Epithelial and bone tissue mast cell populations in the female rat as influenced by calcium and vitamin D deficiencies, ...

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Title:
Epithelial and bone tissue mast cell populations in the female rat as influenced by calcium and vitamin D deficiencies, ovariectomy, and estrogen
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xiv, 169 leaves : ill. ; 28 cm.
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English
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Tesar, Rogene, 1938-
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Subjects / Keywords:
Osteoporosis   ( lcsh )
Mast cells   ( lcsh )
Rats -- Cytology   ( lcsh )
Vitamin D deficiency   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1982.
Bibliography:
Includes bibliographical references (leaves 162-167).
Statement of Responsibility:
by Rogene Tesar.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000316116
notis - ABU2907
oclc - 08556277
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AA00003446:00001

Full Text









EPITHELIAL AND BONE TISSUE MAST CELL
POPULATIONS IN THE FEMALE RAT AS
INFLUENCED BY CALCIUM AND VITAMIN D
DEFICIENCIES, OVARIECTOMY, AND ESTROGEN








BY

ROGENE TESAR


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



UNIVERSITY OF FLORIDA


1982
















Copyright 1982

by

Rogene Tesar



























to my mother

who would have thoroughly enjoyed

observing this entire experience














ACKNOWLEDGEMENTS


My sincere appreciation is expressed to Dr. John P.

Feaster, chairman of the supervisory committee, for under-

taking that position, directing my academic program, and

giving his time and expertise whenever needed. Very deep

appreciation is also expressed to Dr. Morris Notelovitz,

who served as co-chairman of the supervisory committee

and directed the research with unfailing interest and

support. These two individuals made possible the under-

taking and completion of a very important goal.

Special thanks are also extended to Dr. Clarence

Ammerman, Dr. James Dinning, and Dr. W.C. Thomas for their

willing assistance in serving on the supervisory committee.

Thanks are given to Dr. Moreland, the veterinarians,

and staff at the Department of Animal Resources who helped

with the maintenance of the laboratory animals, especially

Terry who aided me in the most unpleasant aspect. The tech-

nical advice and use of equipment and facilities offered by

Dr. David Chen are also very much appreciated. Much thanks

goes to I.C. Song for teaching me the surgical and other

skills necessary in carrying out the experiments. Specially

remembered are Pat Ruzakowski and Liu for their assistance.







Terry Ansman and staff at the Department of Pathology

are thanked for their time and efforts in teaching me the

histology techniques required to carry out the work.

Sincere thanks are given to Dr. Hans Burchardt and

staff, Department of Orthopaedics, for technical guidance in

working with skeletal tissue. The assistance and expertise

Dr. Gary Miller has provided for the biomechanical tests is

also appreciated.

Inestimable thanks are extended to John Funk, Ray Smith,

Pam Miles, John Easley, Dr. John Moore and others at the

Nutrition Lab for providing knowledge, assistance, equipment

and supplies, all necessary to complete the laboratory

analyses within the project. Closely felt is the friendship

of the graduate students there, especially Dawn, Oswaldo,

Roberto and Joshua.

Remembered also is Lynda McKenzie who assisted with the

densitometry and gave her time to listen and help. Bill

Noffsinger is thanked for all the statistical analysis

assistance. Pam Victor is thanked for the typing of this

dissertation. Her proficiency has helped immeasurably.

The cooperation and financial assistance provided by the

Department of Animal Science and The Center for Climacteric

Studies, University of Florida, in order to carry out the

research, are gratefully acknowledged. Sigma Xi, the

Scientific Research Society, has partially supported the

study by a Grant-In-Aid of Research. Appendix A contains

information relative to this grant.







Lastly, but of great importance, sincere gratitude is

also expressed to my children and husband for their patience,

interest and support, which were entirely necessary.
















TABLE OF CONTENTS

PAGE
ACKNOWLEDGEMENTS .................................. ... iv

LIST OF TABLES ........................................ ix

LIST OF FIGURES ....................................... xi

ABSTRACT ................................ .............. xii

CHAPTER

I INTRODUCTION ................................. 1

II REVIEW OF THE LITERATURE ..................... 3

Description of the Mast Cells ................. 3
Mast Cells In Bone Marrow .................... 6
Effects of Diet ............................ 9
Mast Cells in Vaginal Tissue and Influence
of Gonadal Hormones .......................... 11

III MATERIALS AND METHODS ......................... 14

Animals and Treatments ........................ 14
Analytical Methods ........................... 16
Histology ..................................... 18
Data Analysis ............................... .. 19

IV RESULTS AND DISCUSSION ........................ 21

Body Weights .. ............................... 22
Serum Calcium ................................ 25
Bone Densitometry ............................ 28
Femur lengths ........................... 28
Bone mineral content .................... 31
Bone density ............. ............. .. 33
Biomechanical Tests ............................ 35
Torque ................................. 35
Deformation ............................. 38
Bone Ash ...................................... 38


vii








Table of Contents continued

CHAPTER PAGE

Mast Cells ..................................... 41
Bone Marrow Mast Cells ..................... 41
Normal diet group ....................... 44
Calcium-deficient group ................. 44
Vitamin D-deficient group ............... 45
Calcium- and vitamin D-deficient group .. 45
Vaginal Tissue Mast Cells .................. 47
Normal diet group ...................... 47
Calcium-deficient group ................. 49
Vitamin D-deficient group ............... 49
Calcium- and vitamin D-deficient group .. 49

V CONCLUSIONS ................................. 51

APPENDICES

A SIGMA XI GRANT-IN-AID OF RESEARCH ............. 55

B LABORATORY ANIMAL USE ......................... 66

C EXPERIMENTAL ANIMAL BODY WEIGHTS ............. 70

D COMPOSITION OF EXPERIMENTAL DIETS ............. 74

E SERUM CALCIUM ANALYSIS ........................ 76

F DENSITOMETRIC BONE ANALYSIS ................... 83

G BIOMECHANICAL TESTING ......................... 96

H ASH ANALYSIS .................................... 103

I MAST CELLS ...................................... 113

REFERENCES ............. ............................... 162

BIOGRAPHICAL SKETCH .................................. 168


viii


















LIST OF TABLES


PAGE


Study Design .................................

