully survived and integrated with the host spinal cord. In behavioral
studies of animals with surviving transplants, peak amplitude of the
electrocutaneous reflex was less than observed for animals with a chronic
transection. The duration of the electrocutaneous reflex for animals with
transplants was less than observed for transected animals without transplants and
was similar to normal animals. These findings indicate that the presence of
transplant tissue can modulate the cutaneous hyperreflexia that otherwise develops
after spinal cord transection.
XII

CHAPTER 1
INTRODUCTION AND BACKGROUND
Improved treatment strategies for spinal cord injury represent a major goal
for the health care industry. Approximately 10,000 new cases of spinal cord injury
are reported each year in the United States. The causes of spinal cord injuries are
numerous; however, they primarily involve automobile accidents, acts of violence,
falls and sports related injuries. Typically, these individuals are men between the
ages of 19 and 26. On a cost basis, including lost wages, billions of dollars are
spent each year on medical intervention, rehabilitation and chronic health care of
individuals with spinal cord injuries (U.S. Congress, 1990).
Consequences of Human Spinal Cord Injury
Initially after a spinal cord injury, the patient goes through a period of spinal
shock when all motor functions of spinal segments below the lesion are depressed.
After spinal shock recedes the neurological effects of spinal cord injury can be
dichotomized in terms of either negative (i.e., loss of functions) or positive (i.e.,
abnormal behaviors) signs (Young and Shahani, 1986).
Negative signs that are present below the spinal level of injury can include
a complete loss of voluntary movement (e.g., paraplegia or quadriplegia), weakness
1

2
or paresis of a limb, and a loss of bowel and bladder control. In addition, cutaneous
sensibility for fine touch, proprioception, pain and temperature sensations can be
disrupted or lost. Problems associated with sensory loss are significant, since a
patient with a lowered pain response to a noxious stimulus (hypoalgesia) or a
complete absence of pain (analgesia), may unknowingly experience a higher
incidence and severity of accidental injury (e.g., from a bum, cut, or pressure sore).
Positive signs after a lesion to the spinal cord can involve the development
of postural abnormalities, exaggerated flexion and tendon reflexes, muscle spasms,
and painful sensations. Both hyperreflexia and pain can interfere with the spinal
cord injured patient’s ability to function. The different manifestations of
hyperreflexia that can occur after a spinal cord lesion are known collectively as the
spastic syndrome (Young and Shahani, 1986).
Alterations in muscle tone can include a fixed postural abnormality of a limb
(a dystonia or contracture), rigidity, and changes in stretch reflexes. The
exaggeration of segmental stretch reflexes is considered a crucial or defining
feature of spasticity. The alteration of segmental stretch reflexes is thought to (1)
underlie changes in threshold and amplitude of the tendon jerk (myotactic reflex),
(2) trigger a radiation of a local myotactic reflex to the entire limb, (3) trigger rapid
oscillatory movements of a limb (clonus), and (4) include modifications of tonic
stretch reflexes that cause abnormalities in posture (dystonia) of a limb (Dimitrijevic
and Nathan, 1967). Spasticity has been characterized as a velocity sensitive
increase in resistance to passive stretch of a limb with an accompanied increase

3
in tendon reflexes and hypertonia (Landau, 1974). In human patients with cortical
lesions, quantitative behavioral studies have characterized spasticity as a shift in
threshold to elicit stretch reflexes without an increase in magnitude or gain of the
response (Lee et al., 1987). However, similar studies have not been performed to
characterize spasticity in patients with spinal cord injuries.
After spinal cord injury in humans, cutaneous hyperreflexia is evident for
forms of stimulation other than muscular stretch. Cutaneous stimulation normally
elicits flexion reflexes only when the level of stimulation is nociceptive. However,
in a spastic patient, an innocuous cutaneous stimulus on the leg can produce a
brisk, stereotyped, involuntary, flexor movement that can include triple flexion at the
hip, knee and ankle, and crossed extension (Dimitrijevic and Nathan, 1967; Roby-
Brami and Bussel, 1987). The flexion reflex is normally a limited response in one
muscle or limb, but in spastic patients the response spreads to other muscles or
other limbs (Dimitrijevic and Nathan, 1968). In addition, involuntary flexor
movements of a limb can occur in the absence of a known cutaneous stimulus.
Because flexor spasms can increase in frequency in the presence of cutaneous
stimuli, it would appear that central flexor reflex mechanisms are involved (Shahani
and Young, 1973).
Another positive sign after spinal cord injury is the possible occurrence of a
central pain syndrome, despite diminution or loss of cutaneous sensations after
damage to the long ascending spinal pathways. Ninety percent of spinal cord
injured patients report some unpleasant sensory experiences; fifty percent of these

4
patients experience some pain sensations, and twenty-five percent report disabling
pain (Davis and Martin, 1947; Botterell et al., 1953; Kaplan et al., 1962;
Nepomuceno et al., 1979). Pain sensations can involve (1) a phantom limb pain
where the limb is sensed to be in a cramped, twisted position; (2) a visceral pain
where the patient perceives a fullness of the abdomen, fecal urgency and/or
bladder disturbances; (3) a dysesthesia, where the patient feels an unpleasant
sensation that can include diffuse burning or tingling; and (4) a neuralgia, where the
patient feels a hot, sharp, knifelike pain localized to a specific dermatome (Merskey
et al., 1986). With respect to sensations elicited by cutaneous stimulation, pain
perception varies in patients following spinal cord injuries (Lindblom, 1985). A
patient can have a lowered threshold for painful sensations (allodynia), where a
painful sensation is elicited by a stimulus that normally does not provoke pain. In
addition, a patient can report that a noxious stimulus elicits an increased sensation
of pain (hyperalgesia).
Treatments for Spinal Cord Injury
Multiple approaches are employed in treatment of the acute and chronic
effects of spinal cord injuries. In an attempt to control spasticity and pain following
a spinal cord injury, pharmacological and surgical interventions are used with
varying degrees of success (Dimitrijevic and Nathan, 1967; Davidoff, 1985).
Physical therapy and specialized mechanical devices (e.g., wheel chairs, crutches,
prosthetic devices, specialized hand splints) are utilized to improve independence

5
and provide some form of mobility (Trieschmann, 1988). However, there are
currently no available treatments to compensate for the loss of cutaneous sensibility
or development of chronic pain after spinal cord injury.
A new strategy that might ameliorate some effects of spinal cord injury is the
grafting of embryonic neural tissue. An example of the success of embryonic neural
tissue transplantation is found with Parkinson's disease, where grafting of cells from
embryonic brainstem has been reported to have measurable therapeutic benefits
for some patients (Lindvall et al., 1990; Freed et al., 1992). With this approach, the
goal can be to replace lost neurons, connections or neural pathways, to rescue
neurons at risk of cell death, or to modulate neural activity in the host nervous
system. Anatomical evidence suggests that some of these mechanisms may be
associated with the benefits derived from embryonic grafts in patients with
Parkinson's disease.
With respect to the spinal cord, anatomical studies have provided evidence
for the ability of embryonic spinal tissue to integrate and form axonal projections
with the host (Jakeman and Reier, 1991; Reier et al., 1994). Behavioral studies in
cats and rats have shown that fetal spinal transplants can promote recovery in
locomotor behavior (Yakoleff et al, 1989; Kunkel-Bagden and Bregman, 1990;
Anderson et a I., 1991; Goldberger, 1991; Kunkel-Bagden et al., 1991; Stokes and
Reier, 1992). In other experiments in acutely spinalized animals, long-latency
components of flexion reflexes have been shown to be enhanced by previous
transplants of fetal brain stem tissue (Moorman et al., 1990). Similarly, sexual

6
reflexes can be enhanced by transplantation of brain stem tissue (Privat et al.,
1989).
These studies are encouraging in the sense that neural grafts can modulate
or restore certain locomotor behaviors and reflexes that are diminished or lost as
a consequence of spinal cord injury. Improved recovery of locomotion as a result
of fetal cell transplantation could represent, at least in part, an attenuation of
abnormal reflexes, but little or no information is available to evaluate this possibility.
Thus, it is important to determine the effects of fetal transplantation on the
development of spasticity after spinal cord injury.
Behavioral Evaluation of Spinal Cord Injury
Evaluation of new treatment strategies for spinal cord injuries will require
quantitative evaluation in animals of both volitional and reflex behaviors in order to
fully assess the functional utility of these methodologies (Goldberger et al., 1990).
In animals and humans, evaluation of behavioral consequences of spinal cord injury
has relied primarily on subjective rating scales (e.g., Ashworth, 1964; Gale et al.,
1985). This approach has been successful in characterizing consequences of
spinal cord injuries and treatment strategies (Kerasidis et al., 1987). These rating
methods can be effective in specifying qualitative changes, but quantitative
changes in a behavioral function over time remain uncharacterized, irrespective of
issues of test sensitivity and interexaminer reliability.

7
Several different quantitative behavioral assays have been applied to
describe consequences of spinal cord injuries on volitional behaviors. Some of
these behavioral assays include the inclined plane test (Rivlin and Tator, 1977),
foot-falls during grid walking (Bresnehan et al., 1987; Kunkel-Bagden and Bergman,
1990), and quantitative footprint analysis of locomotion (Kunkel-Bagden and
Bergman, 1990). In addition, quantitative behavioral assays have been applied to
forelimb reaching and grasping after spinal cord lesions (Schrimsher and Reier,
1992,1993). Most of these tests evaluate gross behavioral performance by noting
whether an animal achieved the goal of a task. An issue with these behavioral
measures is that an animal with a lesion may utilize an abnormal motor strategy to
achieve the goal. Only a few behavioral paradigms have used electromyographic
and kinematic analyses (Barbeau and Rossignol, 1987; Goldberger, 1988) in
attempts to describe, quantitatively, changes in volitional performance.
Animal Model Systems to Study Spinal Cord Injury
A number of different types of spinal cord lesion have been performed in
order to determine which pathways must be interrupted to produce spasticity and/or
a loss of specific volitional behaviors (Wiesendanger, 1985). However, in the
attempt to understand spasticity in animals, there have been few studies that
quantify the different components of the spastic syndrome in order to relate to the
human condition. In evaluating the consequences of specific spinal cord lesions

8
on the development of spasticity, most animal studies have focused on alterations
of stretch reflexes.
Spinal transection. In cats with chronic spinal transection, behavioral signs
of spasticity are readily and consistently apparent (Fulton and Sherrington, 1932;
McCouch, 1947; Kozak and Westerman, 1966; Afelt, 1970; Bailey et al., 1980), but
rarely have been quantified (Little, 1986). As determined by subjective methods of
neurological evaluations of cats with complete thoracic transection (Bailey et al.,
1980), the limbs are hypotonic for the first few days below the level of the lesion.
The patellar tendon reflex is slightly exaggerated, and the cutaneous flexor reflex
is mildly exaggerated, with the possible presence of cross extension. By the end
of the first week, an increase in tone during passive stretch of a limb is apparent,
and a clasp-knife reflex can be elicited. Tendon reflexes are exaggerated with the
possible presence of clonus. Cutaneous flexor reflexes are exaggerated, and
flexor-extensor spasms might be present. Reflex stepping and standing begin to
appear. By 14 days, muscle tone is only mildly increased, yet all spinal reflexes are
exaggerated. Several early phases of reflex exaggeration have been distinguished
by quantitative methods (Little, 1986), but after 30 days muscle tone is markedly
increased, and spinal reflexes are easily elicited and markedly exaggerated.
A similar time-course has been reported for spinal monkeys. However,
depending on the quality of post surgical care, the lower limbs can become
contracted, rigid, and atrophied (Sahs and Fulton, 1940; Liu et al., 1966). In
paraplegic humans the sequence of hyporeflexia followed by hyperreflexia is similar

9
to that observed in the cat and monkey, except that the time-course of these events
is expanded.
Hemisection. The clinical picture of animals with a hemisection is less clear.
Some studies in rats and cats have reported signs of spasticity (Teasdall et al.,
1958, lesions at T4-L1; Murray and Goldberger, 1974, lesions at T13; Carter et al.,
1991, lesions at L5); whereas, others have not (Hultborn and Malmsten 1983,
lesions at T12 and L2; Carter et al., 1991, lesions at T12). When spasticity is
present after incomplete spinal lesions, the time-course of events is similar to that
of an animal with a transection except that weight bearing occurs in two weeks and
locomotion occurs by three weeks. In addition, hyperreflexive responses can
resolve at later post-injury time periods.
Staggered lateral hemisections. The consequences of staggered lateral
hemisection on reflexes and locomotion also have been investigated (Kato et al.,
1985). Initially there is a depression of segmental reflexes at 1 to 2 days. From 3
to 7 days, segmental reflexes are exaggerated, and unusual reflexes are observed
with the presence of airstepping and alternating limb movements. However, from
7 to 53 days, when the animal begins to stand and walk, abnormal reflexes subside
and segmental reflexes become less exaggerated. In primates, staggered bilateral
hemisection results in a clinical picture similar to that observed after complete
spinal transection (Turner, 1891; Lassek and Anderson, 1961). This difference
among species after staggered lateral hemisections may reflect differences in the

10
organization of the descending systems that control the spinal cord (Holstege and
Kuypers, 1987; Chung and Coggeshall, 1988).
Contusion injury. In animals, a weight drop technique (Allen, 1911) has been
used to model the rapid and traumatic contusive or compressive sort of insult that
causes damage in most patients with spinal cord injury. After a compression or
contusion injury, a hemorrhagic necrosis begins at the center of the insult and is
followed by neural degeneration and the development, over a period of weeks, of
a central cavitation. Modifications of the original technique have improved upon the
reproducibility of the injury (Wrathall et al., 1985; Anderson et al., 1988; Beattie et
al., 1988, Stokes et al., 1992). The behavioral consequences of contusion depend
on the degree of the injury (Gale et al., 1985). With respect to spasticity, both
cutaneous hyperreflexia (Gale et al., 1985) and alterations of physiologically
recorded monosynaptic reflexes (Thompson et al., 1992), develop over a period of
weeks after a contusion injury of the rat spinal cord.
Lesion factors in spasticity. The factors that are critically important to the
development of signs of spasticity in an animal include size, spinal level, and time
after the lesion. Together, these factors determine which pathways are spared, the
extent of deafferentation of the spinal cord, and the excitability of neurons caudal
to the lesion.
Variability in the chronic manifestation of hyperreflexia is apparent in humans
(Mailis and Ashby, 1990). For example, spasticity can be differentiated on the
bases of whether the lesion is spinal or supraspinal (Burke et al., 1972;

11
Wiesendanger, 1985). Following cortical lesions, extensor reflexes are released
over flexors, whereas flexor reflexes become exaggerated after spinal cord lesions.
In addition, not all hemiplegias from cortical or capsular lesions involve a sequential
development from early hypotonia to chronic hypertonia. And, recovery of volitional
control usually results in a decrease in hypertonia in these patients.
Overview of Dissertation
Preliminary to this dissertation research, Rhoton et al. (1988) adapted
traditional methods of human neurological examination to investigate the
consequences of caudal spinal lesions on the tails of cats. Soon after a caudal
spinal transection, the tip of the tail was flaccid. Then, over a period of weeks, the
tail developed heightened cutaneous flexion reflexes and increased tone
(resistance to stretch of tail muscles). Rigidity of the tail appeared, as did clonus¬
like responses to cutaneous stimulation. Subsequently, quantitative measurements
confirmed the impression from neurological testing that tone was greater for tail
muscles of lesioned cats than for normal cats (Ritz et al., 1992).
The dissertation research developed quantitative behavioral methods for
testing cutaneous reflexes. These procedures were evaluated and utilized to study
functional consequences of spinal cord injury and potentially therapeutic
interventions. The key aspect of this research was utilization of sacrocaudal spinal
lesions to evaluate effects of spinal cord lesions on cutaneous reflexes of the tail.
The advantage of a sacrocaudal spinal lesion is that effects of spinal transection

12
can be studied in the absence of bowel and bladder dysfunctions or disruptions in
gait or hindlimb reflexes. The lesions are made caudal to the lumbar enlargement
and are one to two segments caudal to the parasympathetic region (S1 and S2) of
the spinal cord that controls the bowel and bladder (Thor et al., 1989).
Chapter 2 describes studies designed to acquire precise measurements of
cutaneous reflexes in order to determine the time-course and magnitude of any
changes in cutaneous reflexes of the tails of cats after sacrocaudal spinal lesions.
Cutaneous reflexes of the tail were evaluated in normals and animals with spinal
cord lesions. This research determined that lesions of the sacrocaudal cord (S3-
Ca1) provide an appropriate model for investigating effects of transection of the
spinal cord on segmental reflexes of the limbs.
In the studies described in Chapter 3, this animal model was utilized to
evaluate a potential treatment for some of the debilitating effects of spinal cord
injury. Fetal spinal tissue was transplanted into an acute spinal cord transection
injury. The effects of transection and transplantation on cutaneous reflexes were
compared with effects of transection without introduction of a transplant.
In concluding, Chapter 4 discusses possible mechanisms for the cutaneous
hyperreflexia that is typically observed after a spinal cord injury. Also, mechanisms
for influences of fetal spinal transplantation on the consequences of spinal cord
injury are discussed.

CHAPTER 2
CUTANEOUS REFLEXES OF THE TAIL OF CATS BEFORE
AND AFTER SACROCAUDAL SPINAL LESIONS
Introduction
The consequences of a spinal cord transection, following a period of
areflexia (spinal shock), include a gradual development of heightened reflexes
caudal to the site of the lesion (Riddock, 1917; Dimitrijevic and Nathan, 1967). Both
phasic and tonic stretch reflexes are affected and are clinically defined as
hyperreflexic by the appearance of exaggerated tendon reflexes and a heightened
resistance to passive stretch of a muscle, respectively (Landau, 1974; Lance,
1980). Additionally, a limb can exhibit hyperreflexive cutaneous reflexes (Riddoch,
1917; Kuhn and Macht, 1949; Rushworth, 1966; Dimitrijevic and Nathan, 1968;
Shahani and Young, 1971, 1973; Meinck et al., 1985; Roby-Brami and Bussel,
1987). Clinically, cutaneous hyperreflexia is observed when an innocuous stimulus
to the skin of the foot or leg induces an exaggerated triple flexion of the hip, knee
and ankle. The Babinski reflex (dorsiflexion of the great toe with a fanning of the
other toes) is a cutaneous flexion reflex of clinical importance that can be exhibited
after a lesion to the spinal cord (Kugelberg et al., 1960; Roby-Brami et al., 1989).
13

14
Hyperreflexive reflex responses to cutaneous stimulation or muscle stretch are two
components of the spastic syndrome (Young and Shahani, 1986).
In order to study neural mechanisms underlying the development of
spasticity after spinal cord injury, studies in animals have induced hyperreflexia with
complete (Fulton and Sherrington, 1932; Afelt, 1970; Burke et al., 1972; Nelson and
Mendell, 1979; Bailey et al., 1980; Munson et al., 1986; Ritz et al., 1992) and
incomplete lesions (Turner, 1891; Wagley, 1945; Teasdall et al., 1958; Lassek and
Anderson, 1961; Fujimori et al., 1966; Murray and Goldberger, 1974; Kato et al.,
1985; Little, 1986; Carter et al., 1991; Thompson et al., 1992). These studies
primarily have characterized alterations in stretch reflexes. Only a few animal
studies have examined chronic changes in cutaneous (or polysynaptic) reflexes
after spinal cord lesions (Ranson and Hinsey, 1930; McCouch, 1947; Kozak and
Westerman, 1966; Liu et al., 1966; Hultborn and Malmsten, 1983).
Previously, the consequences of a lesion in the cat sacrocaudal spinal cord
which only affects the tail were investigated to develop a minimally disruptive model
of spinal cord injury. Rhoton et al. (1988) observed that a hemisection or complete
transection in the cat sacrocaudal spinal cord produced clinical signs of hypertonia,
cutaneous hyperreflexia, and clonus-like movements. These classical signs of
spasticity occurred in the absence of bowel and bladder dysfunctions or disruptions
in gait and hindlimb reflexes. Subsequent quantitative measurement (Ritz et al.,
1992) confirmed the changes in muscle tone. After a complete transection of the
spinal cord, the tail becomes actively ventroflexed in a midline position, and there

15
is a loss of sensation and volitional control. These functional alterations are akin
to paraplegia with spasticity that occurs in human patients with spinal cord injuries
(Young and Shahani, 1986).
To further investigate alterations in tail reflexes after lesions in the cat
sacrocaudal spinal cord, quantitative measurements of cutaneous reflexes of the
tail to electrocutaneous and mechanical stimuli were performed in normal animals
and in animals with lesions of the sacrocaudal spinal cord. Specifically, this study
addressed whether cutaneous reflexes are hyperactive after spinal transection, as
observed caudal to spinal cord injuries of human patients.
Methods
Subjects. A total of 11 adult cats (10 female, 1 male) served as subjects.
For 8 animals, data were collected before and after lesions of the sacrocaudal
spinal cord. Three animals were part of a prior study (Ritz et al., 1992) and had
previously received either a complete sacrocaudal transection (n=2) or a left
hemisection (n=1). When the animals were not performing in the behavioral
experiments, they were housed at the University of Florida Animal Resources
Facilities.
Surgery. All surgeries were performed under sterile conditions and gas
anesthesia (Flalothane or Isoflourane). Some cats received 50 mg/kg of ketamine
as a preanesthetic. The animal was intubated and a venous catheter was inserted
for infusion of fluids. Dorsal laminectomies were performed at the L6 and L7

16
vertebrae. Cats received a complete transection or a subpial transection cavity (~2-
3 mm long) at the S3/Ca1 level. The lesions were made with precision forceps and
suction. Four to 6 dural sutures secured the spinal cord after the lesion, and when
a subpial transection was performed, 4 pial sutures were added. Visual inspection
at the time of surgery confirmed the completeness of the lesion.
Postoperative care included antibiotic treatments (Amoxicillin, 50 mgs) for
7 days. Canned food was added to the diet. If necessary, the bladders were
manually expressed, but in most cases normal bladder function returned by the
second postoperative day.
At the termination of the behavioral experiments, the cats were anesthetized
with a lethal dose of sodium pentobarbital (100mg/kg) and transcardially perfused
with isotonic saline solution (0.9% with sodium nitroprusside; 0.5g/L) and heparin
sulfate (1.5cc/L), followed by a solution of 4.0% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.6). Upon extraction of the spinal cord, the completeness
of the lesion was determined, and the level of the lesion was identified.
Testing apparatus. The testing apparatus provided comfortable restraint of
the body and tail of each cat (Figure 2-1). Either a cat sack or a restraint harness
was used to situate the body of the cat, and the tail was bracketed at the base. To
provide quantitative measures of cutaneous reflexes, the tip of the tail was tethered
to a copper-beryllium leaf (2.5 cm by 8 cm by 0.5 mm) with an attached strain
gauge. The strain gauge was incorporated into a Wheatstone bridge circuit, and
the signal was amplified 1000 times by a direct current (DC) amplifier. The tether

Figure 2-1. The testing apparati. The cats were positioned with a cat sack (top) or
a restraint harness (bottom). The tail was bracketed at the base. Cutaneous
reflexes of the tail were measured by tethering the tip of the tail to a copper-
beryllium leaf containing a strain gauge.

18
Strain
Gauge

19
tension was held at 1.5 Newtons (N), which held the tail straight in an axial position.
Muscle contractions that moved any portion of the tail away from the axial position
were detectable as an increase in force as measured by the strain gauge. The
measured force assessed the strain produced by a tail reflex. Since the tail was
tethered in parallel, the setup was incapable of measuring the torque of the tail
reflex, which depends on the length of the tail distal to the pivot point (acting as a
moment arm), the angle of the movement, and the reflex force exerted. Attempts
to directly measure torque would encounter an inherent problem in that the pivot
point of tail reflexes were not fixed, but instead depended on stimulus intensity,
since recruited motoneurons course along the length of the tail. The response
forces were collected at a 1 KHz sampling rate and stored digitally in a file, using
computer hardware and software (RC-Electronics).
The timing of all stimulation and event triggering were performed by the
Stoelting Protege System's Stimulus Laboratory Controller with Laboratory
Automation System software.
Training paradigm. Cats were adapted to the testing room and then to the
testing apparatus. For restraint in cat sacks, adaptation to the testing apparatus
included rewards of dry food (BONKERS Cat-Treats or POUNCE Treats for Cats)
as well as physical affection. Training cats restrained with the harness entailed
rewards of liquid food. Subsequently, the proximal tail was braced, and then the
distal tail was tethered. Next, the cats were accustomed to passively receive
mechanical and electrocutaneous stimulation.

20
Testing regimen. When possible, the animals were adapted and tested prior
to receiving a lesion (n=8). Following a sacrocaudal lesion, to evaluate possible
changes in the reflex over time, these animals were tested repeatedly over a period
of at least 7 months and up to a year. Five post-lesion time periods were
specifically examined: 7-14 days, 21-35 days, 70-90 days, 140-165 days, and
greater than 210 days. Due to the methods of restraint and concern for the welfare
of the animal, testing was only rarely attempted during the first week after surgery.
Three other animals were tested only at 3 years post-lesion.
Mechanical stimulation and reflex testing. All mechanical stimulation was
presented on the dorsal surface of the tail at dermatomal level Ca3/Ca4,
approximately 5 cm from the tip of the tail. Mechanical stimulation was presented
with a solenoid and consisted of a 50 ms cutaneous tap elicited through a blunt
metallic probe (1 mm in dia.) or an edge (a Steel T Pin, Create-A-Craft). The probe
was either in continuous contact with the skin or first made contact with the skin
during the tap. In all testing sessions, each stimulus condition was repeated 8
times with an interstimulus interval of 8 s.
Cutaneous response measures and data analysis. Different characteristics
of the response to the mechanical stimuli were tabulated. Individual trials were
evaluated with respect to (1) occurrence of a response, (2) reflex latency, (3) peak
reflex amplitude, and (4) complexity of the response. To tabulate reflex latency,
reflexes shorter than 30 ms were scored in one bin, responses greater than 30 ms
but less than 250 ms were placed in another bin, and reflexes occurring later than

21
250 ms after stimulus onset but less than 750 ms later were scored in a third bin.
Initial responses that occured at latencies greater than 750 ms were considered as
responses unrelated to the mechanical stimulus. The duration of the reflex was
tabulated into 4 bins: (1) less than 500 ms, (2) greater than 500 ms but less than
1 s, (3) greater than 1 s but less than 2 s, and (4) greater than 2 s. The amplitude
of the reflex was also tabulated into 4 bins: (1) less than 0.15 N, (2) greater than
0.15 N but less than 0.25 N, (3) greater than 0.25 N but less than 0.5 N, and (4)
greater than 0.5 N. To characterize the complexity of the reflex response 3 bins
were created. Simple responses, reflexes with less than 3 peak envelopes were
placed in one bin. Responses with greater than 3 peak envelopes but less than
7 peak envelopes were placed in a second bin. And responses with greater than
6 peak envelopes were placed in a third bin.
For each animal 3 to 4 blocks of trials were grouped together and scored to
acquire frequency values for the reflex measures. The population of frequency
values from pre-lesion and post-lesion animals with chronic transection lesions
were statistically compared with the Mann-Whitney Rank Sum Test.
Electrocutaneous stimulation. Innocuous electrocutaneous stimulation
consisted of a 5 ms pulse of constant current (DC) delivered via two wells (5 mm
in dia.) of electrode paste (EC 2 Electrode Cream; Grass Instrument Co.) which
were separated by 1 cm. The wells were punched out of a 1/8" thick polycushion
pad (Smith Nephew Rolyan Inc.) and were secured on the tail with elastic tape
(Elastikon, Johnson and Johnson Medical, Inc.). The stimulation site was the

22
dorsal surface of the tail at dermatomal level Ca3, approximately 8 to 10 cm from
the tip of the tail. Electrocutaneous stimulation was presented by Grass Instrument
Company equipment: a stimulator (Model S8), a stimulus isolation unit (Model SIU
5B), and a constant current unit (Model CCU 1A). The amplitude of the
electrocutaneous stimulation was manually set. A current monitor (built in house)
was utilized to measure the amount of current delivered by each stimulus.
Electrocutaneous reflex testing. Tail reflexes were elicited by a single DC
pulse of electrocutaneous stimulation. To determine intensity-response
relationships, stimuli were presented in blocks of 6 to 8 trials at each of 6 different
stimulus intensities in each testing session. Intensities of 2, 3, 4, 6, 8, and 10.5
mAmps/mm2 (in mAmps, these values were 0.4, 0.6, 0.8, 1.2, 1.6, and 2.1) were
presented in an ascending order of blocks. The intertrial intervals were 7 s.
Occasionally, reflex responses were recorded at various intensities of a 10 pulse,
50 Hz train of electrocutaneous stimulation.
Electrocutaneous response measures and data analysis. The stability of
reflex responses to repeated stimulation was evaluated by comparing the force of
responses on the first trial in a block of trials at one intensity with responses on all
subsequent trials presented at that intensity. For this purpose the force of the reflex
was determined by summing the amplitudes of responses over the first 500 ms after
stimulus onset. For a block of trials to be characterized as showing either wind-up
or wind-down to repeated stimulation, two criteria had to be satisfied. First, the
average change in reflex force had to be greater than 20 percent of the reflex force

23
estimate of the first response. And second, either the average change in reflex
force had to be greater than one standard deviation away from the reflex force
estimate of the first trial, or a consistently increasing or decreasing trend in reflex
force must be present.
For all other further characterizations of the reflex, averaged waveforms were
constructed for each stimulus intensity and period of testing. Individual trials were
not included in the average if there was an unstable baseline due to a volitional
body movement. Starting 10 ms prior to stimulus onset and ending 110 ms later,
data were entered into a waveform data file at the 1 KHz sampling rate. For the
next 400 ms of the response, the file received data at 5 ms intervals. From 400
msec until the end of the data collection window (1.5 s, or 3.5 s), the file received
data at 20 ms intervals.
Different characteristics of the averaged reflex waveforms were evaluated.
Reflex latency was measured by extrapolating back to the X-intercept of a line
made through the 10 and 90 percent peak amplitude points on the ascending limb
of the reflex. Peak reflex amplitude and the latency at peak reflex amplitude were
measured directly. The rise time of the ascending limb of the reflex was estimated
by calculating the slope of a line through the 25 and 75 percent peak amplitude
points on the ascending limb of the reflex. Two measures of reflex duration were
the latency of the reflex amplitude to decay to 50 percent and to 25 percent of the
peak reflex amplitude. An estimate of the overall force of the reflex was determined
by summing the reflex amplitudes from the time of stimulus offset until the decay of

24
the reflex response reached 25 percent of the peak reflex amplitude. The different
characteristics of the averaged waveform were calculated by a computer program
written in the C programming language.
Statistical analysis was performed with SigmaStat computer software (Jandel
Scientific Software). A mixed repeated-measures design was utilized for
comparison of a given characteristic of the tail reflex for normal animals and
animals with chronic sacrocaudal transection. The presence or absence of the
sacrocaudal lesion was treated as the categorical between-subjects factor.
Stimulus intensity was treated as a continuous within-subjects factor. Each
characteristic of the reflex was treated as a continuous dependent variable.
Multiple linear regression analysis was used to evaluate the results (Pedhazur,
1982). To perform the multiple linear regression analysis, the independent and
dependent variables were transformed, when appropriate, to pass tests of normality
(Kolmogorov-Smirnov test) and homoscedasticity (constant variance) and to
linearize the relationship between stimulus intensity and a characteristic of the
reflex. Criterion scaling was used to code for the subjects.
Multiple linear regression analysis was used to evaluate changes in the
reflex over time after sacrocaudal lesions. In the repeated-measures design, the
pre-lesion and 5 post-lesion time periods were treated as a categorical within-
subjects factor. Effect coding was utilized to vector code the categorical variable
for the multiple linear regression analysis. Stimulus intensity was treated as a
continuous within-subjects factor. Each characteristic of the reflex was treated as

25
a continuous dependent variable. Transformations of the independent and
dependent variables were the same as those used for comparisons of pre-lesion
and late post-lesion reflex characteristics.
Results
For 7 of the animals tested, spinal transactions were successfully placed at
sacrocaudal levels between the caudal half of S2 and the caudal half Ca1. These
7 animals comprised a group for analysis of chronic changes in cutaneous reflexes
after sacrocaudal transection. For 5 of these animals, pre-lesion and acute and
chronic post-lesion data were collected to evaluate the time-course of the effects
of the lesions. The reflex responses from 4 additional animals were treated
separately. For one animal, the sacrocaudal transection was located at rostral Ca1,
but evidence of an ischemic episode was present in caudal segments, where cresyl
violet staining revealed an abundance of macrophages and few neurons. In
another animal, the sacrocaudal transection was mistakenly placed at the Ca3
segment. In another animal, an incomplete sacrocaudal lesion at S3 cut through
the dorsal aspect of the spinal cord, disrupting the dorsal columns, Lissauer's tract,
and the dorsal aspect of the dorsolateral white matter, bilaterally (Figure 2-2,
bottom). A near perfect left hemisection at S3 was histologically confirmed for the
fourth animal (Figure 2-2, top).

Figure 2-2. Histological reconstructions of the sacrocaudal hemisections. The top
drawing shows the lesion from the animal with the left hemisection. The bottom
drawing shows the lesion from the animal with the bilateral dorsal hemisection.

2?

28
Responses to Mechanical Stimuli in Normal and Chronic Transection Animals
Examples of tail responses to mechanical stimuli are shown in Figure 2-3.
When tail reflexes could be elicited, various types of responses were expressed
which depended on both the type of mechanical stimulation and the specific animal
tested. Due to the variability observed in the reflexes produced by mechanical
stimuli, different characteristics of the reflex response were tabulated. And,
because of the inability of a single type of mechanical stimulus to consistantly elicit
reflexes in all animals, 4 different types of mechanical stimulation were used.
Reflex threshold to the mechanical stimuli in normal animals (n=7) and
animals with a chronic transection (n=7) was evaluated by determining the reflex
response frequency (Figure 2-4). The trend observed for a decrease in mechanical
reflex threshold for 3 of the 4 mechanical stimuli, as measured by an observed
increase in response frequency, was significant for stimulation with the blunt probe
that remained in contact with the skin (Mann-Whitney Rank Sum test; T = 24.0, P
= 0.008). Over 80 percent of the reflexes elicited were made up of less than 3
envelope peaks, and no differences were observed in the complexity of the reflex
between pre-lesion and chronic post-lesion animals. In post-lesion animals
increases were observed when compared with pre-lesion animals in the frequency
of reflexes with latencies between 30 and 250 ms which paralleled a decline in the
frequency of longer latency responses in post-lesion animals (e.g., edge stimulus,
on skin; P = 0.026). Long latency responses to the blunt probe (off skin) declined
in post-lesion animals (median frequency 0.00) from the frequency observed (0.26)

Figure 2-3. Representative traces of tail reflexes to mechanical stimulation. From
a normal (top) and an animal with a chronic sacrocaudal transection (bottom), the
traces show various reflex responses of the tail to mechanical stimulation. For
clarity, the reflex traces are offset. The bottom trace (top diagram) shows the
stimulus artifact when the mechanical probe displaces a passive tail.

Force (0.65 N/div) Force (0.65 N/div)
30
0.0 ms
800 ms/div
4.0 s

Figure 2-4. Frequency of responses to mechanical stimuli. For 4 different mechanical stimuli, the bar graph shows the
median probability of occurrence of a reflex response for the pre-lesion (grey columns, n=7) and chronic post-lesion periods
(n=7). A decrease in reflex threshold is observed post-lesion, as shown by an increase in probability of responding to the
mechanical stimuli. The lines represent 75 percent and 25 percent confidence intervals.

Blunt Probe On
Blunt Probe Off
Edge Probe On
Mechanical Stimulus

33
in pre-lesion animals (Mann-Whitney Rank Sum test; T = 54.5, P = 0.0086). This
finding confirmed that the responses with latencies longer than 250 ms were most
likely volitional tail movements in response to the mechanical stimuli. And although,
in comparison to pre-lesion animals, there were trends in post-lesion animals for an
increase in reflex duration and peak reflex amplitude in response to the mechanical
stimuli, none were found to be statistically significant (Mann-Whitney Rank Sum
test).
Responses to Electrocutaneous Stimuli in Normal and Chronic Transection Animals
Representative traces of tail reflexes to single electrocutaneous stimuli are
shown in Figure 2-5. Reflexes occurred at short latencies with rapid rise times that
dissipated with a gradual decay. For normal animals and animals with a chronic
sacrocaudal transection, the tail responded similarly, allowing direct statistical
comparisons between pre- and post-lesion measurements of different features of
the reflex. For the following comparisons, averages from 4 pre-lesion sessions
were compared with averages from 2 or 3 sessions at the end of the testing period
for each animal.
Stimulation at 2 mA/mm2 was near threshold for elicitation of the reflex, and
responses were variably absent. Therefore, this intensity was not utilized for the
analyses of different characteristics of the reflex across stimulus intensities.
Comparing response probabilities for stimulation at 2 mA/mm2 during the pre-lesion
and chronic post-lesion periods, significant differences in reflex threshold were not

Figure 2-5. Representative traces of tail reflexes to single DC pulses of
electrocutaneous stimulation. From a pre-lesion recording (top) and after a chronic
sacrocaudal transection (bottom), the top 3 traces show reflex responses of the tail
to electrocutaneous stimulation at different stimulus intensities. For clarity, the
reflex traces are offset. The bottom trace shows the timing of the electrocutaneous
stimulus.