Body Weight Gain as Affected by
Diet and Treatment ...........................
Effect of Diet and Treatment on
Serum Caclium ................................
Effect of Diet and Treatment on Densitometric
Measurements of Rat Femurs ...................
Effect of Diet and Treatment on Biomechanical
Properties of Rat Femurs .....................
Effect of Diet and Treatment on Bone Ash ......
Mast Cell Populations as affected by Diet and
Treatment in the Female Rat ..................

Experimental Body Weights: Normal Diet ........
Experimental Body Weights: -Ca Diet ...........
Experimental Body Weights: -D Diet ............
Experimental Body Weights: -Ca, -D Diet .......


Serum Calcium
Serum Calcium
Serum Calcium
Serum Calcium
Serum Calcium

Densitometric
Densitometric
Densitometric
Densitometric
Densitometric


Standards ....
Analysis Data:
Analysis Data:
Analysis Data:
Analysis Data:

Bone Analysis:
Bone Analysis:
Bone Analysis:
Bone Analysis:
Bone Analysis:


...................
Normal Diet ......
-Ca Diet .........
-D Diet ..........
-Ca, -D Diet.....

Normal Diet ......
-Ca Diet .........
-D Diet ..........
-Ca, -D Diet .....
Bone Length ......


TABLE

III.1

IV. 1

IV.2

IV. 3

IV. 4

IV.6
IV. 6


C.1
C.2
C.3
C.4


E. 1
E.2
E.3
E.4
E.5

F.1
F.2
F.3
F.4
F.5












List of Tables continued


TABLE PAGE

G.1 Biomechanical Properties: Normal Diet ......... 99

G.2 Biomechanical Properties: -Ca Diet ............ 100
G.3 Biomechanical Properties: -D Diet ............ 101
G.4 Biomechanical Properties: -Ca, -D Diet ........ 102

H.1 Bone Ashing Analysis Data: Normal Diet ........ 105
H.2 Bone Ashing Analysis Data: -Ca Diet ........... 107
H.3 Bone Ashing Analysis Data: -D Diet ............ 109
H.4 Bone Ashing Analysis Data: -Ca, -D Diet ....... 111
I.1 Mast Cell Count--Vaginal Tissue: Normal Diet .. 113
1.2 Mast Cell Count--Vaginal Tissue: -Ca Diet ..... 117
1.3 Mast Cell Count--Vaginal Tissue: -D Diet ...... 122
1.4 Mast Cell Count--Vaginal Tissue: -Ca, -D Diet 126
1.5 Mast Cell Count--Bone Marrow: Normal Diet ..... 129
1.6 Mast Cell Count--Bone Marrow: -Ca Diet ........ 133
1.7 Mast Cell Count--Bone Marrow: -D Diet ......... 138
1.8 Mast Cell Count--Bone Marrow: -Ca, -D Diet .... 142
















LIST OF FIGURES


FIGURE PAGE

IV.1 Body Weight Gains in Female Rats .............. 24

IV.1 Femur Length by Photonabsorptiometry .......... 30

IV.3 Bone Mineral Content of Rat Femurs ............ 32

IV.4 Linear Bone Density of Rat Femurs ............ 34

IV.5 Torque Required for Fracture .................. 37

IV.6 Femoral Deformation Prior to Fracture ......... 40

IV.7 Bone Marrow Mast Cell Populations in
the Female Rat ............................... 43

IV.8 Vaginal Tissue Mast Cell Population in
the Female Rat .............................. 48
F.1 The Norland Digital Bone Densitometer ......... 86
F.2 Printout Display of Rat Femur Profile ......... 86

G.1 The Rapid Loading Torsional Testing Machine ... 98

G.2 Representative Torque-Deflection Curves ....... 98

I.1 Photomicrograph of Mast Cells--Bone Marrow:
Normal Diet .................................. 147
1.2 Photomicrograph of Mast Cells--Bone Marrow:
-Ca Diet ..................................... 149
1.3 Photomicrograph of Mast Cells--Bone Marrow:
-D Diet ...................................... 151
1.4 Photomicrograph of Mast Cells--Bone Marrow:
-Ca, -D Diet ................................ 153
1.5 Photomicrograph of Mast Cells--Vaginal Tissue:
Normal Diet ................................... 155
1.6 Photomicrograph of Mast Cells--Vaginal Tissue:
-Ca Diet ..................................... 157
1.7 Photomicrograph of Mast Cells--Vaginal Tissue:
-D Diet ...................................... 159
1.8 Photomicrograph of Mast Cells--Vaginal Tissue:
-Ca, -D Diet .............................. ... 161















Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


EPITHELIAL AND BONE TISSUE MAST CELL POPULATIONS IN THE
FEMALE RAT AS INFLUENCED BY CALCIUM AND VITAMIN D
DEFICIENCIES, OVARIECTOMY AND ESTROGEN


By

Rogene Tesar

May 1982


Chairman: Dr. J.P. Feaster
Cochairman: Dr. Morris Notelovitz
Major Department: Animal Science


Effects of dietary deficiencies on mast cell popula-

tions in the bone marrow and vaginal epithelial tissue of

rats were investigated. Additionally, effects of exogenous

estrogen, bilateral ovariectomy, and a combination of the

two treatments on mast cell populations were observed in

these two tissues in rats on normal and deficient diets.