Force (0.65 N/div) Force (0.65 N/div)
35
.0 ms
800 ms/div
4.0 s
0.0 ms
800 ms/div
4.0 s

36
indicated (Mann-Whitney Rank Sum Test (T=60.0, P=0.694). For pre-lesion testing
at 2 mA/mm2, the median response probability was 0.6 (n=8), and the post-lesion
response probability was 0.75 (n=7).
In an overall evaluation of the electrocutaneous reflex, the force of the reflex
was observed to be larger for animals with a chronic transection than for normal
animals. The force of the reflex increased for both groups with higher levels of
stimulus intensity (Figure 2-6). Multiple regression analysis (Table 2-1) was
performed after the inverse exponential relationship was linearized by applying an
inverse transformation to the independent variable and a natural log transformation
to the dependent variable. The multiple linear regression analysis, which contained
stimulus intensity as a repeated continuous variable and the presence or absence
of the sacrocaudal lesion as the between-subject variable accounted for over 90
percent of the variance and was significant (r2 = 0.906; F = 30.59, P < 0.01). The
possible interaction between stimulus intensity and the presence of a lesion was not
significant (F= 3.04, P > 0.05). The main findings of the analysis were that: (1)
reflex force significantly increased with increases in stimulus intensity (F = 70.79,
P < 0.01); and (2) over this stimulus intensity range, there was a post-lesion
increase in reflex force (F = 7.34, P < 0.05).
An increase in peak reflex amplitude in animals with a chronic transection in
comparison to normal animals was found to be contributing factor for the
differences observed in reflex force between the two groups. Peak reflex amplitude
was observed to increase for both groups with higher levels of stimulus intensity,

Figure 2-6. The relationship of reflex force to stimulus intensity. Total reflex force at 5 different stimulus intensities is shown
pre-lesion (n=8; left) and after a chronic (>5 months) transection (n=7; right). Total reflex force was determined by summing
the reflex amplitudes from the time of stimulus offset until the decay of the reflex response reached 25 percent of peak
amplitude. The wider smooth lines (inverse exponential functions) represent the average response for each testing period.
Multiple regression analysis found that: (1) for both periods, reflex force significantly increased with increases in stimulus
intensity (P < 0.01); (2) over the stimulus intensity range, there was an overall post-lesion increase in reflex force (P < 0.05);
and (3) pre- and post-lesion differences in the increase in reflex force with increases in stimulus intensity were not significant
(P > 0.05).

Reflex Force (N)
Intensity (mA/mm2)
Reflex Force (N)
Intensity (mA/mm2)
CO
00

39
Table 2-1. Summary of the multiple linear regression analysis of reflex force of
normal animals and after a chronic sacrocaudal transection.
Reflex Force
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
1.33
0.234
Treatment
1
39.4
7.34
<0.05
0.279
Subject
13
69.8
20.44
<0.01
0.773
0.94
0.041
Intensity
1
18.6
70.79
<0.01
0.904
-6.05
0.638
Treatment X
Intensity
1
0.8
3.04
>0.05
0.906
Residual
51
13.4
Total
67
127.8
30.59
<0.01
0.906
Transformations: Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Failed; Kurtosis=13.3, Skewness=2.34.
Homoscedasticity Test: Passed.

40
in what appeared to be a saturating nonlinear function (Figure 2-7). Multiple
regression analysis (Table 2-2) was performed after the relationship was linearized
by taking the reciprocal of the independent variable. The multiple linear regression
analysis, which contained stimulus intensity as a repeated continuous variable, the
presence or absence of the sacrocaudal lesion as the between-subject treatment
variable, and an interaction term between stimulus intensity and the possession of
a sacrocaudal lesion, accounted for over 90 percent of the variance and was
significant (r2 = 0.924; F = 39.06, P < 0.01). The main findings of the analysis were
that: (1) peak reflex amplitude significantly increased with increases in stimulus
intensity (F = 82.7, P < 0.01); (2) over this stimulus intensity range, there was a
post-lesion increase in the peak reflex amplitude (F = 9.13, P < 0.01); and (3) for
animals with a lesion, peak amplitude increased at a greater rate with increases in
stimulus intensity (F = 13.13, p < 0.01). The point of intersection of the ordinal
interaction was estimated be at 1.8 mA/mm2. Between normal animals and animals
with a chronic transection, peak reflex amplitudes were significantly different at
intensities greater than 2.4 mA/mm2 (Johnson-Neyman Technique).
Along with the differences observed between the two groups in peak reflex
amplitude, the latency at peak reflex amplitude was observed to remain relatively
constant in normal animals, but for animals with a lesion, latency at peak reflex
amplitude increased with higher levels of stimulus intensity (Figure 2-8). Multiple
regression analysis (Table 2-3) was performed after the relationship was linearized
by applying an inverse transformation to the independent variable and log

Figure 2-7. The relationship of peak reflex amplitude to stimulus intensity. Peak reflex amplitudes at 5 different stimulus
intensities are shown pre-lesion (left) and after a chronic transection (right). The larger smooth lines represent the average
response at each testing period. Multiple regression analysis found that: (1) for both periods, peak amplitude significantly
increased with increases in stimulus intensity (P < 0.01); (2) over the range of stimulus intensities, there was a post-lesion
increase in peak amplitude (P < 0.01); and (3) post-lesion, peak amplitude increased at a greater rate with increases in
stimulus intensity (P < 0.01).

Intensity (mA/mm2) Intensity (mA/mm2)
Peak Amplitude (N)
poo ooooooo
o !-»• k> to A. cn b> co co
Peak Amplitude (N)
oooooooooo
o -»■ Ko co A- cn bi -o bo co
ZV

43
Table 2-2. Summary of the multiple linear regression analysis of peak reflex
amplitude of normal animals and after a chronic sacrocaudal transection.
Peak Amplitude
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.186
0.025
Treatment
1
1.230
9.13
<0.01
0.323
Subject
13
1.751
23.90
<0.01
0.781
1.015
0.039
Intensity
1
0.466
82.70
<0.01
0.904
-0.965
0.094
Treatment X
Intensity
1
0.074
13.13
<0.01
0.924
0.382
0.094
Residual
52
0.293
Total
68
3.521
39.06
<0.01
0.924
Transformations: Reciprocal of Intensity.
Normality Test: Failed; Kurtosis=0.105, Skewness=
Homoscedasticity Test: Passed.
0.836.

Figure 2-8. The relationship of latency at peak reflex amplitude to stimulus intensity. Latency at peak reflex amplitude at
5 different stimulus intensities are shown pre-lesion (left) and after a chronic transection (right). The larger smooth lines
represent the average response of each group. Multiple regression analysis found that: (1) latency at peak reflex amplitude
significantly increased with increases in stimulus intensity post-lesion (P < 0.01); (2) over the stimulus intensity range, there
was a post-lesion increase in latency at peak reflex amplitude (P < 0.01); and (3) increases in latency at peak reflex amplitude
with increases in stimulus intensity were significantly different in the pre- and post-lesion periods (P< 0.01).

Intensity (mA/mm2) Intensity (mA/mm2)
Latency at Peak Amplitude (msec)
Latency at Peak Amplitude (msec)
IO
O
-fc.
O
O)
O
00
o
o
o
to
o
â– N
O
O)
O
IO
00 o
o o
200

46
Table 2-3. Summary of the multiple linear regression analysis of latency at peak
reflex amplitude of normal animals and after a chronic sacrocaudal transection.
Latency at peak reflex amplitude
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.609
0.312
Treatment
1
3.39
13.99
<0.01
0.323
Subject
13
3.15
5.30
<0.01
0.781
0.898
0.068
Intensity
1
0.39
8.54
<0.01
0.904
-0.963
0.273
Treatment X
Intensity
1
0.77
16.85
<0.01
0.924
1.258
0.272
Residual
51
2.33
Total
67
10.30
10.53
<0.01
0.924
Transformations: Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Passed.

47
transformation to the dependent variable. The multiple linear regression model,
which contained stimulus intensity as a repeated continuous variable, the presence
or absence of the sacrocaudal lesion as the between-subject treatment variable,
and an interaction term between stimulus intensity and the presence of a
sacrocaudal lesion, accounted for over 90 percent of the variance and was
significant (r2 = 0.92; F = 10.53, P < 0.01). The main findings of the analysis were
that: (1) latency at peak reflex amplitude significantly increased with increases in
stimulus intensity for animals with a lesion (F = 8.54, P < 0.01); (2) over this
intensity range, there was a post-lesion increase in latency at peak reflex amplitude,
compared to pre-lesion values (F = 13.99, P < 0.01); and (3) for animals with a
lesion, latency at peak reflex amplitude increased at a greater rate with increases
in stimulus intensity (F = 16.85, P < 0.01). The point of intersection of the ordinal
interaction was estimated to be at 2.65 mA/mm2. Between normal animals and
animals with a chronic transection, the latency at peak reflex amplitude values were
significantly different at intensities greater than 3.1 mA/mm2 (Johnson-Neyman
Technique).
The rise time of the reflex was measured in order to investigate a possible
contribution to observed increases in peak amplitude at higher stimulus intensities
and in animals with a sacrocaudal lesion. Reflex rise time was observed to
increase for both groups with higher levels of stimulus intensity in what appeared
to be a saturating nonlinear function (Figure 2-9). Multiple regression analysis
(Table 2-4) was performed after the relationship was linearized by taking the

Figure 2-9. The relationship of reflex rise time to stimulus intensity. Reflex rise times at 5 different stimulus intensities are
shown for pre-lesion (left) and after a chronic transection (right). Reflex rise time was measured by calculating the slope
between the 25 and 75 percent peak amplitudes on the ascending limb of the reflex. The larger smooth lines represent the
average response of each group. Multiple regression analysis found that: (1) for both testing periods, reflex rise time
significantly increased with increases in stimulus intensity (P < 0.01); (2) over the intensity range, there was not an overall
difference in the reflex rise time between the pre- and post-lesion periods (P > 0.05); but (3) reflex rise time increased with
increases in stimulus intensity at a greater rate pre-lesion than post-lesion (P < 0.05).

Intensity (mA/mm2) Intensity (mA/mm2)
Rise Time (dynes/sec)
o o p o p nj ro ro ro
ok>ÍNb>book>itwb)óook}^b>
Rise Time (dynes/sec)
OOOOO—L—*■—'•pppp
oroj!k.b)bobrsjj^b)bobkjik.b)

50
Table 2-4. Summary of the multiple linear regression analysis of reflex rise time
of normal animals and after a chronic sacrocaudal transection.
Rise Time
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.082
0.012
Treatment
1
0.1410
2.52
>0.05
0.140
Subject
13
0.7280
55.57
<0.01
0.861
0.991
0.030
Intensity
1
0.0833
82.66
<0.01
0.943
-0.402
0.040
Treatment X
Intensity
1
0.0048
4.76
<0.05
0.949
0.096
0.040
Residual
52
0.0524
Total
68
1.0095
59.36
<0.01
0.949
Transformations: Reciprocal of Intensity, log of the dependent variable plus one.
Normality Test: Passed.
Homoscedasticity Test: Passed.

51
inverse of the independent variable and applying a log transformation to the
dependent variable. The multiple linear regression analysis, which contained
stimulus intensity as a repeated continuous variable, and an interaction term
between stimulus intensity and the possession of a sacrocaudal lesion, accounted
for over 90 percent of the variance and was significant (r2 = 0.95; F = 59.36, P <
0.01). The main findings of the model were that: (1) reflex rise time significantly
increases with increases in stimulus intensity (F = 82.7, P < 0.01); and (2) for the
between-subject variable, significant differences were not obtained for the presence
or absence of a lesion (F= 2.52, P > 0.05); and (3) for normal animals reflex rise
time increased at a greater rate with increases in stimulus intensity than in animals
with a lesion (F = 4.76, P < 0.05). The point of intersection of the ordinal interaction
was estimated be at -3.4 mA/mm2. Between normal animals and animals with a
chronic transection, the rise time values were significantly different at intensities
greater than -0.4 mA/mm2 (Johnson-Neyman Technique).
Two different measures of reflex duration were obtained in order to
determine the contribution of reflex duration to the reflex force estimate. It took
longer for animals with a lesion to reach half-maximal reflex duration when
compared to normal animals. Half maximal reflex duration was observed to
increase for both groups with higher levels of stimulus intensity (Figure 2-10). The
nonlinear relationships between stimulus intensity and half maximal reflex duration
were fit with hyperbola functions. Multiple regression analysis (Table 2-5) was
performed after the hyperbola relationship was linearized by applying an inverse

Figure 2-10. The relationship of half maximal reflex duration to stimulus intensity. Half maximal durations at 5 different
stimulus intensities are shown pre-lesion (left) and for animals with a chronic transection (right). The larger smooth curved
lines (hyperbola functions) represent the average response for each testing period. Multiple regression analysis found that:
(1) for both groups, half maximal reflex duration significantly increased with increases in stimulus intensity (P < 0.05); (2) over
this stimulus intensity range, there was a post-lesion increase in half maximal reflex duration (P < 0.05); and (3) increases
in half maximal reflex duration with increases in stimulus intensity were not significantly different pre- and post-lesion (P >
0.05).

Duration 0.5 max (msec)
Intensity (mA/mm2)
Duration 0.5 max (msec)
Intensity (mA/mm2)
CD
CO

54
Table 2-5. Summary of the multiple linear regression analysis of half maximal
reflex duration of normal animals and after a chronic sacrocaudal transection.
Half Maximal Duration
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
-1.04E-3
3.41 E-4
Treatment
1
1.01E-4
8.21
<0.05
0.298
Subject
13
1.60E-5
12.94
<0.05
0.769
0.933
5.01E-2
Intensity
1
2.97E-5
31.23
<0.05
0.857
7.65E-3
1.21 E-3
Treatment
X Intensity
1
1.40E-6
1.47
>0.05
0.861
Residual
51
4.85E-5
Total
67
2.92E-4
19.20
<0.01
0.861
Transformations: Reciprocal of Intensity minus 1; reciprocal of the dependent variable.
Normality Test: Failed; Kurtosis=7.45, Skewness=2.16.
Homoscedasticity Test: Passed.

55
transformation to both the independent variable and the dependent variable. The
multiple linear regression analysis, which contained stimulus intensity as a repeated
continuous variable and the presence or absence of the sacrocaudal lesion as the
between-subject variable, accounted for over 80 percent of the variance and was
significant (r2 = 0.86; F = 19.2, P < 0.01). The interaction between stimulus
intensity and the presence of a lesion was not significant (F = 1.47, P > 0.05). The
main findings of the analysis were that: (1) half maximal reflex duration significantly
increased with increases in stimulus intensity (F = 31.23, P < 0.05); and (2) over
this stimulus intensity range, there was a post-lesion increase in half maximal reflex
duration (F = 8.21, P < 0.05).
One quarter maximal reflex duration values were observed to be greater in
animals with lesions in comparison to normal animals (Figure 2-11). Multiple
regression analysis (Table 2-6) was performed after the relationship was linearized
by taking the reciprocal of the independent variable and performing a natural log
transformation of the dependent variable. The multiple linear regression analysis,
which contained stimulus intensity as a repeated continuous variable, the presence
or absence of the sacrocaudal lesion as the between-subject treatment variable,
and an interaction term between stimulus intensity and the possession of a
sacrocaudal lesion accounted for 90 percent of the variance and was significant (r2
= 0.899; F = 27.92, P < 0.01). The main findings of the analysis were that: (1)
quarter maximal reflex duration significantly increased with increases in stimulus
intensity (F = 3.54, P < 0.01); (2) over this stimulus intensity range, there was a

Figure 2-11. The relationship of quarter maximal reflex duration to stimulus intensity. Quarter maximal reflex durations at
5 different stimulus intensities are shown pre-lesion (left) and after a chronic transection (right). The larger smooth lines
(inverse exponential functions) represent the average response for each testing period. Multiple regression analysis found
that: (1) for both periods, quarter maximal reflex duration significantly increased with increases in stimulus intensity (P < 0.01);
(2) over this stimulus intensity range, there was a post-lesion increase in the quarter maximal reflex duration (P < 0.05); and
(3) post-lesion, quarter maximal duration increased with increases in stimulus intensity at a greater rate than pre-lesion (P<
0.01).

Duration 0.25 max (msec)
Duration 0.25 max (msec)
Intensity (mA/mm2)

58
Table 2-6. Summary of the multiple linear regression analysis of quarter
maximal reflex duration of normal animals and after a chronic sacrocaudal
transection.
Quarter Maximal Duration
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.595
0.280
Treatment
1
11.60
5.55
<0.05
0.242
Subject
13
27.19
21.70
<0.01
0.810
0.965
0.043
Intensity
1
3.54
36.72
<0.01
0.884
-1.601
0.234
Treatment X
Intensity
1
0.07
7.68
<0.01
0.899
0.723
0.233
Residual
50
4.82
Total
66
43.07
27.92
<0.01
0.899
Transformations: Reciprocal of Intensity minus one; natural log of the dependent
variable.
Normality Test: Passed.
Homoscedasticity Test: Passed.

59
post-lesion increase in quarter maximal reflex duration (F = 5.55, P < 0.05); and (3)
for animals with a lesion, quarter maximal reflex duration increased at a greater rate
with increases in stimulus intensity (F = 7.68, P < 0.01). The point of intersection
of the ordinal interaction was estimated be at 2 mA/mm2. Comparing normal
animals and animals with a chronic transection, the quarter maximal durations were
significantly different at intensities greater than 2.3 mA/mm2 (Johnson-Neyman
Technique).
Reflex latency was observed to be shorter in normal animals in comparison
to animals with a transection of the sacrocaudal spinal cord. The latency of reflex
onset was observed to decrease for both groups with higher levels of stimulus
intensity in what appeared to be a saturating nonlinear function (Figure 2-12). The
nonlinear relationships between stimulus intensity and reflex latency were fit with
inverse exponential functions. Multiple regression analysis (Table 2-7) was
performed after the inverse exponential relationship was linearized by applying a
natural log transformation to the dependent variable. The multiple linear regression
analysis, which contained stimulus intensity as a repeated continuous variable and
the presence or absence of the sacrocaudal lesion as the between-subject variable
accounted for over 80 percent of the variance and was significant (r2 = 0.834; F =
16.24, P < 0.01). The possible interaction between stimulus intensity and the
presence of a lesion was not significant (F= 0.53, P > 0.05). The main findings of
the analysis were that: (1) reflex latency significantly decreased with increases in

Figure 2-12. The relationship of reflex latency to stimulus intensity. Reflex latency estimates at 5 different stimulus intensities
are shown pre-lesion (left) and after a chronic transection (right). Reflex latency was determined by extrapolating back to
the x intercept of a line through the 10 and 90 percent peak amplitude time points. The larger smooth lines (inverse
exponential functions) represent the average response for each testing period. Multiple regression analysis found that: (1)
for both periods, reflex latency significantly decreased with increases in stimulus intensity (P < 0.01); and (2) over the
intensity range, there was a post-lesion increase in reflex latency (P < 0.05).

Latency (msec)
2345678 9 10 11
Intensity (mA/mm2)

62
Table 2-7. Summary of the multiple linear regression analysis of reflex latency
of normal animals and after a chronic sacrocaudal transection.
Reflex Latency
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
4.403
1.158
Treatment
1
202.5
12.66
<0.01
0.343
Subject
13
208.0
8.46
<0.05
0.697
0.985
0.060
Intensity
1
80.3
42.43
<0.01
0.834
-5.664
0.772
Treatment X
Intensity
1
1.0
0.53
>0.05
0.834
0.382
0.094
Residual
51
98.4
Total
67
589.2
16.24
<0.01
0.834
Transformations: Log of Intensity.
Normality Test: Passed.
Homoscedasticity Test: Passed.

63
stimulus intensity (F = 42.43, P < 0.01); and (2) over this stimulus intensity range,
there was a increase in post-lesion reflex latency (F = 12.66, P < 0.01).
Flow the different measured characteristics of the reflex waveform were
related was examined with Spearman rank order correlation coefficients as shown
in Table 2-8. In normal prelesion animals, measures of force, half maximal
duration, quarter maximal duration and peak amplitude were significantly
intercorrelated. Reflex rise time was significantly correlated with reflex force, peak
amplitude and latency at peak reflex amplitude, but not with the measures of
duration. Latency at peak reflex amplitude was significantly correlated with the
measures of duration, but not with peak amplitude or reflex force. Reflex latency
was not significantly correlated with any of the other measured characteristics. In
contrast, all of the reflex characteristics were significantly intercorrelated in chronic
post-lesion animals. The correlations among the reflex characteristics were
significantly greater in the chronic post-lesion animals (Mann-Whitney Rank Sum
Test; T = 306.0, P =< 0.0001).
The Time-course of Changes in Reflex Characteristics
The time-course of changes in the electrocutaneous tail reflex after a
sacrocaudal spinal transection was evaluated for 5 animals that were tested
preoperatively and at 5 different post-lesion time periods. Post-lesion, alterations
in the electrocutaneous reflex progressively developed over a period of months.

64
Table 2-8. Spearman rank order correlation coefficients between
measured characteristics of the reflex waveform.
Latency at
Peak
Amplitude
Duration
0.25 max
Duration
0.5 max
Rise
Time
Reflex
Latency
Reflex
Force
Normal
Peak
Amplitude
0.013*
0.937
0.451
0.008
0.297
0.082
0.840
<0.001
-0.255
0.133
0.840
<0.001
Latency at Peak
Amplitude
0.650
<0.001
0.610
<0.001
-0.448
0.006
-0.004
0.980
0.208
0.082
Duration
0.25 max
0.710
<0.001
0.038
0.829
-0.311
0.073
0.740
<0.001
Duration
0.5 max
-0.112
0.520
-0.211
0.223
0.620
<0.001
Rise
Time
-0.238
0.162
0.518
0.002
Reflex
Latency
-0.326
0.056
Postlesion
Peak Amplitude
0.750
<0.001
0.870
<0.001
0.810
<0.001
0.740
<0.001
-0.470
0.006
0.940
<0.001
Latency at Peak
Amplitude
0.730
<0.001
0.750
<0.001
0.540
0.002
-0.720
<0.001
0.730
<0.001
Duration
0.25 max
0.950
<0.001
0.640
<0.001
-0.650
<0.001
0.970
<0.001
Duration
0.5 max
0.570
<0.001
-0.630
<0.001
0.930
<0.001
Rise
Time
-0.394
0.023
0.660
<0.001
Reflex
Latency
-0.542
0.001
‘For each cell, the top value is the correlation coefficient while
the bottom number is the corresponding P value.

65
The force estimate of the reflex was observed to increase over time after the
lesion for individual animals (Figure 2-13). For the group of subjects, a multiple
linear regression analysis (Table 2-9) accounted for over 70 percent of the variance
and was significant (r2 = 0.747; F = 30.8, P < 0.0001). Interactions between
stimulus intensity and reflex force at different times after the lesion were not
significant (P > 0.05). The main findings of the multiple regression analysis were:
(1) reflex force significantly increased with increases in stimulus intensity (t = -9.18,
P < 0.0001), and (2) the grouping of reflex force values into different post-lesion
time periods accounted for a significant proportion of the variance (Table 2-9). A
post hoc Scheffe analysis found that pre-lesion reflex force values were not
significantly different from post-lesion values at 7-14 and 21-35 days (P > 0.05), but
the reflex force at those 3 time periods was significantly less than at 70-90 days,
140-165 days, and >210 days (P < 0.05).
Peak reflex amplitude was observed to increase over time after the lesion for
individual animals (Figure 2-14). For the group of subjects, the multiple linear
regression analysis (Table 2-10) accounted for over 70 percent of the variance and
was significant (r2 = 0.743; F = 30.6, P < 0.0001). Interactions between stimulus
intensity and peak amplitude at different times after the lesion were generally not
significant (P > 0.05; except for the period 70-90 days, P = 0.0217). The main
findings of the multiple regression analysis were: (1) peak reflex amplitude
significantly increased with increases in stimulus intensity (t = -10.142, P < 0.0001),
and (2) the grouping of peak reflex amplitudes into different post-lesion time periods

Figure 2-13. The time course of changes in reflex force. Reflex force, averaged
across intensities for each of 5 animals (symbols), is shown at 6 different time
periods. The columns represent the average reflex force determined across
animals. A post hoc Scheffe analysis found that pre-lesion values were not
significantly different from the 7-14 and 21-35 day values (P > 0.05), but reflex force
at those 3 time periods was significantly less than the mean force at 70-90 days,
140-165 days, and >210 days (P < 0.05).

Reflex Force (N)
67
Time period (days)

68
Table 2-9. Summary of the multiple linear regression analysis of changes in
reflex force over time after sacrocaudal transection.
Reflex Force
Source
DF
SS F
P
R2
Regression
15
167.6
Residual
122
56.7
Total
137
224.3 30.8
<0.0001
0.747
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
7.236
<0.0001
2.120
0.2930
Subject
12.825
<0.0001
0.703
0.0548
Intensity
-9.181
<0.0001
-6.069
0.6610
7-14 days
-3.857
0.0002
-0.487
0.1263
21-35 days
-2.444
0.0159
-0.314
0.1285
70-90 days
3.648
0.0004
0.498
0.1366
140-165 days
4.233
<0.0001
0.535
0.1263
210 days plus
4.811
<0.0001
0.608
0.1263
Intensity X
7-14 days
3.374
0.7088
0.546
1.4586
Intensity X
21-35 days
1.126
0.2622
1.647
1.4620
Intensity X
70-90 days
-1.727
0.0867
-2.695
1.5610
Intensity X
140-165 days
-1.211
0.2281
-1.747
1.4421
Intensity X
>210 days
-0.319
0.7501
-0.460
1.4421
Transformations:
Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Failed.

Figure 2-14. The time course of changes in peak reflex amplitude and latency at
peak reflex amplitude. Peak amplitudes (top) and latency at peak amplitude
(bottom) for each of 5 animals (symbols) are shown at 6 different time periods,
averaged across intensities. The columns represent the average peak amplitude
and latency at peak reflex amplitude determined across animals. A post hoc
Scheffe analysis found that pre-lesion peak amplitudes were significantly less than
post-lesion amplitudes at 21-35, 70-90, 140-165 and >210 days (P < 0.05). Post
hoc Scheffe analysis found that pre-lesion latencies at peak amplitude were
significantly less than post-lesion latencies at 70-90, 140-165 and >210 days (P <
0.05).

Latency at Peak Amplitude (msec) Peak Amplitude (N)
70
Time Period (days)
Time Period (days)

71
Table 2-10. Summary of the multiple linear regression analysis of changes in
peak reflex amplitude over time after sacrocaudal transection.
Peak Amplitude
Source
DF
SS F
P
R2
Regression
15
3.75
Residual
124
1.29
Total
139
5.04 30.6
<0.0001
0.743
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
6.220
<0.0001
0.2030
0.0326
Subject
13.413
<0.0001
0.9837
0.0733
Intensity
-10.142
<0.0001
-0.9987
0.0985
7-14 days
-1.946
0.0538
-0.0368
0.0189
21-35 days
0.093
0.9258
0.0018
0.0189
70-90 days
1.599
0.1123
0.0328
0.0205
140-165 days
2.962
0.0037
0.0560
0.0189
210 days plus
4.133
<0.0001
0.0781
0.0189
Intensity X
7-14 days
0.982
0.3281
0.2144
0.2184
Intensity X
21-35 days
-0.416
0.6780
-0.0909
0.2184
Intensity X
70-90 days
-2.323
0.0217
-0.5430
0.2337
Intensity X
140-165 days
0.012
0.9897
0.0028
0.2159
Intensity X
>210 days
-0.253
0.8004
-0.0547
0.2159
Transformations
: Reciprocal of Intensity.
Normality Test:
Passed.
Homoscedasticity Test: Failed.

72
accounted for a significant amount of the variance (Table 2-10). Scheffe analysis
found that pre-lesion values were significantly less than post-lesion peak amplitude
levels at 21-35 days, 70-90 days, 140-165 days, and >210 days (P < 0.05).
Latency at peak reflex amplitude was observed to increase over time after
the lesion for the individual animals (Figure 2-14). The multiple linear regression
analysis (Table 2-11) for latency at peak reflex amplitude accounted for over 50
percent of the variance and was significant (r2 = 0.537; F = 12.1, P < 0.0001).
Interactions between stimulus intensity and time after the lesion were generally not
significant (P > 0.05; except for the period of >210 days, P = 0.0067). The main
findings of the multiple linear regression analysis were: (1) latency at peak reflex
amplitude significantly increased with increases in stimulus intensity (t = -3.736, P
= 0.0003), and (2) the grouping of latency at peak reflex amplitude values into post¬
lesion time periods accounted for a significant amount of the variance (Table 2-11).
Post hoc analysis found that pre-lesion values were significantly less than the mean
peak reflex latencies at 70-90 days, 140-165 days, and >210 days (Scheffe, P <
0.05).
Changes over time for several measures of reflex duration are shown in
Figure 2-15. Multiple linear regression analyses were performed on the measures
of half maximal reflex duration (Table 2-12, R2 = 0.539, F = 12.3, P < 0.0001) and
quarter maximal reflex duration (Table 2-13, R2 = 0.599, F = 15.7, P < 0.0001). For
half maximal reflex duration, interactions between stimulus intensity and duration
for different times after the lesion were generally not significant (P > 0.05; except

73
Table 2-11. Summary of the multiple linear regression analysis of changes in
latency at peak reflex amplitude over time after sacrocaudal transection.
Latency at peak reflex amplitude
Source
DF
SS F
P
R2
Regression
15
6.17
Residual
122
5.31
Total
137
11.48 12.1
<0.0001
0.537
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
1.408
0.1615
0.9063
0.6435
Subject
5.668
<0.0001
0.8200
0.1447
Intensity
-3.736
0.0003
-0.7634
0.2043
7-14 days
-1.813
0.0723
-0.0701
0.0386
21-35 days
0.269
0.7887
0.0104
0.0386
70-90 days
1.913
0.0580
0.0795
0.0416
140-165 days
3.005
0.0032
0.1161
0.0386
210 days plus
4.299
<0.0001
0.1689
0.0393
Intensity X
7-14 days
1.666
0.0981
0.7452
0.4472
Intensity X
21-35 days
0.867
0.3875
0.3879
0.4472
Intensity X
70-90 days
-1.304
0.1947
-0.6240
0.4786
Intensity X
140-165 days
-1.380
0.1700
-0.6103
0.4422
Intensity X
>210 days
-2.757
0.0067
-1.2758
0.4628
Transformations: Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Failed.

Figure 2-15. The time course of changes in reflex duration. Half maximal (top) and
quarter maximal (bottom) durations for each of 5 animals (symbols) are shown at
6 time periods, averaged across intensities. The columns represent the average
half maximal and quarter maximal durations determined across animals. A post hoc
Scheffe analysis found that pre-lesion half maximal durations were significantly less
than the values at 70-90, 140-165 and >210 days (P < 0.05). Post hoc Scheffe
analysis found that pre-lesion quarter maximal durations were not significantly
different from the 7-14 and 21-35 day values (P > 0.05), but quarter maximal
durations at these 3 time periods were significantly less than at 70-90 days, 140-
165 days, and >210 days (P < 0.05).

Duration 0.25 max (msec) Duration 0.5 max (msec)
75
Time Period (days)
Time Period (days)

76
Table 2-12. Summary of the multiple linear regression analysis of changes over
time in the duration of decay to 0.5 maximal amplitude after sacrocaudal
transection.
Duration at Half Maximal
Source
DF
SS
F
P
R2
Regression
15
0.000320
Residual
123
0.000274
Total
138
0.000594
12.3
<0.0001
0.539
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
3.741
0.0003
0.0015
0.0004
Subject
5.906
<0.0001
0.4847
0.0821
Intensity
6.549
<0.0001
0.0057
0.0009
7-14 days
2.136
0.0346
0.0006
0.0003
21-35 days
0.523
0.6019
0.0001
0.0003
70-90 days
-1.833
0.0692
-0.0005
0.0003
140-165 days
-2.954
0.0037
-0.0080
0.0003
210 days plus
-3.999
0.0001
-0.0011
0.0003
Intensity X
7-14 days
-0.943
0.3475
-0.0018
0.0019
Intensity X
21-35 days
-1.102
0.2724
-0.0021
0.0019
Intensity X
70-90 days
2.276
0.0245
0.0047
0.0021
Intensity X
140-165 days
1.002
0.3180
0.0019
0.0019
Intensity X
>210 days
-0.160
0.8733
-0.0003
0.0019
Transformations: Reciprocal of Intensity-1; reciprocal of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Failed.

77
Table 2-13. Summary of the multiple linear regression analysis of changes over
time in the duration to decay to 0.25 maximal amplitude after sacrocaudal
transection.
Duration at Quarter Maximal
Source
DF
SS
F
P
Ft2
Regression
15
55.0
Residual
123
36.8
Total
138
91.8
15.7
<0.0001
0.599
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
4.069
<0.0001
2.100
0.5160
Subject
8.743
<0.0001
0.698
0.0798
Intensity
-5.033
<0.0001
-1.603
0.3185
7-14 days
-3.359
0.0010
-0.340
0.1013
21-35 days
-3.303
0.0012
-0.334
0.1013
70-90 days
4.191
<0.0001
0.457
0.1091
140-165 days
2.967
0.0036
0.300
0.1012
210 days plus
3.536
0.0006
0.358
0.1012
Intensity X
7-14 days
2.093
0.0384
1.470
0.7023
Intensity X
21-35 days
1.788
0.0762
1.256
0.7023
Intensity X
70-90 days
-1.917
0.0575
-1.446
0.7543
Intensity X
140-165 days
-2.384
0.0186
-1.661
0.6966
Intensity X
>210 days
-0.754
0.4520
-0.526
0.6966
Transformations: Reciprocal of Intensity-1; natural log of the dependent variable.
Normality Test: Passed.
Homoscedasticity Test: Failed.

78
for the time period of 70-90 days, P = 0.0245). For quarter maximal reflex duration,
some of the interactions between stimulus intensity and duration at different times
after the lesion approached significance (Table 2-13). The main findings of the
multiple regression analyses were that: (1) reflex duration significantly increased
with increases in stimulus intensity (Table 2-12, t = 6.549, P < 0.0001; Table 2-13,
t=-5033, P < 0.0001), and (2) the grouping of reflex durations at most of the post¬
lesion time periods accounted for a significant amount of the variance (Tables 2-12,
2-13). For the increasing trend over time in half maximal reflex duration, a post hoc
Scheffe analysis found that pre-lesion values were significantly less than post¬
lesion values at 70-90 days, 140-165 days, and >210 days (P < 0.05). For quarter
maximal duration, a post hoc Scheffe analysis found that pre-lesion values were not
significantly different from post-lesion values at 7-14 and 21-35 days (P > 0.05).
The quarter maximal durations at these 3 time periods were significantly less than
the mean quarter maximal reflex durations at 70-90 days, 140-165 days, and >210
days (P < 0.05).
The changes over time in reflex latency and rise time are shown in Figure 2-
16. For reflex latency, the multiple linear regression analysis accounted for over
60 percent of the variance and was significant (Table 2-14; r2 = 0.634; F = 18.2, P
< 0.0001). Interactions between stimulus intensity and latency at different times
after the lesion were not significant. The main findings of the multiple regression
analysis were: (1) reflex latency significantly decreased with increases in stimulus
intensity (t = -8.504, P < 0.0001), and (2) the grouping of reflex latency values into

Figure 2-16. The time course of changes in reflex latency and rise time. Reflex
latency (top) and rise time (bottom) for 5 animals (symbols) are shown at 6 time
periods, averaged across intensities. The columns represent the average reflex
latencies and reflex rise times determined across animals. A post hoc Scheffe
analysis found that pre-lesion latencies were significantly less than at all post-lesion
time periods (P < 0.05). Pre-lesion rise times were not significantly different from
post-lesion values at any time period after the lesion.