Two-month old, female Sprague-Dawley rats were fed cal-

calcium- and vitamin D-deficient diets for five weeks. One

group of rats was given estradiol by injection (100 pg/.l ml)

three times a week for the duration of the experiment, another

group was ovariectomized, a third group received both treat-

ments, while a fourth, untreated group served as a control

group.


I








Dietary-induced osteopenia was evidenced by densitomet-

ric measurements and bone ash of the rat femur. Significant

decreases in bone mineral content (P<.005) due to diet were

observed. Bone ash values were also significantly low due

to diet (P<.0005).

Femur length, measured by photonabsorptiometry, was

found to be decreased due to (a) dietary deficiencies of

calcium and vitamin D (P<.005); (b) estrogen administration

(P<.005); and (c) ovariectomy (P<.005).

Contrary to expectations, bone marrow mast cell popula-

tions were not altered by dietary deficiencies in untreated

rats. This result may be due to the age of the rat when

begun on the deficient diet (two months old) and to the

duration of the imposed diet deficiencies (five weeks).

Dietary deficiencies reduced marrow mast cell counts in

estrogen treated rats, however (P<.05). Ovariectomy induced

a reduction of mast cells in the bone marrow of calcium- and

vitamin D-deficient rats (P<.01), which suggests that bone

is an estrogen sensitive tissue even though estrogen recep-

tors are not present in bone.

Vaginal tissue mast cells were not significantly

altered in number by dietary deficiencies except when rats

were estrogen treated (P<.05). The most significant finding

was that of mast cell population increases in vaginal tissue

as a result of ovariectomy (P<.01) in all dietary groups.


xiii









The removal of endogenous estrogen by ovariectomy in

the female rat was found to affect both bone marrow and

vaginal tissue mast cell populations. The relevance of this

finding remains to be determined.


xiv














CHAPTER I
INTRODUCTION


Rapid bone loss with resultant osteoporosis affects 25

to 30% of all postmenopausal women (Urist, 1971) and about

75% of women who have undergone a bilateral ovariectomy. The

significance of this condition is associated not only with

pathological fractures and potential invalidism but also with

the premature death of these women. The early diagnosis of

this condition and measurement of treatment response present

two problem areas in clinical management. To date there is

no universally applicable, noninvasive method for detecting

the early stages of rapid bone loss in humans.

Research has shown that mast cells, found in increased

numbers in the bone marrow of osteoporotic and calcium-defi-

cient subjects, accompany local loss of bone mass. The

present study seeks to determine whether vaginal epithelial

mast cell populations, as well as bone marrow populations are

affected by dietary deficiencies of calcium and vitamin D,

exogenous estrogen, and ovariectomy in the female rat.

Should such alterations be found in the female rat, similar

changes in populations might be expected in the osteoporotic

human female. Tissue biopsy of vaginal epithelium to deter-

mine mast cell populations could provide a relatively nonin-

vasive method for detecting osteomalacia and conditions








involving increased bone resorption such as osteoporosis.

The procedure might also be used to assess treatment

efficacy.

Therefore, two hypotheses were tested in this

investigation:

(a) Dietary calcium- and vitamin D-deficiencies produce

changes in mast cell populations in female rat bone

marrow and vaginal epithelium.

(b) Exogenous estrogen, bilateral ovariectomy, or a

combination of the two treatments alters mast cell

populations in bone marrow and vaginal tissue of

calcium- and vitamin D-deficient female rats.

Other parameters considered and investigated in this

study were:

(a) weight changes of the laboratory animals during the

experimental period

(b) serum calcium concentration

(c) bone mineral content, density, and length

(d) torsion and deformation of the rat femurs

(e) ash content of the femurs.


L













CHAPTER II
REVIEW OF THE LITERATURE


Addressed in the literature review are a description of

the mast cell and the activity of two constituents, histamine

and heparin. Research involving the mast cell in bone marrow

of the rat and human is reviewed. Effects of dietary defi-

ciencies, specifically those of calcium and vitamin D, on

the rat mast cell in bone tissue are examined. Also cited

are the few studies concerning mast cells in vaginal tissue

and those relating to the effects of gonadal hormones on

skeletal and vaginal tissues.


Description of the Mast Cell

Naming the specialized histiocytes as mast cells has

been credited to Paul Ehrlich when he suggested, in 1877,

that the cells arose from connective tissue cells which had

been well-fed, or mastedd' (Wilhelm et al., 1978). The German

masten (to feed) or mastzellen (mast cells), from which the

name originated, appropriately describes their usually full

appearance. This is due to a high content of cytoplasmic

granules.

Pathak and Goyal (1973) state that two separate types

of mast cells occur (1) large spindle shaped, fusiform or

cylindrical cells with or without elongated processes and

(2) small, round, or elliptical cells. Riley (1959) also








described differences in rat mast cells and distinguished

between two types based on granule maturation. Differences

also exist when mast cells are examined with electron micro-

scopy (Combs et al., 1965).

Rat peritoneal fluid, the most common source of mast

cells for laboratory investigation, exhibits round mast cells,

13.5 to 17 pm in diameter, each with a round or oval nucleus.

Cytoplasmic granules are approximately .7 pm in diameter

(Yong et al., 1975).

In an extensive quantitative analysis of rat mast cell

structure, Helander and Bloom (1974) report an average mast

cell diameter as 11 pm, granule diameter as .78 pm, with the

nucleus occupying 10.7% of the cell. The size, shape, stain-

ing properties, and distribution of mast cells vary with the

tissue and species of animal studied (Wilhelm et al., 1978).

Rat tissues abound in mast cells, whereas tissues of the

rabbit contain related basophils often referred to as blood

mast cells. Tissues in man and the guinea pig exhibit both

mast cells and basophils.