Rise Time (dynes/sec) Latency (msec)
25
20
15
10
5 -
A
♦
o
A
Prelesion 7-14 21-35 70-90 140-165
Time Period (days)
>210
Time Period (days)

81
Table 2-14. Summary of the multiple linear regression analysis of changes in
reflex latency after sacrocaudal transection.
Reflex Latency
Source
DF
SS
F
P
R2
Regression
15
817
Residual
123
472
Total
138
1289
18.2
<0.0001
0.634
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
3.104
0.0024
6.3303
2.040
Subject
8.560
<0.0001
0.9445
0.110
Intensity
-8.504
<0.0001
-7.1937
0.846
7-14 days
-0.104
0.9170
-0.0379
0.363
21-35 days
0.005
0.9958
0.0019
0.363
70-90 days
1.660
0.0994
0.6473
0.390
140-165 days
3.393
0.0009
1.2295
0.362
210 days plus
2.527
0.0128
0.9156
0.362
Intensity X
7-14 days
0.184
0.8544
0.3441
1.871
Intensity X
21-35 days
1.709
0.0900
3.1984
1.872
Intensity X
70-90 days
-1.380
0.1700
-2.7382
1.984
Intensity X
140-165 days
-0.486
0.1627
-0.8901
1.831
Intensity X
>210 days
0.084
0.9329
0.1545
1.831
Transformations
: Log of Intensity.
Normality Test:
Failed; Passed.
Homoscedasticity Test: Passed.

82
post-lesion time periods accounted for a significant amount of the variance (Table
2-14). A post hoc Scheffe analysis of latency found that pre-lesion values were
significantly less than that observed for all post-lesion time periods (P < 0.05).
Reflex rise time at the different time periods is shown in Figure 2-16. The
multiple linear regression analysis accounted for over 70% of the variance and was
significant (Table 2-15, R2 = 0.74, P < 0.0001). Except for a significant effect of
intensity (P = < 0.0001) and an interaction between intensity and rise time at >210
days (P = 0.0387), no other significant differences were obtained for changes in rise
time.
Stability of the Electrocutaneous Reflex with Repetitive Stimulation
When electrocutaneous stimulation was presented in blocks of trials at a
single intensity, there were instances of wind-down (a decrease) or wind-up (an
increase) of reflex force estimates, when comparing the response to the first
stimulus with subsequent responses. The frequency of wind-down varied in normal
animals, ranging from 10 percent to 36 percent, with a median of 13.9 percent
(Figure 2-17). Post-lesion, wind-down increased for some animals and decreased
for others. For the population of 5 animals there was a slight decrease in the
occurrence of wind-down over the 5 different time periods after the lesion.
In normal animals wind-up occurred less often than wind-down, varying from
0 percent to 14 percent, with a median of 8 percent across subjects (Figure 2-18).
Post-lesion, the frequency of wind-up increased for most animals. Also, as early

83
Table 2-15. Summary of the multiple linear regression analysis of changes in
reflex rise time after sacrocaudal transection.
Rise Time
Source
DP
SS F
P
R2
Regression
15
1.021
Residual
123
0.361
Total
138
1.382 29.7
<0.0001
0.739
Variable
t
P
Equation
Coefficient
Standard
Error
Constant
4.632
<0.0001
0.0949
0.0205
Subject
15.896
<0.0001
0.9712
0.0611
Intensity
-8.828
<0.0001
-0.4633
0.0525
7-14 days
-0.377
0.7068
-0.0038
0.0100
21-35 days
-1.648
0.1019
-0.0165
0.0100
70-90 days
0.762
0.4473
0.0083
0.0109
140-165 days
0.446
0.6561
0.0045
0.0100
210 days plus
1.897
0.0601
0.0190
0.0100
Intensity X
7-14 days
-0.662
0.5091
-0.0767
0.1159
Intensity X
21-35 days
-1.549
0.1240
-0.1795
0.1159
Intensity X
70-90 days
-1.397
0.1648
-0.1733
0.1240
Intensity X
140-165 days
1.840
0.0681
0.2109
0.1146
Intensity X
>210 days
2.089
0.0387
0.2394
0.1146
Transformations: Reciprocal of Intensity; log of the dependent variable+1.
Normality Test: Passed.
Homoscedasticity Test: Passed.

Figure 2-17. Frequency of wind-down of the reflex with repetitive stimulation. For
the 6 time periods, the graphs show the median probability of occurrence of a
diminished reflex response with repetitive stimulation at one stimulus intensity for
each animal (top) and for the group of 5 subjects (bottom). A decreased frequency
of wind-down was observed following sacrocaudal lesions. The lines represent 75
percent and 25 percent confidence intervals.

Wind-down Probability
85
0.70
0.60
Time Period (days)
â– 
Pre-lesion
â–¡
7 to 14
hd
21 to 35
â–¡
70 to 90
â–¡
140 to 165
s
>210
Time Period (days)

Figure 2-18. Frequency of wind-up with repetitive stimulation. For the 6 time
periods, the graphs show the median probability of occurrence of wind-up of the
reflex response with repetitive stimulation at a constant intensity for each subject
(top) and for the group (bottom). The increase in frequency of wind-up developed
progressively over time after sacrocaudal lesions. The lines represent 75 percent
and 25 percent confidence intervals.

Wind-up Probability
87
0.70
0.60
>.
S
ro
-O
o
Q_
Q.
D
i
"O
c
£
â– 
Pre-lesion
â–¡
7 to 14
21 to 35
â–¡
70 to 90
B
140 to 165
Q
>210
Subject 1 Subject 2 Subject 3 Subject 4 Subject 5
Time Period (days)
Time Period (days)

88
as 70 to 90 days post-lesion, a new form of wind-up was observed, involving
increases in reflex force over a number of stimulus presentations (Figure 2-19).
This contrasted with the normal pattern, involving stable reflex force values after the
first response. The percent change in the magnitude of reflex force during wind-up
were similar pre- and post-lesion but varied across the 5 post-lesion periods (Figure
2-19). The percent change in the magnitude of the reflex force during wind-down
was generally reduced post-lesion (Figure 2-20).
Post-lesion responses of the tail to trains of low intensity electrocutaneous
stimulation (50 Flz for 100 msec) sometimes included a late response at latencies
on the order of 400 to 500 ms (Figure 2-21). The late onset reflex was observed
in half (3/6) of the animals tested, with an average frequency of 26 percent. When
the late reflex occurred, it was present on the first few trials of a block and then
dissipated. At high stimulus intensities, the responses to trains of stimulation
occurred at latencies similar to those observed for a single pulse (Figure 2-21).
Reflex Characteristics in Animals with Variants of the Standard Sacrocaudal Lesion
Changes in properties of the electrocutaneous reflex were examined for
lesions other than the intended sacrocaudal transection. A decrease in reflex force
was observed for animals that received either a dorsal hemisection at S3, or a S3
transection with evidence for an ischemic episode, or a transection at rostral Ca3
(Figure 2-22). This effect was opposite to the post-lesion increase in reflex force
observed for other animals with sacrocaudal transactions.

Figure 2-19. Representative responses showing wind-up of the tail reflex. For an
animal with a sacrocaudal transection (5 months post-lesion), the waveforms show
a series of reflex responses of the tail to electrocutaneous stimulation at a single
stimulus intensity. For clarity, the reflex waveforms are offset. The top waveform
shows the first reflex. The next waveforms show responses of the tail to the
second, fourth, and seventh presentations of the stimulus. The bottom trace shows
the timing of the stimulus.

Force (0.65 N/div)
90
0.0 ms
800 ms/div
4.0 s

Figure 2-20. The magnitude of changes in the reflex response with repetitive
stimulation. For the 6 time periods, the graphs show the percent change in
magnitude of the reflex during the occurrences of wind-up (top) and wind-down
(bottom) of the reflex response with repetitive stimulation. The error bars represent
the standard error of the mean.

Time Period (days)
Wind-down Magnitude (percent decrease)
V
O
70%
Time Period (days)
Wind-up Magnitude (percent increase)
l\) -&â–  05 00 O
o o o o o o
Vp vP ^
co
N)
120%

Figure 2-21. Reflex responses of the tail to trains of electrocutaneous stimulation.
From an animal with a sacrocaudal transection (3 months post-lesion), 4 waveforms
(top) show sequential responses of the tail to electrocutaneous stimulation at 2
mA/mm2. For clarity, the waveforms are offset. Note that the onset latencies of the
late response range between 420 and 470 ms from stimulus onset. By the fifth
presentation of the stimulus (not shown), the reflex habituated. The bottom trace
shows the timing of the stimulus. Two other traces (bottom) show the response to
trains of stimulation at 3 and 4 mA/mm2. The onset latency is about 18 ms. The
bottom trace shows the timing of the stimulus.

94
>
T3
z
Stimulus
0.0 ms
800 ms/div
4.0
s
0.0 ms
800 ms/div
4.0 s

Figure 2-22. Reflex force in animals with variants of the sacrocaudal lesion. Average reflex force (top) and average reflex
force-intensity relationships (bottom) at different testing periods are shown for animals that received a dorsal hemisection
at S3 (left), an S3 transection with an ischemic episode (middle), or a Ca2-Ca3 transection (right). A decrease in reflex force
was produced by these lesions.

Reflex Force (N)
iv) -u o> 03 o ro
o o o o o o o
o
3
CD
3
% *.
3"
£
3 ®
3
IV)
Reflex Force (N)
10 O) co o to
o o o o o o o
3
CD
3
CO
3
£
3
3
rv)
Reflex Force (N)
—*■ ro co -u en o
o o o o o o o
Reflex Force (N)
Pre-lesion
3
CD
~0
CD
o'
CL
CL
CD
•<
CO
7-14
21-35
70-90
140-165
J M U L OI ffi
o o o o o o o
-J
o
Reflex Force (N)
Reflex Force (N)
to CO L OI O)
oooooooo
1—I—I—I—I—I—(■
Pre-lesion
H 7-14
1
3
CD
21-35
o'
Q.
70-90
140-165
â–¡
96

97
To fully characterize changes in the electrocutaneous reflex following dorsal
hemisection at S3, the six other reflex characteristics were evaluated (Figure 2-23).
Decreases in peak reflex amplitude and latency at peak reflex amplitude were
observed for all post-lesion testing periods. In contrast, the slope of the ascending
limb of the reflex (rise time) increased after the lesion. Half maximal duration was
reduced at the 7-14 day post-lesion period. Quarter maximal duration recovered
from a transient post-lesion decrease, and duration at the 70-90 day period was
greater than at the 7-14 day and 21-35 day periods. Additionally, reflex latency at
the 70-90 day post-lesion period was less than at the 2 preceding post-lesion
periods and the >210 day post-lesion period.
Following the sacrocaudal transection with an ischemic episode (Figure 2-
24) a post-lesion decrease was observed for peak amplitude and rise time for the
reflex at all 5 post-lesion periods. Latency at peak reflex amplitude at the 21-35 day
post-lesion period was greater than at the pre-lesion and other post-lesion time
periods. The increases in reflex latency at the 21-35 and 70-90 post-lesion periods
were different from the pre-lesion value and the 7-14 day post-lesion value. Small
post-lesion increases were observed in reflex duration.
For the animal with a caudal transection at the Ca3 spinal segment (Figure
2-25) a post-lesion reduction was observed for peak reflex amplitude. In addition,
peak amplitude at the 140-165 post-lesion testing period was less than at the
preceding post-lesion periods. A post-lesion reduction was observed for reflex rise

Figure 2-23. Reflex characteristics following dorsal hemisection at S3. The 6 graphs show changes in reflex characteristics
at 6 different time periods. Persistent decreases in peak amplitude and latency at peak reflex amplitude were observed after
the lesion. Reflex duration was transiently reduced. Rise time progressively increased after the lesion.

Time Period (days) Time Period (days) Time Period (days)
Peak Latency (msec)
i\i -u cn> a> o
o o o o o o
Pre-lesion
7-14
21-35
70-90
140-165
>210
Peak Amplitude (N)
p o o o o
o i\3 co Ln
P re-lesion
H
3
CO
7-14
I
"0
CD
“*
21-35
I
o
Q.
70-90
I
0)
*<
C/>
140-165
I
>210
I
Duration (1/4 max, msec)
Pre-lesion
7-14
21-35
70-90
140-165
>210
O
O
ro
CO
cn
CD
o
o
O
o
O
o
o
o
o
o
o
o
Duration (1/2 max, msec)
—‘MU
o o o
o o o o
—I—I—I—I—I—I—
1 1 1
Pre-leslon
üHüimw
-â– 
H
I
3 7-14
CD
I
"0
I
© 21-35
I
o'
Q.
I
— 70-90
I
Cl
0)
I
m, 140-165
I
I
>210
I
Rise Time (Dynes/sec)
OOOOpj-*-‘-‘-iM
bro^cncookj^cnboc)
Latency (msec)
o ro o> oo o
1 1 1 1
Pre-leslon
OH
H
3
7-14
I
CD
T)
CD
21-35
I
o'
CL
70-90
I
0)
<
W
140-165
|
>210
66
400

Figure 2-24. Reflex characteristics following sacrocaudal transection with an ischemic episode. The 6 graphs show changes
in reflex characteristics at 5 testing periods. Persistent decreases in peak amplitude and rise time were observed after the
lesion. Half maximal duration, reflex latency and latency at peak reflex amplitude increased after the lesion.

Time Period (days) Time Period (days) Tjme period Peak Latency (msec)
—1 tO
cn o cn o
o o o o o
Pre-lesion
7-14
21-35
70-90
140-165
Peak Amplitude (N)
o o o o
b b ^ 1*.
o cn o cn
r— | 1
Pre-lesion
I
H
3
CD
7-14
I
"0
CD
o
o
21-35
I
"2
CD
<
cn
70-90
140-165
Duration (1/4 max, msec)
M ^ O) ffl O
o o o o o
o o o o o o
Pre-lesion
7-14
21-35
70-90
140-165
Duration (1/2 max, mse
-* -r M to U U
cn o cn o cn o cn
oooooooo
Rise Time (Dynes/sec)
p p o o o
o ^ ro b 4^ cn
Latency (msec)
ro
O) 00
ro
o
0.20 i — . o' 400

Figure 2-25. Reflex characteristics following caudal spinal transection at Ca3. The 6 graphs show changes in the reflex
characteristics at the 5 different time periods. Persistent decreases in peak amplitude and rise time were observed after the
lesion.

ecu
Peak Latency (msec)
M Ü1 n| O M U1
o cn o cn o cn o
Pre-lesion
H
3
CD
7-14
Tl
CD
“1
o'
21-35
CL
CD
><
70-90
CD
140-165
Peak Amplitude (N)
o
o
b
cn
cn
o
ro
Duration (1/4 max, msec)
ro 4^ CD CD o
o o o o o
o o o o o o
Pre-lesion
H
3
CD
7-14
"0
CD
o'
CL
21-35
"S
CD
*<
CD
70-90
140-165
—~
Duration (1/2 max, msec)
->â–  n> co ^
o o o o
o o o o o
P re-lesion
Rise Time (Dynes/sec)
o
o b.
o
ho
o
co
o
4^
o
cn
Latency (msec)
o ro ^ cn oo o
Pre-lesion
3
CD 7-14
TJ
(D
O 21-35
'El
0)
*< 70-90
C/)
140-165
H h
fjíV.,-;
¿
500

104
time, and rise time at 140-165 days was less than at the preceding 2 post-lesion
periods. Only small differences were observed for the other reflex characteristics.
To characterize the chronic changes in the electrocutaneous reflex following
a left hemisection at S3, the reflex characteristics from this animal were compared
to the reflex measures from prelesion animals and animals with a chronic
transection lesion (Table 2-16). In general, the reflex characterisitics from the
animal with the left hemisection were more similar to those obtained from normal
pre-lesion animals than from chronic post-lesion animals. For example, with
respect to the estimate of reflex force, the average value across intensities for this
animal (39.09 N) compared more favorably with pre-lesion values (44.41 N) than
with the average post-lesion force (398.18 N). The slope of the regression line
relating reflex force with stimulus intensity was less than that determined for the
pre-lesion and post-lesion animals which suggests that after the hemisection lesion
the force of the reflex was less sensitive to stimulus intensity. The average peak
reflex amplitude of 0.31 N fell in between the values observed for pre-lesion (0.22
N) and post-lesion animals. For the animal with the left hemisection, the slope of
the regression line (-0.54) relating reflex peak amplitude with stimulus intensity was
nearly identical to that observed for pre-lesion animals (-0.58) and therefore less
than half of that calculated for post-lesion animals (-1.25). And latency at peak
reflex amplitude (55.48 ms) was even less than that observed for both pre-lesion
(68.20 ms) and chronic post-lesion (116.16 ms) animals. The duration to decay to
half maximal peak reflex amplitude is less for the animal with the left hemisection

105
Table 2-16. Comparison of 5 reflex characteristics for one animal after a left
hemisection and a group of 7 animals pre- and post-transection.
Left Hemisection
Normal
Chronic Transection
Coefficent
Std.
Error
Coefficent
Std.
Error
Coefficent
Std.
Error
Reflex Force
Ave (y)a
36.09
44.41
10.96
398.16
149.86
Y-int (ln(y))b
3.88
0.17
5.12
0.23
7.31
0.23
Slope (1/x)
-1.20
0.81
-6.05
0.64
-6.05
0.64
Peak Amplitude
Ave (y)
0.31
0.22
0.03
0.48
0.09
Y-int
0.43
< 0.01
0.33
0.02
0.74
0.02
Slope (1/x)
0.54
0.03
-0.58
0.09
-1.25
0.09
Latency at peak
reflex amplitude
Ave (y)
Y-int (ln(y))
Slope (1/x)
55.48
4.02
0.02
0.02
0.08
68.20
4.16
0.29
4.60
0.31
0.27
116.16
5.12
-2.22
13.17
0.31
0.27
Duration (1/4 max)
Ave (y)
251.08
193.45
20.08
565.84
160.54
Y-int (ln(y))
5.35
0.16
6.07
0.28
7.59
0.28
Slope (1/(x-1))
0.52
0.53
0.88
0.23
-2.32
0.23
Rise Time
Ave (y)
1.48
0.76
0.16
1.19
0.28
Y-int (log(y+1))
0.47
0.02
0.34
0.01
0.40
0.01
Slope (1/x)
-0.39
0.12
-0.50
0.04
-0.30
0.04
aThe average score is the mean response across intensities for the different animals.
bThe Y-intercept was determined for the population of normal and chronic transection
animals by applying the mean subject reference code to the regression equation.