A well-known characteristic of both mast cell and baso-

phil leukocyte granules is an exhibition of metachromasia

upon treatment with certain basic dyes such as toluidine

blue, methylene blue, alcian blue, and azure A. These

stains are used to identify and demonstrate the mast cells

in tissues. Since many differences in mast cell reactivity

towards these dyes occur within and between species, an exten-

sive investigative interaction among histological studies,

identification of cell constituents, and physiological








functions of the mast cells exists. The constituents of the

mast cell granules, specifically the acidic mucopolysaccha-

rides, are responsible for the various staining properties

of the cells. However, partly because of the chemical diver-

sity of the granular contents, mast cell functions in health

or disease remain an enigma.

In 1937, the metachromatic component of the mast cell

was reported to be heparin (Jorpes et al., 1937); and since

then, the mast cell in hepatic tissue has been considered as

the only endogenous source of heparin production (Riley, 1962).

A protein matrix is thought to bind anionic heparin in the

mast cell granule, possibly by sulfate groups. Heparin, in

turn, binds histamine and other basic nitrogen-containing

compounds such as serotonin (Schubert, 1968). Histamine

levels in tissues correlate with the mast cell count and a

very large proportion of rat histamine formation takes place

in the bone marrow.

The close histological relationship of mast cells and

blood and lymph vessels is well-known. Small vessels are

the prime target of histamine-mediated inflammatory reactions.

Histamine is known to cause contraction of part of smooth

muscle (mainly the bronchioles), dilate blood capillaries,

and increase their permeability (Rahima and Soderwall, 1977).

In-vitro laboratory studies of the mast cells (in cer-

tain tissues and species) demonstrate that histamine is

released by liberators such as stilbamidine, 48/80, or pro-

tamine sulfate by displacing the heparin-bound histamine

(Schubert, 1968).







Sudden degranulation of mast cells may cause adverse

reactions since large amounts of histamine are released into

the extracellular space. This release usually occurs in

response to a type I antigen-antibody reaction on the surface

of mast cells that have been previously sensitized by cell-

bound Ig E antibody (Coombs and Gell, 1975). Allergic rhin-

itis, allergic asthma, urticaria, angioedema, and mastocyto-

sis constitute some manifestations of extensive degranulation.

An editorial by Kaliner (1979) details this aspect of the mast

cell's varied activities.


Mast Cells in Bone Marrow

In addition to its role in immediate hypersensitivity

reactions, histamine has been reported to affect bone remodel-

ing and maturation (Norton et al., 1969). Systemic mastocyto-

sis in urticaria pigmentosa has been accompanied by marked

bone remodeling, bone hypertrophy (Sagher et al., 1956), and

osteosclerosis (Kruse et al., 1973).

In this same regard, the acid glycosaminoglycan, heparin,

in addition to its anticoagulant property, has been considered

as a bone resorbing and osteoporosis producing agent. This is

especially true with the use of high doses of the anticoagu-

lant for long periods of time (Goldhaber, 1965; Griffith et

al., 1965; Jaffe and Willis, 1965; Wise and Hall, 1980).

Heparin has been reported to stimulate bone collagenase activ-

ity in the rat (Asher and Nichols, 1965) and to potentiate the

action of parathyroid hormone (Goldhaber, 1965), suggesting

inducement of osteoporosis. A review of the relationship








between mast cells, heparin, and osteoporosis has been

provided by Hegsted (1969).

Many reports of heparin use with resulting skeletal

problems involved the use of heparin for control of blood

coagulation. It has been documented that heparin extracted

and purified from tissues rich in mast cells and reinjected

by the physician behaves differently from endogenous heparin

(Jaques et al., 1977). Only in the dog has the hepatic re-

lease of heparin been shown to have a rapid anticoagulant

effect in the circulation. Several species have no heparin

in the mast cell; its metachromasia is attributed to other

sulfated mucopolysaccharides.

Osteoporosis has been viewed by some as a sequel to

diminished blood flow through the marrow (Burkhardt, 1973).

Contracted arterioles in mastocellular lesions of the bone

marrow in human osteoporotics provide evidence for this

concept (te Velde et al., 1978).

Increased numbers of bone marrow mast cells have been

reported in osteoporosis (Frame and Nixon, 1968; Kruse et

al., 1973; Peart and Ellis, 1975). Two theories have been

have been expressed (1) mast cells induce porosis (Frame

and Nixon, 1968) and (2) mast cells oppose the porosis

(Kruse et al., 1973).

Increased mast cell numbers have been associated with

bone resorption in regenerating parts of the marrow. Gillman

(1958) noted increased mast cells in long bone marrow of rats

fed sweet pea seeds containing a lathyrogenic agent. He








distinguished between newly formed and old femoral shafts,

with the increased number found in the marrow of the newly

formed shaft. Severson (1969) showed that mast cells secrete

a factor necessary for hydrolytic enzyme release in regions

of increasedresorption and remodeling. Walker (1970) reported

an eight-fold increase of mast numbers in regenerating rat

femoral marrow after mechanical disruption when compared to

the unoperated contralateral femur. Hypophysectomy resulted

in an even greater increase and longer effect.

Extensive studies in bone repair (Lindholm et al., 1967,

1969) demonstrated active involvement by mast cells, increased

mast cell numbers in callus formation, and mast cell provision

of alkaline phosphatase, phosphorylase, and other enzymes es-

sential for endochondral ossification.

Human alveolar bone resorption in chronic periodontal

disease is associated with increased mast cell counts in

gingival tissue (Shapiro et al., 1969; Riley, 1959; Sognnaes,

1965). Other investigators (Carranza and Cabrini, 1955;

Calonius, 1960; Dummet et al., 1961) failed to confirm this

finding.

In hyperparathyroidism, both the resorption and the for-

mation of bone are stimulated, but greater increases in bone

resorption occur (Bonucci et al., 1978). Mast cells have

been reported in fibrotic marrow spaces in human hyperpara-

thyroid patients. Other researchers (Rockoff and Armstrong,

1970) found that low doses of parathyroid hormone chronically

administered to rats produced mast cell hyperplasia in the








tibial metaphyseal marrow, without alterations of serum

calcium or phosphorous.

Secondary hyperparathyroidism is known to result from

lowered serum calcium levels and is thought to be a mechanism

whereby low dietary calcium intakes promote mast cell in-

creases in bone.


Effects of Diet

As early as 1922, increase of tissue basophils in the

immediate vicinity of the bone trabeculae and marked resorp-

tion of bone in rats on calcium deficient diets were reported

(Shipley and Park, 1922). Urist and McLean (1957) identified

those basophils as mast cells. They also maintained rats on

low calcium, low vitamin D, and high phosphorus diets which

produced rickets, osteoporosis, and osteitis fibrosa as well

as increased endosteal mast cells. Cass et al. (1958) con-

firmed the results of increased bone mast cells in rats fed

calcium-deficient diets and found an increase in bone marrow

content of histamine and 5-hydroxytryptamine, another mast

cell mucopolysaccharide. Rockoff and Armstrong (1970) also

administered a calcium-deficient but vitamin D-adequate diet

to a group of rats, with bone marrow mast cell hyperplasia

resulting in all test animals. In providing hypocalcemia-

inducing vitamin D-deficient and calcium- and vitamin D-

deficient diets to rats, Rasmussen (1972) observed signifi-

cant increases in tibial metaphyseal bone marrow mast cells.

Parathyroidectomy caused a significant reduction in mast

cells, again suggesting secondary hyperparathyroidism as a








mechanism for increased mast cell populations in bone. Other

rats given low calcium and high phosphorus diets with and

without vitamin D exhibited hypocalcemia, rachitic bone

changes, increased bone resorption and increased mast cells

in metaphyseal areas of long bone but not in the epiphyses or

caudal vertebrae (Feik and Storey, 1979); however, it was not

possible to relate the mast cell increases to specific areas

of bone formation or resorption, as had been planned.

In dietary calcium- and vitamin D-deficient rats with

induced fracture callus, mast cell counts in the callus

approximated 200 to 300 cells per mm2 which rose to 1,900

cell per mm2 until 35 days after fracture. These mast

cells were mostly degranulated. Normal rats exhibited

strongly granulated mast cells, 2,000 to 4,000 mm2 for the

first two-month period with remarkably decreased levels

thereafter. Mast cell numbers were correlated with mineral-

ization after fracture (Lindholm et al., 1972).

Accumulations of mast cells in healing sockets or

extracted mandibular first molars were found in rats fed

calcium-deficient diets, with control rats exhibiting only

an occasional mast cell (Smith, 1974). Besides the fact that

mast cell numbers were examined in different bones in the two

studies, the contradictory findings were not explained.

Other dietary deficiencies have also affected mast cell

populations in bone marrow. Belanger (1978) found a signif-

icant increase of bone marrow mast cells in rats on zinc








deficient diets, and also on magnesium-deficient diets (1977).

Concurrent decreases in skin mast cell numbers of the magnesium-

deficient rats agree with the findings of Bois (1962).


Mast Cells in Vaginal Tissue and Influences of Gonadal Hormones


Vaginal tissue mast cell population studies in the rat

are essentially nonexistent. Salvi (1952) found mast cells

more abundant and with greater metachromatic properties in

the mouse vagina than in the uterus. After daily estrogen

administration, adult mouse vaginal tissue revealed a con-

siderable increase in the number of mast cells (Arvy, 1955).

Westin and Odeblad (1956) also investigated the influence

of ovarian hormones on mast cells in the mouse vagina.

Darker metachromasia in the vagina than in the uterus and

difficulty in detecting granules were experienced. The con-

trol group had the highest number of vaginal tissue mast

cells and also the highest variation in number per field

examined. The estrogen treated groups had a significantly

reduced number; intermediate numbers were observed when

estrogen plus progesterone was administered. Mast cells

of the skin remained constant. The estrogen effect on the

mast cells was considered a local process within the repro-

ductive organs. Zwillenberg (1958) noted a variable occur-

rence of mast cells in the vaginal epithelium of human

subjects.

Discrepancies in results among studies may be due, in

part, to differences in estrogen dosage. Iversen (1962)








notes that, while small doses of estradiol decreased the

number of uterine mast cells in the guinea pig, prolonged

treatment with large doses had an opposite effect.

Although there are mast cell studies which investigated

the effect of gonadal hormones in various tissue (Constan-

tinides and Rutherdale, 1954; Asboe-Hansen, 1956; Johansson

and Westin, 1958; Smith and Lewis, 1958; Schiff and Burn,

1961; Kameswaren et al., 1978), few reported on bone tissue.

Belanger (1977), in his study with magnesium deprived rats,

administered large doses of testosterone to males and estra-

diol to females. This treatment depressed the mast cell popu-

lation increase in the bone marrow and moderated skin mast

cell loss.

Similarly, there is no reference in the literature con-

cerning the effect of ovariectomy on mast cells in bone or

vaginal tissue. The mice in the study of Westin and Odeblad

(1956) were all spayed so that effect of ovariectomy could

not be compared to control groups. Two studies reported

that ovariectomy has no effect on uterine mast cells in

guinea pigs or hamsters (Iversen, 1962; Harvey, 1964).