106
(122.85 ms) than for pre-lesion animals (193.45 ms) and animals with a chronic
transection (565.84). These last two comparisons of the electrocutaneous reflex
characterisitics might suggest that a slight hyporeflexia for cutaneous stimulation
might be the consequence of a lateral hemisection at sacrocaudal levels. However,
this judgement might be premature since the values for quarter maximal reflex
duration and reflex rise time are greater for the animal with the left hemisection than
that observed for normal prelesion animals (Table 2-16).
Discussion
The present study was undertaken to determine whether changes in
cutaneous reflexes of the tail occur after sacrocaudal spinal lesions. Prior research
characterized a paralysis and increased muscle tone of the tail after sacrocaudal
spinal lesions (Ritz et al., 1992). Results presented here show that cutaneous
hyperreflexia develops after a sacrocaudal spinal transection and is similar to that
observed in humans following spinal cord injury.
Characteristics of Electrocutaneous Reflexes with Chronic Spinal Cord Injury
Spinal cord injuries alter the characteristics of cutaneous flexion reflexes of
humans. In the chronic spinal cord injured patient a pronounced cutaneous
hyperreflexia to mechanical and electrocutaneous stimulation (Dimitrijevic and
Nathan, 1967) can override the normal organization of cutaneo-muscular reflexes.
Instead of a short latency local response to a cutaneous stimulus, with reciprocal
inhibition of other muscle groups (Hagbarth, 1952; Hagbarth and Finer, 1963), the

107
flexion reflex to cutaneous stimulation is of greater magnitude and longer duration,
and the reflex activity radiates to distant muscle groups (Pedersen, 1954;
Dimitrijevic and Nathan, 1967). Similar alterations in the cutaneous flexion reflex
of the tail was observed after sacrocaudal spinal transection (Table 2-17).
After sacrocaudal transection, the electrocutaneous reflex of the tail
exhibited an increase in magnitude, as measured by the force of the response.
Since reflex force was determined by summing the amplitude of the response over
the duration of the reflex, it was not unexpected that this increase in overall force
was accompanied by increases in peak amplitude and duration.
To examine factors contributing to increases in peak amplitude after
sacrocaudal transection, the slope of the ascending limb of the reflex (rise time) and
latency at peak reflex amplitude were evaluated for the pre- and post-lesion
periods. In comparison to pre-lesion measurements, post-lesion peak reflex
amplitude was greater at all stimulus intensities and increased at a greater rate with
stimulus intensity. Latency at peak reflex amplitude also was greater after
sacrocaudal transection. Latency at peak reflex amplitude remained stable with
increases in stimulus intensity during pre-lesion testing, but post-lesion latency at
peak reflex amplitude increased with higher levels of stimulation. Additionally, the
post-lesion slope of the ascending limb of the reflex was greater than the pre-lesion
slope (although this difference was not statistically significant). These findings
suggest that there is a post-lesion change in the recruitment pattern and duration
of activation of motoneurons mediating the reflex.

108
Table 2-17. Summary of the consequences of a transection of the sacrocaudal
spinal cord on the response characteristics of the electrocutaneous reflex.
Overall Change3 Interaction6 Change Over Time
Reflex Force
Increase
No
Increase
Peak Amplitude
Increase
Yes
Increase
Latency at peak
reflex amplitude
Increase
Yes
Increase
Duration (1/4 max)
Increase
Yes
Increase
Duration (1/2 max)
Increase
No
Increase
Rise Time
None
Yes
None
Latency
Increase
No
Increase
aAn overall change indicates that over this stimulus intensity range there was a significant
difference between the two groups irrespective of stimulus intensity.
bAn interaction indicates a significant change in the intensity-response relationship
between the two groups.

109
Changes in reflex duration between the pre- and post-lesion periods
contributed to the increase in reflex force that was seen after transection of the
sacrocaudal spinal cord. Post-lesion, the time to decay to quarter maximal and half
maximal amplitude was greater than that observed during pre-lesion testing. The
decay to quarter maximal amplitude was increased to a greater extent for post¬
lesion animals, and quarter maximal duration increased at a greater rate with
stimulus intensity after transection. Similarly, prolonged stretch and withdrawal
reflexes have been observed for the legs of human spinal cord injured patients
(Dimitrijevic and Nathan, 1967) and for the hindlimbs of spinal cats with lesions at
thoracic and lumbar levels (Kozak and Westerman, 1966).
While an increase in the magnitude of the reflex was observed for the all the
animals with a transection lesion of the sacrocaudal spinal cord, the magnitude of
cutaneous hyperreflexia varied considerably from animal to animal. This increase,
between animals after lesions, in the variability of the flexion reflex most likely was
a result of differences in the sacrocaudal lesion that occurred at the time of surgery.
Even when the transection lesion of the sacrocaudal spinal cord was complete,
differences in the possible hemorrhagic damage to caudal segments, acute and
chronic alterations in the blood supply to the caudal cord, and potential caudal
spinal root damage could contribute to the differences observed between animals.
Another source for the variability could be that there are inherent differences
between animals in the plasticity of the cutaneous reflex.

110
For a number of characteristics of the electrocutaneous reflex, there was an
overall shift in response values and a post-lesion increase in slope of the
relationship between the response measures and the intensity of the stimulus. The
change in reflex gain contrasts with studies on stretch reflexes in hemiplegic
subjects, where a lowering of reflex threshold without a change in gain has been
observed (Lee et al., 1987; Powers et al., 1988). This distinction found between
electrocutaneous reflexes after sacrocaudal transection and stretch reflexes in
patients with an upper motor neuron syndrome suggests either that muscle and
cutaneous reflexes are differentially affected in spasticity, or that different lesions
produce distinct effects on both types of reflex, or there is a species difference
between humans and cats.
In spinal cord injured patients, there is a dramatic decrease in reflex
threshold (Dimitrijevic and Nathan, 1967; Shahani and Young, 1973; Roby-Brami
et al., 1989). Similarly, after sacrocaudal transection, a decrease in reflex threshold
was observed with mechanical stimulation, as has been seen for other animal
models involving spinal transection (Ranson and Hinsey, 1930; Afelt, 1970). After
sacrocaudal transection, the latency of the electrocutaneous reflex was significantly
increased. Comparable increases in flexion reflex latency has been observed with
electrical stimulation of the skin (Dimitrijevic and Nathan, 1967), but not for
percutaneous nerve stimulation (Pederson, 1954). Increased latencies have been
noted for the affected side of hemiplegic patients (Dimitrijevic, 1973).

111
Development of Cutaneous Hyperreflexia
Changes in electrocutaneous tail reflexes could have occurred shortly after
the spinal lesion, without changing thereafter, as for decerebrate rigidity
(Sherrington, 1898; Walshe, 1923). However, the results presented in this study
show that cutaneous hyperreflexia increased over an extended time period after
sacrocaudal transection, which is consistent with the progressive nature of human
spasticity.
Two time periods appeared in the development of cutaneous hyperreflexia
after sacrocaudal transection. During the second week after the lesion, there was
a significant change in reflex latency, with a significant increase in peak amplitude
by 21 to 35 days post-lesion. A second time period occurred at 70 to 90 days,
when significant increases from pre-lesion values occurred for reflex force and
duration. Other studies with different lesions have described phases of reflex
exaggeration (Murray and Goldberger, 1974; Little, 1986) that occur over similar
time periods.
As expected, cats did not show the extended period of hyporeflexia that is
observed in human spinal cord injured patients. Hyperreflexia was detectable soon
after the first post-lesion week for the electrocutaneous response measures. Early
hyperreflexia is consistant with other studies with cats (Murray and Goldberger,
1974; Bailey et al., 1980; Kato et al., 1985; Rhoton et al., 1988), in which the period
of hyporeflexia was on the order of a few days, with hyperreflexive responses
appearing by the end of the first week.

112
Stability of the Reflex
Reflex responses to repetitive stimulation of animals following spinal
transection have provided important model systems for investigation of neurological
mechanisms of habituation and sensitization (Thompson and Spencer, 1966;
Groves et al., 1969). In the present study, the electrocutaneous tail reflex at times
exhibited both wind-down and wind-up to repetitive stimulation in animals prior to
and after sacrocaudal transection. An increase in the frequency of windup and an
alteration of its form were produced by spinal transection. Similar effects of
repetitive electrocutaneous stimulation have been observed for human patients
following spinal cord injury (Dimitrijevic and Nathan, 1970; Führer, 1977). In human
patients, expression of wind-up or a wind-down of the flexion reflex was dependent
on the intensity, rate, and number of stimulations.
The Late Reflex
After sacrocaudal transection, a long latency reflex was sometime elicited
with low intensity trains of electrocutaneous stimulation. Similar reflexes have been
observed in humans after spinal cord injuries. A long latency reflex (130 to 300 ms)
was produced by a normally subthreshold electrocutaneous stimulus (Shahani and
Young, 1971; Roby-Brami and Bussel, 1987) or mechanical stimulus (Roby-Brami
et al., 1989). However, this response occurred infrequently in cats with a spinal
lesion in contrast to human patients. This difference may be due to the use here
of electrocutaneous stimulation rather than percutaneous nerve stimulation used

113
in the human study. And, with increases in electrocutaneous stimulus intensity, the
latency of the electrocutaneous response decreased in the present study in a
similar manner as that observed in humans after spinal cord injury (Shahani and
Young, 1971; Roby-Brami and Bussel, 1987). In acute spinal cats injected with
DOPA (Anden et al., 1966a), a long latency reflex can also be elicited, which may
be related to the long latency reflex observed after chronic lesions of the spinal
cord. Physiological studies (Anden et al., 1966b) have suggested that a lowered
threshold for the long latency reflex results from active inhibition of the short latency
flexion reflex by flexor reflex afferents (Lunberg, 1979). A similar change in the
segmental circuitry mediating the flexion reflex may be a consequence of a spinal
cord injury (Roby-Brami and Bussel, 1990).
Other Types of Sacrocaudal Lesions
Cutaneous hyperreflexia did not develop in four animals with lesions which
differed from the main group of sacrocaudal transections. Two animals received
lesions that directly damaged caudal spinal segments. Post-lesion
electrocutaneous reflexes of these animals were reduced and were reminiscent of
the reduction in amplitude of reflexes that is observed in patients with lower motor
neuron disease. For an animal with a chronic sacrocaudal hemisection on the left,
electrocutaneous reflex values were similar to normal pre-lesion values and differed
from the reflex responses characteristic of animals with sacrocaudal transection.
This result in one cat provides quantitative verification of other studies in which

114
reflex responses were not enhanced by hemisection of the spinal cord (Hultborn
and Malmsten, 1983; Carter et al., 1991). In contrast, the bilateral dorsal
hemisection performed on one cat attenuated cutaneous reflexes throughout the
post-lesion testing period. This result differs from reports that acute dorsal
hemisection exaggerates hindlimb stretch reflexes (Powers and Rymer, 1988).
These findings suggest either that section of a specific combination of spinal
pathways is required to produce spasticity, or the extent of deafferentation dictates
whether cutaneous hyperreflexia develops for segments caudal to a spinal lesion
(Wagley, 1945).
In conclusion, classical signs of spasticity can be detected and evaluated
quantitatively by measuring tail reflexes of cats following transection of the
sacrocaudal spinal cord. The reflex effects of sacrocaudal transection on the tail,
in the absence of bowel and bladder dysfunctions or major alterations in
locomotion, makes this an ideal and minimally disruptive animal model for
investigations of intervention strategies and the neurobiology of spinal cord injury.

CHAPTER 3
EFFECTS OF FETAL SPINAL TISSUE TRANSPLANTS
ON CUTANEOUS REFLEXES OF THE CAT TAIL
Introduction
Multiple approaches are being employed in the treatment of acute and
chronic effects of spinal cord injuries. In an attempt to minimize damage from the
initial insult to the spinal cord, marked clinical success has been observed with
methylprednisolone treatment if given shortly after spinal cord injury (Young, 1991).
To control spasticity and pain following a spinal cord injury, pharmacological and
surgical interventions are used with varying degrees of success (Dimitrijevic and
Nathan, 1967; Davidoff, 1985). Physical therapy and specialized mechanical
devices (e.g., wheel chairs, crutches, prosthetic devices, specialized hand splints)
are utilized to improve mobility (Trieschmann, 1988). Currently, there are no
treatments to compensate for the loss of cutaneous sensibility after spinal cord
injury.
A new strategy to prevent or reverse adverse effects of neural injury involves
transplantation of embryonic neural tissue. For example, grafting of cells from
embryonic brainstem into the brains of individuals with Parkinson's disease has
been shown to have a therapeutic benefit for some patients (Lindvall et al., 1990;
115

116
Freed et al., 1992). With respect to repair of the spinal cord, anatomical studies in
animals have found evidence for the ability of embryonic fetal graft tissue to survive,
differentiate and integrate with the host which may promote functional restoration
of the spinal cord (Reier et al., 1994). By acting as a conduit, spinal cord grafts
might assist in restoration of connectivity between descending systems and spinal
target neurons (Bregman, 1987; Nothias and Peschanski, 1990; Bregman and
Bemstein-Goral, 1991). By providing trophic support, fetal grafts have been shown
to rescue neurons from retrograde cell death due to a spinal injury (Bregman and
Reier, 1986). And in attempts to replace degenerated cells, grafts of fetal
motoneurons have been shown to survive in the host spinal cord and retain the
capacity to innervate muscle (Clowry et al., 1991). Thus, the goals of embryonic
spinal transplantation can include 1) replacement of lost neurons, connections or
neural pathways; 2) rescue neurons at risk of cell death; and 3) modulation of
existing neural activity of the damaged host nervous system.
Behavioral studies in cats and rats have found that fetal spinal transplants
can ameliorate some of the consequences of spinal cord injury. Intraspinal fetal
grafts have been shown to promote recovery in locomotor behavior (Anderson et
al., 1991; Goldberger, 1991; Kunkel-Bagden and Bregman, 1990; Kunkel-Bagden
et al., 1992; Stokes and Reier, 1992). However, a late component of reflex activity
has been shown to be enhanced by the presence of fetal graft tissue (Buchanan
and Nornes, 1986), suggesting that an exaggeration of spasticity might result.;
whereas, another study has found otherwise (Thompson et al., 1993). Thus, it is

117
important to determine whether a neural graft provides beneficial or deleterious
modulatory effects on the hyperreflexia that often results from spinal cord injury.
Prior research has show that after transection of the cat sacrocaudal spinal
cord, the tail exhibits hyperactive cutaneous and proprioceptive reflexes and loss
of sensation and volitional control of the tail (Chapter 2; Ritz et al., 1992). These
functional alterations are akin to the spastic syndrome and losses of sensory and
motor function that occur in humans with spinal cord injuries. Consequences of the
sacrocaudal lesion occur in the absence of bowel and bladder dysfunctions or
disruptions of locomotion. Thus, functional studies of the tail provide a minimally
disruptive animal model system to study intervention strategies and the
neurobiology of spinal cord injury.
The present study utilized the cat tail model to investigate functional
consequences of transplantation of embryonic grafts of fetal tissue into the injured
spinal cord. Specifically, this study addressed whether embryonic tissue can
survive and differentiate in an acute transection cavity at sacrocaudal levels and
whether electrocutaneous reflexes of the tail are affected by transplantation of
embryonic tissue into spinal cord lesions.
Methods
Subjects. A total of 11 adult cats (female) served as experimental subjects
and were recipients of fetal graft tissue. For 9 of these animals, normal pre-lesion
data were collected. Subsequently, these animals received a complete transection

118
of the sacrocaudal spinal cord followed by placement of embryonic tissue into the
lesion cavity. When the animals were not performing in the behavioral experiments,
they were housed at the University of Florida Animal Resources Facilities.
Surgery and transplantation. All surgeries were performed under sterile
conditions and gas anesthesia (Halothane or Isoflourane). The animal was
intubated and a venous catheter was inserted for the infusion of fluids. Dorsal
laminectomies were performed at the level of the L6 and L7 vertebrae. A subpial
transection cavity (3-5 mm long) was made with precision forceps at the S3/Ca1
level.
Fetal cat spinal cord and brainstem tissue was obtained at E21-E23. Care
was taken to strip the surrounding meninges, as well as to remove any attached
dorsal root ganglia. Pieces of fetal spinal tissue were placed in each cavity in a
longitudinal orientation. In 4 instances, fetal brainstem and spinal cord tissue were
placed in the transection cavity. Four pial sutures and 6 to 8 dural sutures secured
the transplant.
Postoperative care. Postoperative care included antibiotic treatments
(Amoxicillin, 50 mgs) for 7-10 days. Canned food was added to the diet. If
necessary, the bladders were manually expressed, but in most cases normal
bladder function returned by the second postoperative day. In the one case where
normal bladder function did not return, the bladder was manually expressed 3 times
a day for 6 weeks, and then the cat was euthanized by a lethal injection of sodium
pentobarbital.

119
Beginning one day prior to transplantation, the animals received a daily dose
of Cyclosporine A (Sandoz Pharmaceuticals; 10 mg/kg) until the day of sacrifice.
One animal was removed from daily Cyclosporine A treatment 4 days after
receiving transplant tissue, because it was discovered the animal had previously
been exposed to the toxoplasmosis virus.
Anatomy. At the termination of the behavioral experiments, the animals were
anesthetized with a lethal dose of sodium pentobarbital and transcardially perfused
with isotonic saline solution (0.9% with sodium nitroprusside (0.5g/L) and Heparin
(1.5cc/L), followed by a solution of 4.0% paraformaldehyde in 0.1 M phosphate
buffer (pH 7.6). The tissue was either cut with a vibratome at 30 pm or paraffin
embedded and serially sectioned at 7 pm. A cresyl violet stain was used to
evaluate the tissue.
Behavior. Quantitative behavioral measures of cutaneous reflexes were
performed as previously described (Chapter 2). In brief, reflex testing was
performed with the tail bracketed at the base while the cat was situated in a cat
sack or restrained by a harness placed around the torso. The tip of the tail was
tethered to a strain gauge device to provide quantitative measures of cutaneous
reflexes. Innocuous electrocutaneous stimulation consisted of a 5 ms pulse of
constant current (DC) delivered to the dorsal surface of the tail at dermatomal level
Ca3. Relationships between stimulus intensity and reflex responses were
determined for 6 different stimulus intensities: 2, 3, 4, 6, 8, and 10.5 mAmps/mm2.

120
Individual trials within each block of trials at a given stimulus intensity were
averaged together. Seven different aspects of the averaged reflex waveform were
evaluated: (1) latency, (2) peak amplitude, (3) latency at peak reflex amplitude, (4)
rise time of the ascending limb of the reflex, (5) half maximal duration, (6) quarter
maximal duration, and (7) the overall force of the tail reflex. The overall force of the
tail reflex was determined by summing the response amplitudes from the time of
stimulus offset until the decay of the response reached 25 percent of the peak
amplitude.
Statistical analysis. A mixed repeated-measures design was used to
compare at chronic time periods (>5 months) electrocutaneous tail reflexes of
animals with surviving transplants and animals that received a sacrocaudal
transection without transplantation. Multiple linear regression analysis (Pedhazur,
1982) was used to evaluate changes in the reflex. The presence or absence of
fetal tissue in the transection cavity was treated as the categorical between-subjects
factor. Stimulus intensity was treated as a continuous within-subjects factor. Each
characteristic of the reflex was treated as a continuous dependent variable. To
perform the multiple linear regression analysis, the independent and dependent
variables were transformed, when appropriate, in attempts to pass tests of normality
(Kolmogorov-Smirnov test) and homoscedasticity (constant variance) and to
linearize the relationships between stimulus intensity and the measured
characteristics of the reflex. Criterion scaling was used to code for the subjects.