Because of the differences in mast cell populations,

structure, function and activity in various species (and

in tissues within the same species), information cannot be

extrapolated from one species to another. Because of the

various mast cell constituents and their resulting diverse

functions and actions in tissues, inconsistencies in results

will continue to be reported. However, in the recent past,





13


much new information on the mast cell has been brought forth.

The particular role of the mast cell in the pathogenesis of

the osteoporoses and other demineralizing bone diseases re-

mains in need of further investigation.














CHAPTER III
MATERIALS AND METHODS


Animals and Treatments

To monitor the care, treatment, and use of laboratory

animals at the University of Florida, the All University

Committee on the Care and Use of Laboratory Animals requires

specific information pertaining to research involving labor-

atory animals. The application requesting use of laboratory

animals for this particular research project as submitted to

the Committee and its approval are found in Appendix B.

One hundred thirty-six female Sprague Dawley rats,l

9 weeks of age and weighing approximately 180 g at the

start, were used for the research.

The Health Center Animal Resources Department, Univer-

sity of Florida, provided housing for the animals. The rats

were kept in galvanized wire cages, two to a cage, in a room

maintained constantly at 240C and 60% humidity. The rats

were weighed at least once a week for the 4 to 5 week exper-

imental period. Appendix C contains data on the animal

weights.

The animals were divided into the following dietary

groups:



Outbred laboratory Sprague-Dawley rats were obtained from
Harlan-Sprague-Dawley, Madison, WI 53711.








I. Normal (control) rats (semipurified complete diet)

II. Calcium-deficient (-Ca) rats (calcium-deficient diet)

III. Vitamin D-deficient (-D) rats (rachitogenic diet;

calcium to phosphorus ratio of 4.2:1)

IV. Calcium- and vitamin D-deficient (-Ca, -D) rats

(custom formulated calcium- and vitamin D-deficient

diet).

Appendix D contains information on dietary formulations.

Each dietary group consisted of 32 rats, except for group

II, in which there were 40. The diets (pelleted) and dis-

tilled water were offered ad libitum. Group III rats were

borderline vitamin D-deficient at the beginning of the experi-

mental period. One rat in group IV was rejected because of a

malfunctioning eye.

Upon receipt of the animals and prior to further treat-

ment, each rat was tested by vaginal smear daily for an 8 day

period to determine presence of estrous cycling. Each rat

presented with at least once cycle, demonstrating reproductive

capability and ascertaining endogenous circulating estrogen.

Half the rats in each group were bilaterally ovariectom-

ized. Sham ovariectomies were performed on the remaining rats

in each group. The combination of ketamine and xylazine (Van

Pelt, 1977), at a concentration of 87 mg ketamine and 13 mg

xylazine per kg body weight of rat, was used to induce surgi-

cal anesthesia. One rat in group II died as a result of the

surgery.







One-half of the ovariectomized and one-half of the sham-

operated rats in each group were injected three times per week

under the dorsal skin with .1 ml of a solution of estradiol

valerate (100 pg/.1 ml) in sesame oil (12 injections per rat

total). Concurrently, the remaining rats in each group were

injected with .1 ml of sesame oil as a control measure.

After the five to six week experimental period, all ani-

mals were killed by decapitation.

Table III.1 summarizes the research design described.


TABLE III.1
STUDY DESIGN

Diet Group

Normal -Ca -D -Ca, -D
(N) (N) (N) (N)
Treatment

Ovariectomized + Estrogen 8 12 8 8

Ovariectomized 8 12 8 8

Estrogen 8 8 8 8

Normal (no treatment) 8 8 8 8




Analytical Methods

Blood was obtained at the time of decapitation by

exsanguination. Total calcium in serum was determined by

atomic absorption spectrophotometry (AAS). The procedure

followed was that of Fick et al. (1979). Data on serum

calcium values are found in Appendix E.








Immediately after the animals were killed, uterine and

vaginal tissues were removed, hind extremities were disarti-

culated at the acetabulum, and femurs were dissected free.

The femurs were cleaned of adherent tissues. The uterine and

vaginal tissues and femurs were fixed for 24 hours in 10%

aqueous formalin.

Bone densitometric measurementsI using direct photon

absorptiometry were made on one femur from each animal.

Values of bone mineral content in grams per centimeter length

of bone, linear bone density in grams per square centimeter,

and bone length in centimeters were obtained. Appendix F

contains information on these paremeters.

The same femurs were subjected to torque and deformation

testing2 to determine the effect of treatment modality on

these biomechanical properties. The procedure followed was

that of Puhl et al. (1972). Explanation of this testing

procedure is found in Appendix G.

Ashing of these same femurs was done as described by

Fick et al. (1979). Appendix H contains data on ash

analysis.





1Norland Digital Bone Densitometer, Model 278, Norland Cor-
poration, Ft. Atkinson, WI 53538.

2Rapid Load Torsional Testing Machine, Biomechanics Labora-
tory, Department of Mechanical Engineering, University of
Florida, Gainesville, FL 32611.








Histology

The alternate femurs of all animals were demineralized

in a 10% solution of di-sodium-ethylene-diamine-tetracetic

acid (EDTA) for a 7 to 14 day period. The solution was kept

at 50F with changes of solution every 2 to 3 days (Belanger

et al., 1965).

The demineralized femurs and vaginal tissues were dehy-

drated in 80% acetone for one-half hour and in 100% acetone

for another half-hour. Clearing was accomplished with two

changes of xylene (15 and 45 minutes); subsequently the tis-

sues were embedded in paraffin. Medial sagittal sections of

bone and cross sections of vaginal tissue were cut at 8 im

in a microtome-cryostat, floated on water, slipped onto

slides prepared with Haupt's solution, and air-dried.