121
Results
The level of a sacrocaudal lesion and the presence and integration of graft
tissue were confirmed for all animals at the end of the experiment (Table 3-1). For
8 of the animals, the sacrocaudal transection cavity contained graft tissue. Six of
these animals were combined to form a group to study cutaneous reflexes in the
presence of graft tissue after a sacrocaudal transection lesion at chronic time
periods. For two animals with surviving transplant tissue, only early post-lesion
behavioral data were collected. One of these was sacrificed at 6 weeks post-lesion,
after an acute outbreak of viral pneumonia (toxoplasmosis virus) that resulted from
a weakened immune system, due to Cyclosporine intake. The other self-mutilated
a portion of the insensate tail, beginning 1 month post-lesion and resulting in
termination of data collection for this animal. Autotomy of the tail occurred in
another animal, beginning 1 month post-lesion, but wrapping the tail in veterinary
bandages and placement of an Elizabethan Collar allowed the animal to finish the
study without critical damage to the tail. The 3 other animals received embryonic
fetal spinal tissue that was not present at the time of sacrifice.
Anatomical Integration of the Transplant
Survival of grafted embryonic tissue was initially evaluated at a gross
anatomical level (Figure 3-1). Superficially, transplant tissue usually formed a
continuous bridge between the rostral and caudal host spinal cord (Figure 3-1 d-f).
Coronal sections through the center of the transplant demonstrated a lack of
residual host spinal cord, confirming that the subpial transection was complete

Table 3-1. Anatomical evaluation of transplant survivability and integration.
Animal
Number
Donor
Tissue
Survival
Time (days)
Level of
lesion
Viable
Graft
Size
Neural
Maturation
Rostral
Integration
Caudal
Integration
Oil 8
FSC
145
Ca1
yes
medium
yes
yes
yes
1c7
FSC
145
Ca1
yes
medium
yes
yes
yes
20
FSC + BSt
230
S3/Ca1
yes
large
yes
yes
yes
1f13
FSC
37
S3
yes
small
yes
yes
yes
1 ¡17
FSC + BSt
60
S2
yes
large
yes
yes
yes
2c12
FSC
313
S3/Ca1
no
200
FSC + BSt
181
S3
no
1 ¡19
FSC
209
Ca1/Ca2
yes
large
yes
yes
yes
1 ¡5
FSC + BSt
385
Ca1/Ca2
yes
large
yes
yes
nd
1 ¡14
FSC
331
S3
no
1i2
FSC
198
Ca1
yes
large
yes
yes
yes
FSC: fetal spinal cord; BSt: brainstem; nd: not determined

Figure 3-1. The survival of transplant tissue.
Composite series of photographs show dorsal (A and D), ventral (B and E) and lateral (C and F) views of transplants from
two animals who received embryonic spinal grafts at the time of sacrocaudal spinal transection. The animals were sacrificed
at 7 months post-lesion. The transplant is in the middle, with caudal host cord on the left and sacral host spinal segments
on the right. The white bar represents 5 mm.

124

125
(Figure 3-2). Within the graft, neurons were present and showed an adult
morphology (Figures 3-2c and 3-4c). Integration of host and graft tissue was
evaluated at the rostral and caudal interfaces (Figures 3-3b, 3-3d, and 3-4b).
Transverse sections through the transplant and host spinal cord showed that the
fetal tissue could form a continuous bridge between the host spinal cord at the
rostral and caudal ends of the transection cavity (Figure 3-4). Lack of a definable
glial scar and a normal appearance of the host ventral horn suggested the
possibility that a transplant could interact with the host spinal cord (Figures 3-3b,
3-3d, 3-4b).
Electrocutaneous Reflexes in Animals with Transplant and Transection Lesions
A post-lesion comparison was made of animals with transplants and animals
that received only a transection cavity. The values for the chronic transection-only
animals were the same as those used previously (Chapter 2). The comparisons
between transplanted (n=6) and transection-only animals (n=7) used averages from
2 or 3 sessions at the end of the testing period for each animal.
In an overall evaluation of the electrocutaneous reflex, the force of the reflex
was observed to be larger for animals with a chronic transection than for animals
with a transection and transplant. The force of the reflex increased for both groups
with higher levels of stimulus intensity (Figure 3-5). Multiple regression analysis
(Table 3-2) was performed after the inverse exponential relationship was linearized
by applying an inverse transformation to the independent variable. The multiple

Figure 3-2. Coronal section through the transplant tissue.
In two recipients of embryonic fetal tissue, coronal sections (A and B) through the
transection cavity show that it was filled by grafted embryonic tissue. Notice the
lack of any residual host cord, confirming that the subpial transection was complete.
The higher power view (C) shows the presence of differentiated neurons within a
transplant (B). The bars in A and B represents 500 microns. The bar in C
represents 50 microns.

ro

Figure 3-3. Anatomical integration of transplant tissue with host spinal cord.
Integration between host and graft tissue is shown at the caudal (A and B) and rostral interfaces (C and D) from two recipients
at 5 months post-transplantation. In the higher power views (B and D), notice the apparent lack of a glial scar and the normal
appearance of the host ventral horn neurons. The bars represent 500 microns.

129

Figure 3-4. Longitudinal sections through a transplant.
In a longitudinal section through a transplant (A), integration between host and graft tissue is shown for both the caudal
(bottom) and rostral (top) interfaces. This animal was sacrificed at 2 months post-lesion. At the rostral interface (B), notice
the integration between host (top) and graft (bottom) tissue, without a definable glial scar. A higher power view (C) shows
the presence of differentiated neurons within the transplant. The bars represent 500 microns.

131

Figure 3-5. Relationship of reflex force to stimulus intensity for animals following transection or transection plus
transplantation. Overall reflex force at 5 different stimulus intensities is shown for animals with transplants (n=6, left) and
transection-only (n=7, right). The wider smooth lines represent the average response of each group. Multiple regression
analysis found that: (1) for both groups, reflex force significantly increased with increases in stimulus intensity (P < 0.01); (2)
over this stimulus intensity range, there was not an overall difference in reflex force between the two groups of animals (P
> 0.05); but (3) the interaction between the treatments and stimulus intensity was significant (P < 0.01), indicating that
changes in reflex force with increases in stimulus intensity were different between transplant and transection-only animals.

1300
O'
1200
1100
1000
900
800
700
600
500
400
Reflex Force (N)
Intensity (mA/mm2)
133

134
Table 3-2. Summary of the multiple linear regression analysis of reflex force of
animals following transection or transection plus transplantation.
Reflex Force
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
199.21
34.86
Treatment
1
1.77E6
4.17
>0.05
0.23
Subject
11
4.67E6
30.01
<0.01
0.83
1.01
0.04
Intensity
1
5.10E5
36.05
<0.01
0.90
-1012.66
155.58
Treatment X
Intensity
1
1.10E5
7.78
<0.01
0.91
478.71
155.58
Residual
48
6.79E5
Total
62
7.74E6
35.65
<0.01
0.91
Transformations: Reciprocal of Intensity.
Normality Test: Passed.
Homoscedasticity Test: Failed (P = 0.04).

135
linear regression analysis accounted for over 90 percent of the variance and was
significant (r2 = 0.91; F = 35.65, P < 0.01). Multiple regression analysis found that:
(1) for both groups, force increased significantly with increases in stimulus intensity
(F = 36.05, P < 0.01); (2) over this intensity range, there was not an overall
difference in reflex force between the two groups of animals (F = 4.17, P > 0.05);
but (3) the interaction between the treatments and stimulus intensity was significant
(F = 7.78, P < 0.01). That is, increases in force with increases in stimulus intensity
were significantly greater for transection-only animals than for transplanted animals.
The point of intersection of the ordinal interaction was estimated be at 1.9 mA/mm2.
Reflex force for the transplant and chronic transection-only animals were
significantly different at intensities greater than 2.5 mA/mm2 (Johnson-Neyman
Technique).
An increase in peak reflex amplitude in animals with a chronic transection in
comparison to animals with transplants was found to be contributing factor for the
differences observed in reflex force between the two groups. Peak reflex amplitude
was observed to increase for both groups with higher levels of stimulus intensity
(Figure 3-6). Multiple regression analysis (Table 3-3) was performed after the
relationship was linearized by taking the reciprocal of the independent variable.
The multiple linear regression analysis accounted for over 80 percent of the
variance and was significant (r2 = 0.87; F = 23.04, P < 0.01). Multiple regression
analysis found that: (1) for both groups, peak amplitude increased significantly with
increases in stimulus intensity (F = 78.09, P < 0.01), (2) peak amplitude over this

Figure 3-6. Relationship of peak reflex amplitude to stimulus intensity for animals following transection or transection plus
transplantation. Peak reflex amplitudes at 5 different stimulus intensities are shown for animals with transplants (left) and
transection-only (right) near the time of sacrifice (>5 months). The larger smooth lines represent the average response of
each group. Multiple regression analysis found that: (1) for both groups, peak amplitude significantly increased with
increases in stimulus intensity (P < 0.01); (2) peak reflex amplitude was significantly different (P < 0.01) between animals
with transplants and transection-only over this intensity range; and (3) the possible interaction between stimulus intensity
and the treatments was not significant (P > 0.05).

Intensity (mA/mm2) Intensity (mA/mm2)
Peak Amplitude (N)
oooooooooo
ó-^k)wA.tnb)'vibo(X)
Peak Amplitude (N)
oooooooooo
O -'-MtO -UCnO-'vICDCO

138
Table 3-3. Summary of the multiple linear regression analysis of peak reflex
Peak Amplitude
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.261
0.036
Treatment
1
0.892
5.34
<0.05
0.22
Subject
11
1.838
14.96
<0.01
0.66
1.014
0.577
Intensity
1
0.872
78.09
<0.01
0.87
-1.352
0.138
Treatment X
Intensity
1
0.002
0.18
>0.05
0.87
Residual
48
0.534
Total
62
4.137
23.04
<0.01
0.87
Transformations: Reciprocal of Intensity.
Normality Test: Passed
Homoscedasticity Test: Passed.

139
intensity range was significantly different (F = 5.34, P < 0.05) for transplant and
transection-only animals, and (3) the possible interaction between stimulus intensity
and the treatments was not significant (F = 0.18, P > 0.05).
Along with the differences observed between the two groups in peak reflex
amplitude, the latency at peak reflex amplitude was observed to remain relatively
constant in transplant animals, but for animals with a lesion, latency at peak reflex
amplitude increased with higher levels of stimulus intensity (Figure 3-7). Multiple
regression analysis (Table 3-4) was performed after the relationship was linearized
by applying an inverse transformation to the independent variable and a natural log
transformation to the dependent variable. The multiple linear regression analysis
accounted for over 60 percent of the variance and was significant (r2 = 0.67; F =
9.57, P < 0.01). Multiple regression analysis found that: (1) latency at peak reflex
amplitude significantly increased with increases in stimulus intensity (F = 15.23, P
< 0.01), (2) there was a significant difference in latency at peak reflex amplitude
over this intensity range (F = 4.83, P < 0.01) for transplant and transection-only
animals, and (3) the interaction (F = 10.15, P< 0.01) between treatment and
stimulus intensity was significant. The point of intersection of the ordinal interaction
was estimated to be at 2.75 mA/mm2. Latency at peak reflex amplitude values for
the groups were significantly different at intensities greater than 4.6 mA/mm2
(Johnson-Neyman Technique).
The rise time of the reflex was measured in order to investigate a possible
contribution to observed increases in peak amplitude at higher stimulus intensities

Figure 3-7. Relationship of latency at peak reflex amplitude to stimulus intensity for animals following transection or
transection plus transplantation. Peak reflex latencies at 5 different stimulus intensities are shown for animals with
transplants (left) and transection-only (right) near the time of sacrifice (>5 months). The larger smooth lines represent the
average response of each group. Multiple regression analysis found that: (1) latency at peak reflex amplitude significantly
increased with increases in stimulus intensity (P < 0.01); (2) there was a significant difference in latency at peak reflex
amplitude over this intensity range (P < 0.01) between animals with transplants and transection-only; and (3) the interaction
between treatment and stimulus intensity was significant (P< 0.01), indicating that changes in latency at peak reflex amplitude
with increases in stimulus intensity were different for transplant and transection only animals.

Intensity (mA/mm2) Intensity (mA/mm2)
Latency at Peak Amplitude (msec)
Latency at Peak Amplitude (msec)
200 i i 200

142
Table 3-4. Summary of the multiple linear regression analysis of latency at
peak reflex amplitude of animals following transection or transection plus
transplantation.
â–  â– â–  - - - -- â–  -p
Peak Latency
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.778
0.418
Treatment
1
2.51
11.46
<0.01
0.23
Subject
11
2.41
4.83
<0.01
0.55
0.862
0.090
Intensity
1
0.69
15.23
<0.01
0.60
-1.001
0.351
Treatment X
Intensity
1
0.46
10.15
<0.01
0.67
1.228
0.351
Residual
47
2.13
Total
61
8.21
9.57
<0.01
0.67
Transformations: Reciprocal of Intensity; natural log of the dependent variable.
Normality Test: Failed.
Homoscedasticity Test: Failed.

143
and to determine whether there was a transplant specific effect. Reflex rise time
was observed to increase for both groups with higher stimulus intensities (Figure
3-8). Multiple regression analysis (Table 3-5) was performed after the relationship
was linearized by taking the inverse of the independent variable and applying a log
transformation to the dependent variable. The multiple linear regression analysis
accounted for 80 percent of the variance and was significant (r2 = 0.80; F = 13.65,
P < 0.01). Multiple regression analysis found that: (1) rise time significantly
increased with increases in stimulus intensity (F = 28.94, P < 0.01), (2) over this
intensity range, there was not an overall difference in rise time between transplant
and transection-only animals (F = 2.81, P > 0.05), but (3) the interaction between
treatment and stimulus intensity was significant (F = 15.00, P< 0.01). The point of
intersection of the ordinal interaction was estimated be at 18 mA/mm2. Rise time
values for the groups were significantly different at intensities less than 9.6 mA/mm2
(Johnson-Neyman Technique).
Two different measures of reflex duration were obtained in order to
determine the contribution of reflex duration to the reflex force estimate. It took
longer for animals with a transection lesion to reach half-maximal reflex duration
when compared to animals with a transection and transplant. Half maximal duration
increased for both groups at higher stimulus intensities (Figure 3-9). Multiple
regression analysis (Table 3-6) was performed after the hyperbola relationship was
linearized by applying an inverse transformation to both the independent and
dependent variables. The multiple linear regression analysis accounted for over

Figure 3-8. Relationship of reflex rise time to stimulus intensity for animals following transection or transection plus
transplantation. Reflex rise times at 5 different stimulus intensities are shown for animals with transplants (left) and
transection-only lesions (right) near the time of sacrifice (>5 months). The larger smooth lines represent the average
response of each group. Multiple regression analysis found that: (1) reflex rise time significantly increased with increases
in stimulus intensity (P < 0.01); (2) over this intensity range, there was not an overall difference in the reflex rise time between
transplant and transection-only animals (P > 0.05); but (3) the interaction between treatment and stimulus intensity was
significant (P< 0.01), indicating that changes in reflex rise time with increases in stimulus intensity were different for transplant
and transection-only animals.

Intensity (mA/mm2)
Rise Time (dynes/sec)
OOOOO-*—‘NJMMM
bkj'*.b)bobKjj^b)bobroj^b)
Rise Time (dynes/sec)
N)
CO
â– t*.
cn
Oi
â– nI
oo
co
o
OOOOO-^^^-^^NJKJNJNi
bk)^.bbobN)^.bbobk>J».b
-I I- 1 1 1 1 1 i 1—I 1 1 1
SH

146
Table 3-5. Summary of the multiple linear regression analysis of reflex rise time
of animals following transection or transection plus transplantation.
Rise Time
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.102
0.028
Treatment
1
0.142
2.81
>0.05
0.12
Subject
11
0.555
10.66
<0.01
0.61
0.986
0.073
Intensity
1
0.137
28.94
<0.01
0.73
-0.557
0.089
Treatment X
Intensity
1
0.071
15.00
<0.01
0.80
-0.385
0.089
Residual
49
0.232
Total
63
1.138
13.65
<0.01
0.80
Transformations: Reciprocal of Intensity, log of the dependent variable plus one.
Normality Test: Failed.
Homoscedasticity Test: Passed.

Figure 3-9. Relationship of half maximal reflex duration to stimulus intensity for animals following transection or transection
plus transplantation. Half maximal durations at 5 different stimulus intensities are shown for animals with transplants (left)
and transection-only (right) near the time of sacrifice (>5 months). The larger smooth curved lines (hyperbola functions)
represent the average response of each group. Multiple regression analysis found that: (1) half maximal reflex duration
significantly increased with increases in stimulus intensity (P < 0.01), (2) there was a significant difference (P < 0.01) between
animals with transplants and transection-only animals in half maximal reflex duration over this intensity range, and (3) the
possible interaction between stimulus intensity and the treatments was not significant (P > 0.05).

Intensity (mA/mm2)
Duration 0.5 max (msec)
Intensity (mA/mm2)
148

149
Table 3-6. Summary of the multiple linear regression analysis of half maximal
reflex duration of animals following transection or transection plus
transplantation.
Half Maximal Duration
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
-1.52E-3
5.56E-4
Treatment
1
1.26E-4
9.90
<0.01
0.28
Subject
11
1.40E-4
5.18
<0.01
0.59
0.945
0.081
Intensity
1
6.10E-5
24.81
<0.01
0.73
0.011
0.002
Treatment
X Intensity
1
3.00E-6
1.22
>0.05
0.74
Residual
48
1.18E-4
Total
62
4.48E-4
9.59
<0.01
0.74
Transformations: Reciprocal of Intensity; reciprocal of the dependent variable.
Normality Test: Failed (P = 0.017)
Homoscedasticity Test: Passed.