Toluidine blue stain was chosen for mast cell quantita-

tion purposes (Pathak and Goyal, 1973). With this stain,

mast cells appear purple or reddish-purple against a general

blue background. The slides were subjected to the following

staining procedure:

(1) 2 changes of xylene (4 minutes each)

(2) acetone (several dips)

(3) water rinse

(4) .2% toluidine blue

(5) water rinse

(6) acetone (several dips)

(7) 2 changes of xylene (4 minutes each)








Alternate slides were stained with .1% alcian blue in

3% acetic acid for thirty minutes followed by a water rinse

and .1% safranin in 1% acetic acid for five minutes in place

of step #4 (Spicer, 1960). Using this staining procedure,

the maturity of mast cells can be determined. Analysis of

these slides is planned for a future time.

After air-drying, cover slips were applied to the slides

with mounting medium.

Mast cells were counted in vaginal tissue and in the

distal part of the femoral metaphysis and in the bone marrow

of the diaphysis. Care was taken to avoid bone trabeculae

and sinuses. Counts were made over five fields in each of

five sections (25 fields per rat) for each type tissue at

a magnification of X400. Each field measured .458 mm in
2
diameter, representing a surface area of .165 mm and a
2
total surface area of 4.1 mm for each tissue per rat.

Counts were adjusted to 1 mm2 surface area. Appendix I

contains mast cell quantitation data.


Data Analysis

The statistical evaluations for tests of significance

were carried out on the parameters using analysis of variance

and applying the t test (Steel and Torrie, 1960). The tables

list mean values and standard error of the mean (SE = s/Yn ),

and indicate level of significance.

1Labophot-Laboratory & Clinical Microscope, Nikon Instrument
Division, 623 Stewart Ave, Garden City, NY 11530.





20


Effects of diet were determined statistically by compar-

ing the means in each deficient diet group with means in the

normal diet group for each treatment.

Effects of treatment were determined statistically by

comparing means for each treatment with the mean of the

untreated rats within each diet group.














CHAPTER IV
RESULTS AND DISCUSSION


The experimental animals used in this investigation

were at least three months old at termination of the experi-

ment and regarded as young adults. Reproductive capability

was determined prior to treatments by vaginal cell sampling.

One-half the population underwent ovariectomy, thereby re-

moving the source of estrogen production.

The diet used for the vitamin D-deficient group of rats

was also notably deficient in phosphorus, with a calcium to

phosphorus ratio of 4.2 to 1. Calcium to phosphorus ratios

of all the diets are given in Appendix D.

As a matter of information, the calcium content of the

complete diet (normal diet group) was 11.5 g/kg and the

phosphorus content was 10.1 g/kg. The calcium-deficient

diet contained 1.6 g/kg calcium and 26.6 g/kg phosphorus.

The rachitogenic vitamin D-deficient diet noted as being low

in phosphorus contained 12.4 g/kg calcium and 2.9 g/kg phos-

phorus. The calcium- and vitamin D-deficient diet contained

1.6 g/kg calcium and 26.6 g/kg phosphorus (identical to the

calcium-deficient diet but with omission of vitamin D3).

In addition, the protein in the vitamin D-deficient

diet provided by whole yellow maize and as gluten, was of

poor quality, lacking in essential amino acids. Therefore,








when evaluating effects of this diet, the deficiency in

protein and phosphorus must also be considered. Effects of

diet were not due solely to lack of vitamin D.


Body Weights

Body weight changes were observed in the rats. In

Table IV.1 is recorded the average weight gain for each

cell. The % weight gain is listed below the mean. The

untreated rats fed a normal diet, and used as a control

group, increased their body weight by 34% during the

experiment. Lower weight gains were observed as due to

calcium deficiency (P <.05). The vitamin D-deficient group

also had lower weight gains as an effect of diet (P<.01).

Administration of estrogen to both intact and ovariec-

tomized rats decreased body weight gains in the normal,

calcium-deficient, and the calcium- and vitamin D-deficient

groups (P<.05 to .005). Similar effects occurred with rats

on a normal diet (Cruess and Hong, 1979).

Ovariectomy increased the weight gains in the same

three dietary groups (P<.005) (Fig. IV.1).

This supports the findings of Cruess and Hong (1979)

and Lindgren and Lindholm (1979) in that removal of ovaries

subjected rats to high increases in body weight. This

effect has been associated with a higher food intake (Aitken

et al., 1972). However, significant decreases in body

weight of young castrated male rats have been observed

(Scow, 1952; Gumbreck, 1957; Saville, 1969; Wink and Felts,

1980).


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Results indicate a gain of weight in all cells of the

study. A calcium-deficient diet and a combination protein,

phosphorus, and vitamin D-deficient diet caused rats to gain

less weight.

Estrogen inhibited the rate of weight gain, which is an

effect not well understood. This effect was seen with both

intact and ovariectomized rats. Ovariectomy clearly in-

creased weight gain and estrogen administration reduced that

gain to below the normal gain. The same pattern of this

hormonal effect was observed in the deficient diet groups.


Serum Calcium

Table IV.2 outlines the changes in serum calcium

observed in rats on four diet regimes and four treatments.

Untreated rats on a normal diet had a mean serum calcium of

9.63 mg/100 ml. Rats on deficient diets which had been

given estrogen had significantly lower serum calcium (P<.01

to .0005) than normally fed rats given estrogen. Rockoff

and Armstrong (1970) and Feik and Storey (1979) also

observed significant decreases in serum calcium with

untreated, calcium- and vitamin D-deficient rats (P<.1 to

.01).