150
70 percent of the variance and was significant (r2 = 0.74; F = 9.59, P < 0.01).
Multiple regression analysis found that: (1) half maximal reflex duration increased
significantly with increases in stimulus intensity (F = 24.81, P < 0.01), (2) there was
a significant difference (F = 9.90, P < 0.01) between groups in half maximal reflex
duration over this intensity range, and (3) the possible interaction between stimulus
intensity and the treatments was not significant (F = 1.22, P > 0.05).
One quarter maximal reflex duration values were not significantly different
between transplant animals and transection animals, partially as a consequence of
the variability observed across animals. One quarter maximal duration increased
for both groups with higher stimulus intensities (Figure 3-10). Multiple regression
analysis (Table 3-7) was performed after the relationship was linearized by taking
the reciprocal of the independent variable and performing a natural log
transformation of the dependent variable. The multiple linear regression analysis
accounted for over 80 percent of the variance and was significant (r2 = 0.82; F =
15.51, P < 0.01). Multiple regression analysis found that: (1) for both groups
quarter maximal duration increased significantly with increases in stimulus intensity
(F = 33.37, P < 0.01), (2) over this range of stimulus intensities there was not an
overall difference in quarter maximal duration between the groups (F = 3.44, P >
0.05), and (3) the possible interaction between stimulus intensity and the treatments
was not significant (F = 0.09, P > 0.05).
Comparable reflex latencies were observed in transplant animals and chronic
transection animals. The latency of reflex onset decreased for both groups at

Figure 3-10. Relationship of quarter maximal reflex duration to stimulus intensity for animals following transection or
transection plus transplantation. Quarter maximal reflex durations at 5 different stimulus intensities are shown for animals
with transplants (left) and transection-only (right) near the time of sacrifice (>5 months). The larger smooth lines (inverse
exponential functions) represent the average response of each group. Multiple regression analysis found that: (1) for both
groups, quarter maximal reflex duration significantly increased with increases in stimulus intensity (P < 0.01); (2) over this
stimulus intensity range, there was not an overall difference in quarter maximal reflex duration between the two groups (P
> 0.05); and (3) the possible interaction between stimulus intensity and the treatments was not significant (P > 0.05).

3500
3250
3000
2750
2500
in
¿ 2250
| 2000
cn 1750
d
g 1500
Intensity (mA/mm2)
Duration 0.25 max (msec)
Intensity (mA/mm2)

153
Table 3-7. Summary of the multiple linear regression analysis of quarter
maximal reflex duration of animals following transection or transection plus
transplantation.
Quarter Maximal Duration
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
0.590
0.428
Treatment
1
9.57
3.44
>0.05
0.16
Subject
11
30.63
12.73
<0.01
0.69
0.985
0.065
Intensity
1
7.30
33.37
<0.01
0.82
-2.354
0.368
Treatment X
Intensity
1
0.02
0.09
>0.05
0.82
Residual
48
10.48
Total
62
58.00
15.51
<0.01
0.82
Transformations: Reciprocal of Intensity minus one; natural log of the dependent
variable.
Normality Test: Passed.
Homoscedasticity Test: Passed.

154
higher stimulus intensities (Figure 3-11). Multiple regression analysis (Table 3-8)
was performed after the relationship was linearized by applying a log transformation
to the independent variable. The multiple linear regression analysis accounted for
over 70 percent of the variance and was significant (r2 = 0.74; F = 9.70, P < 0.01).
Multiple regression analysis found that: (1) for both groups latency decreased
significantly with increases in stimulus intensity (F = 25.50, P < 0.01), (2) there was
not an overall difference between the groups in reflex latency (F = 0.12, P > 0.05),
and (3) the possible interaction between stimulus intensity and the treatments was
not significant (F = 0.79, P > 0.05).
Discussion
The present study was undertaken to determine whether transplantation of
fetal spinal cord tissue at the time of sacrocaudal transection can modulate the
cutaneous hyperreflexia that develops following transection without transplantation.
The presented results show that fetal tissue can survive, differentiate and integrate
with the rostral and caudal host spinal cord. Cutaneous reflexes were affected by
the transplantation procedure, and key facets of cutaneous hyperreflexia were
absent in animals with transplant.
Characteristics of Reflexes Following Chronic Spinal Lesions with Transplants
The comparison of electrocutaneous reflexes chronically after transection
and after transection plus transplantation provided a means to evaluate the utility
of the transplantation regimen in the treatment of cutaneous hyperreflexia. This

Figure 3-11. Relationship of reflex latency to stimulus intensity for animals following transection or transection plus
transplantation. Reflex latency estimates at 5 different stimulus intensities are shown for animals with transplants (left) and
transection-only (right) near the time of sacrifice (>5 months). The larger smooth lines (inverse exponential functions)
represent the average response of each group. Multiple regression analysis found that: (1) for both groups, reflex latency
significantly decreased with increases in stimulus intensity (P < 0.01); (2) there was not an overall difference in reflex latency
between the two groups (P > 0.05); and (3) the possible interaction between stimulus intensity and the treatments was not
significant (P > 0.05).

Intensity (mA/mm2) Intensity (mA/mm2)
Latency (msec)
Latency (msec)

157
Table 3-8. Summary of the multiple linear regression analysis of reflex latency
of animals following transection or transection plus transplantation.
Reflex Latency
Source
DF
SS
F
P
R2
Equation
Coefficient
Std.
Error
Constant
5.091
1.783
Treatment
1
3.07
0.12
>0.05
0.01
Subject
11
286.63
9.84
<0.01
0.60
0.940
0.086
Intensity
1
67.50
25.50
<0.01
0.74
-5.316
0.965
Treatment X
Intensity
1
2.10
0.79
>0.05
0.74
Residual
47
124.40
Total
61
483.70
9.70
<0.01
0.74
Transformations: Log of Intensity.
Normality Test: Failed.
Homoscedasticity Test: Passed.

158
comparison has clinical relevance since cutaneous hyperreflexia of the tail after a
sacrocaudal transection is similar to the reflex abnormalities observed in humans
with spinal cord injuries (Chapter 2).
In general, the transplantation of fetal tissue into the transection cavity
reduced the level of cutaneous hyperreflexia observed after a transection only
sacrocaudal lesion (Table 3-9). A difference in reflex gain between transplant and
transection-only animals was observed for reflex force. The tails of animals with a
transection-only lesion exhibited greater increases in magnitude at higher stimulus
intensities than did animals with transplants. Transplant animals also exhibited
significantly smaller peak amplitudes and shorter peak reflex latencies than
transection-only animals. These differences between transplant and transection-
only animals were similar to the differences observed between pre- and chronic
post-lesion sacrocaudal transection animals (Chapter 2). The differences observed
in rise time and half maximal duration between transplant and transection-only
animals were also similar to the differences observed between pre-lesion and
chronic transection-only animals.
Distinctions existed between the comparisons of transplant with transection-
only animals and chronic transection animals with pre-lesion values. For instance,
for quarter maximal duration, a significant difference was not obtained in
comparisons between transplant and chronic transection-only animals. In contrast,
quarter maximal duration was significantly elevated for chronic transection-only
animals, relative to pre-lesion values (Chapter 2). Similar comparisons were

159
Table 3-9. Summary of the consequences of transection plus transplantation in
comparison to the transection-only lesion on the response characteristics of the
electrocutaneous reflex.
Overall Change3 Interaction1*
Reflex Force
None
Yes
Peak Amplitude
Decrease
No
Latency at peak
reflex amplitude
Decrease
Yes
Duration (1/4 max)
None
No
Duration (1/2 max)
Decrease
No
Rise Time
None
Yes
Latency
None
No
aAn overall change indicates that over this stimulus intensity range there was a significant
difference between the two groups irrespective of stimulus intensity.
bAn interaction indicates a significant change in the intensity-response relationship
between the two groups.

160
observed for reflex latency where a significance difference was not found between
transection and transection with transplant animals. These results suggest that
some features of the electrocutaneous reflex were not affected by the presence of
transplant tissue.
Mechanisms for the Functional Consequences of Transplantation
A functional role for transplantation in modulation of electrocutaneous
reflexes is suggested by the alterations observed in cutaneous reflexes of animals
with transplants when compared to animals with only a transection lesion. In data
not shown, it was observed that the cutaneous hyperreflexia failed to develop even
at short time periods after the sacrocaudal lesion with transplantation. This
contrasts with the progressive development of hyperreflexia for animals with a
transection-only lesion (Chapter 2). This suggests that early influences of the
transplant may prevent a gradual progression of secondary neural degeneration
that is triggered by spinal trauma which may lead to the development of the
cutaneous hyperreflexia. Alternatively, the continued reduction of reflex excitability
may reflect a gradual process of maturation and integration of the graft into the host
tissue. Inhibitory influences on segmental reflexes have been observed for fetal
transplants placed in contusion lesions (Thompson et al., 1993). Anatomical
studies have shown the ability of transplants to rescue neurons (Bregman and
Reier, 1986); however, a long-term need for trophic support of host neurons
following spinal trauma has not been examined specifically.

161
It is conceivable that cutaneous hyperreflexia was not present at chronic
post-transplantation time periods because of neuronal degeneration of host
segmental circuitry, produced by encroachment into host tissue of the developing
fetal transplant. In apparent support of this possibility, the reflexes of transplanted
animals in the late periods of testing were in some respects similar to the reflexes
of 2 animals with lesions that involved caudal segmental circuitry (Chapter 2).
However, suprathreshold levels of electrocutaneous stimulation (e.g., 20 mA/mm2)
augmented the reflexes of animals with transplants (data not shown), but not the
reflexes of the two animals with lesions involving the sacrocaudal segments that
were tested. Also, anatomical reconstructions of the lesions did not provide
evidence for extensive caudal growth of the transplants.
The immunosuppressive drug, Cyclosporine A, might have contributed to the
functional consequences of transplantation on electrocutaneous reflexes, since
lesion-only animals did not receive Cyclosporine A. As an immunosuppressive
drug, Cyclosporine A inhibits T-cell proliferation; however, this agent also can affect
neurons directly. In cell culture, axonal elongation is prevented by the action of
Cyclosporine A on a protein called Calcineurin (Ferreira et al., 1993). In addition,
Cyclosporine A can inhibit MPTP neurotoxicity toward nigrostriatal dopaminergic
neurons (Kitamura et al, 1994). However, with respect to spinal cord injury, no
histological changes due to immunosuppressive drug treatments have been
observed at lesion sites (Feringa et al., 1975). Also, an effect of Cyclosporine A on

162
locomotion was not observed in a chronic transplant paradigm (Anderson et al.,
1991).
The finding that transplant tissue can modulate cutaneous hyperreflexia after
spinal cord injury complements other studies suggesting that transplantation of fetal
tissue can alter locomotion in adult animals with spinal injuries (Anderson, et al.,
1991; Kunkel-Bagden et al., 1992; Reier et al., 1992a,b; Stokes and Reier, 1992).
The time-course of improvement in locomotor behaviors in these studies was similar
to the time-course observed here for modulation of cutaneous reflexes. This
suggests that, at least in part, an attenuation of abnormal reflexes contributes to
improved recovery of locomotion. Improvement in locomotor behaviors concomitant
with changes in reflex behaviors has been noted following incomplete spinal cord
lesions (Kato et al., 1985).
Mechanisms for the effects of fetal spinal transplants on the segmental
circuitry controlling cutaneous reflexes have not been identified. Early after
surgery, the transplant might excrete trophic factors that rescue neurons
susceptible to secondary degeneration. Progressive secondary degeneration of
host tissue following a lesion may be responsible for the gradual development of
spasticity following spinal cord injury. Later on, the transplant might act by
synaptically integrating with the segmental circuitry or with propriospinal neurons
that modulate spinal reflexes. Additionally, the transplant might serve as a
functional neural bridge, conveying descending influences or regulatory signals to
spinal levels caudal to the injury. Further studies are needed to evaluate the

163
mechanisms for the modulatory effects of transplants that have been demonstrated
in the present study of spinal reflexes.

CHAPTER 4
OVERALL DISCUSSION
The present study documented effects of sacrocaudal transection on
cutaneous reflexes of the tail. Cutaneous hyperreflexia developed after
sacrocaudal transection, unless allografts of fetal tissue were placed into the
transection cavity. The presence of graft tissue attenuated development of
cutaneous hyperreflexia of the tail that was observed chronically after complete
transection without transplantation. Questions remain as to the neurological basis
of hyperreflexia from spinal lesions and mechanisms underlying the beneficial
effects of transplant tissue.
Neural mechanisms underlying spasticity after a human spinal cord injury are
not completely understood (Ashby and McCrea, 1987; Katz and Rymer, 1989). The
question of mechanisms underlying injury induced hyperreflexia is complicated by
finding that not all neurological lesions produce uniform alterations in stretch
reflexes that make up part of the spastic syndrome (Berger et al., 1984; Mailis and
Ashby, 1990). But for cutaneous reflexes, a more uniform hyperreflexia develops
for a number of different neurological lesions the produce spasticity (Meinck et al.,
1985).
164

165
Characteristics of the cutaneous flexion reflex change in humans after spinal
cord injuries. Instead of the normal short latency local response to a cutaneous
stimulus with reciprocal inhibition of other muscle groups (Hagbarth, 1952;
Hagbarth and Finer, 1963), after a spinal cord injury, the flexion reflex is of greater
magnitude, of longer duration and the reflex activity radiates to distant muscle
groups. Additional changes in the electrocutaneous flexion reflex include a lowered
reflex threshold, an increase in reflex latency, and the presence of a long latency
reflex (Dimitrijevic and Nathan, 1967; Pedersen, 1954; Roby-Brami and Bussel,
1987; Roby-Brami et al., 1989; Shahani and Young, 1973). Chronically after the
sacrocaudal transection lesion, similar changes in cutaneous reflexes were
observed as have been seen for animals after other spinal transection lesions
(Afelt, 1970; Ranson and Hinsey, 1930).
A parsimonious explanation to account for the changes in cutaneous reflexes
is to propose that reflexive responses are being generated by a different segmental
pathway after spinal cord injury. Electrophysiological animal studies have shown
that cutaneous stimuli, as well as proprioceptive afferents from muscles and joints,
evoke common reflex pathways that lead to ipsilateral flexion and crossed extension
(Lundberg, 1979). The polysynaptic pathways for these flexor reflex afferents
(FRAs) might have an active role in motor control, in addition to their role in
nociceptive reflexes, since they are modulated by various supraspinal motor
centers. The neural circuits mediating FRA reflexes have been categorized into
short-latency and long-latency pathways (Baldissera et al., 1981). Cutaneous

166
reflexes characterized in normal animals are thought to be mediated by short-
latency FRA pathways which evoke short-latency polysynaptic EPSPs in flexor and
IPSPs in extensor motoneurones, with reciprocal inhibition. The long-latency FRA
pathways occur at longer latencies with long durations and have a higher threshold
than the short-latency FRA pathways to afferent stimulation. With the administration
of DOPA (3-4 dihydroxyphenylalenine), short-latency FRA pathways are inhibited
and long-latency FRA pathways produce a response that occurs at even longer
latencies and consists of a sustained discharge that can last for several hundred
milliseconds (Anden et al., 1966a).
The long-latency FRA pathway, that can be observed in the acute spinal
preparation treated with DOPA, is thought to mediate hyperreflexive cutaneous
responses after spinal cord injury (Roby-Brami and Bussel, 1987; Roby-Brami et al.,
1989). Direct support for this mechanism comes from a study that observed
modulation of soleus H reflexes in human spinal cord injured subjects with
characteristics that would be expected if cutaneous reflexes were being mediated
by the long-latency FRA pathway (Roby-Brami and Bussel, 1990). In support of this
idea, DOPA induced long-latency FRA reflexes are similar to electrocutaneous
reflexes of the tail after sacrocaudal spinal lesions.
The progressive nature in the development of spasticity and cutaneous
hyperreflexia, and how this segmental reorganization occurs are not answered by
proposing that cutaneous reflexes are mediated by long-latency FRAs after spinal
cord injury. In rats, prolonged afterdischarge of neurons is observed after

167
quisqualic acid lesions of interneurons of the spinal cord (Yezierski and Park,
1993). For transection-only animals, the activation of the muscles that mediate the
electrocutaneous tail reflex is of similar duration to that observed for spinal
interneurons in close proximity to quisqualic acid lesions. This prolonged
afterdischarge is thought to result from the destruction of inhibitory propriospinal
interneurons that normally modulate the response of these neurons to sensory
stimuli (Sandkuhler et al., 1991). It is possible that the functional loss of inhibitory
propriospinal interneurons disinhibits long-latency FRA pathways. Progressive
alterations in cutaneous reflexes after spinal cord injuries might result from
secondary losses of inhibitory propriospinal neurons and underlie the changes over
time in presynaptic inhibition observed after spinal cord injuries (Calancie et al.,
1993; Thompson et al., 1992).
The recent finding that fetal spinal transplants reduce the consequences of
a spinal cord lesion on presynaptic inhibition (Thompson et al., 1993) suggests a
single mechanism might be involved in the functional alterations in cutaneous
reflexes. Thus, placement of fetal tissue into an acute lesion might supply tissue
protective factors and produce effects similar to acute application of drugs like
methylprednisolone, which also have beneficial effects. It would be interesting to
know whether long term survival of transplant tissue is required for normalization
of presynaptic inhibition in injury models such as spinal contusion. Future studies
are needed to specifically examine properties of presynaptic inhibition after
sacrocaudal lesions and transplantation in order to address the idea that alterations

168
in presynaptic inhibition play a key role in the development of cutaneous
hyperreflexia after spinal cord lesions.

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BIOGRAPHICAL SKETCH
Robert M. Friedman was born on February 8, 1961, to Carolyn and Lester
Friedman in Manhassette, New York. He spent his youth in a Cleveland suburb
where he collected butterflies, coins, baseball cards, and generally played around
outdoors. After taking a college preparatory curriculum, he graduated from
Beachwood High School in 1979. Beloit College in Beloit, Wisconsin, was his next
stop, where he, after a few personal detours and a lot of chemistry courses,
received in 1984 a Bachelor of Science degree with majors in biology and
psychology. To combine his interests, his graduate training began in August of
1986 in the Department of Neuroscience at the University of Florida. There, he was
able to pursue his interests in systems neuroscience as well as study the functional
consequences of spinal cord lesions in the laboratory of Dr. Charles J. Vierck, Jr.
During his graduate training, he met and married Laura Errante, conceived Hannah
Concetta Friedman, and eventually completed his doctoral research. Future
paternal duties take place in New Haven, Connecticut, where he plans, as a
postdoctoral fellow, to study the somatosensory system in the laboratory of Dr.
Robert H. LaMotte.
180

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.
Charles
Profess
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
l/j
%
Brian Y. Cooper, Co-Chair
Associate 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 scope and
quality, as a dissertation for the degree of Qector of Philosopf
Louis A. Ritz
Associate 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 scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Christiana M. Leonard
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 scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
mn B. Munson
Vofessor 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.
Pauy^Reier
Mark F. 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, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
^
Alan C. Spector /
Associate Professor of
Psychology
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 1995
Dean, College of Medicine
Dean, Graduate School

UNIVERSITY OF FLORIDA
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