Estrogen increased the serum calcium in the intact and

ovariectomized, normally fed rats (P<.01 to .001) on the

present study. Cruess and Hong (1979) found no consistent

change in serum calcium concentration when estrogen was

administered to intact, normally fed female rats over a 12

month period, but observed significant increases at one and

six months (P<.05).











































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Ovariectomy significantly increased serum calcium in

all dietary groups (P<.05 to .001) except in the vitamin

D-deficient group. Lindgren and Lindholm (1979) did not

observe an increase in serum calcium in normally fed,

ovariectomized rats. Others (Cruess and Hong, 1979) found

ovariectomy to significantly decrease the serum calcium

(P<.05).

Results indicate that calcium- and vitamin D-deficient

diets including deficiencies in phosphorus and protein may

not have an effect on calcium concentration in the blood.

The rat may compensate for lack of dietary nutrients by bone

resorption of calcium and phosphorus to maintain normal

blood calcium levels. Evidence for bone resorption was

found in lower bone mineral content and lower ash values in

rats deficient in calcium and vitamin D in the present study

(Tables IV.3 and IV.5).

Estrogen increased serum calcium in normally fed intact

and ovariectomized rats. However, because of conflicting

findings in the several studies mentioned, no firm conclu-

sions can be made on the effect of estrogen on serum calcium

in normal rats.

Ovariectomy effects on serum calcium have also been

noted to vary among studies so that no conclusions can be

made. Unknown factors may be influencing these two treat-

ments, which causes findings to be inconsistent.








Bone Densitometry

Femur lengths. Statistically significant differences

in femur lengths due to diet and to treatments were

observed. Values are found in Table IV.3.

The bone lengths of the normally fed rats were signifi-

cantly longer than those of calcium-deficient and calcium-

and vitamin D-deficient rats in all treatment groups (P<.05

to .005).

Estrogen administered to intact and ovariectomized rats

reduced bone length in the normally fed group and the cal-

cium- and vitamin D-deficient group (P<.05 to .005).

Ovariectomy increased femur lengths in the normally fed

group (P<.05). Lindgren and Lindholm (1979) found femur

length unaffected by oophorectomy. The sensitive photon-

absorption method (Norland, 1980) used in the present study

may have been responsible for detecting length differences.

Deficient diets used in this study clearly bring about

a decrease in femur length in female rats during the growth

period.

Estrogen tends to cause a shorter bone length, espe-

cially in normally fed rats and rats both calcium- and

vitamin D-deficient. It is thought that estrogen causes the

epiphyses to close prematurely which results in a shorter

bone. Use of estrogen in young human females has this

effect.

Confirming evidence of the preceding is observed in

Fig. IV.2. Ovariectomy, or absence of estrogen, caused bone













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lengths to increase in normal rats. Addition of estrogen

depressed bone length to normal or below normal.

Bone mineral content. Means and standard errors of the

mean for mineral content of the femur as measured by the

photonabsorption method (Norland, 1980) are listed in Table

IV.3. Statistically significant differences in mineral

content of the femur due to dietary deficiencies and to

treatments were found. The calcium-deficient diet and the

vitamin D-deficient diet lowered bone mineral content

(P<.005).

When administered estrogen, the normally fed intact

rats had decreased bone mineral content (P<.05) but other

dietary groups showed no effect from estrogen.

Ovariectomy caused a higher bone mineral content in

calcium-deficient rats (P<.01), and a lower bone mineral

content in vitamin D-deficient rats (P.<005); the latter

effect most probably was due to the multiple deficiencies of

the diet.

When rats were ovariectomized and estrogen was added,

bone mineral content decreased in the normally fed group

(P<.01), and in both the vitamin D and calcium- and vitamin

D-deficient groups (P<.005).

The results indicate that diet does cause decreases in

bone mineral content in the female rat. When the rat is

depleted of certain nutrients, osteopenia results. That the

rat is a suitable model for this premise has been estab-

lished by this and other studies. Extrapolation of the










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conclusion to the human female is difficult, however, the

corollary does exist.

Sanchez et al. (1981) measured bone mineral mass in

vivo in normally fed, untreated rats with a Norland-Cameron

model 178 bone mineral analyzer. They found highly signifi-

cant positive correlations between femoral mineral mass,

femoral ash weight, and body weights. Similar statistical

correlation tests are planned for the data in the present

study.

Bone density. The bone density measurement is a ratio

of the bone mineral content and the femur width, so that

differences between bone mineral content and bone density

were due to bone width and did not vary with bone mineral

content, since the measurements were done simultaneously.

The means and standard errors of the means for bone

density values are found in Table IV.3.

Dietary deficiencies significantly decreased bone den-

sity in several treated and untreated groups (P<.05 to

.005). Calcium- and vitamin D-deficiency especially

affected density (P<.005) (Fig. IV.4). Only one intact

femoral bone from untreated calcium-deficient rats was

available; therefore it was not used for statistical

comparisons.

Estrogen treatment showed no effect on bone density,

whether given to intact or ovariectomized rats.














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Ovariectomy also did not affect bone density. However,

within the vitamin D deficient group, which also was defi-

cient in protein and phosphorus, bone density was decreased

by estrogen and by ovariectomy (P<.005).

Burkhart and Beresford (1978) castrated 1 1/2-year-old

male rats and reported decreased femoral density 3 to 6

months later. A Joyce-Loebel photodensitometer was used to

measure the density. Wink and Felts (1980) also reported

density decreases in male castrates (P<.01) and femoral oste-

oporosis four months after castration in year-old male rats.


Biomechanical Tests

Table IV.4 lists the mean and standard errors of the

torque required to fracture the femurs and of the deforma-

tion undergone by the bones at fracture.

Torque. In all treatments, the calcium-deficient group

and the vitamin D-deficient group required the least torque

(P<.01) for fracture to occur (Fig. IV.5).

Estrogen administered to intact rats significantly

lowered torque in the normally fed group (P<.05) and raised

it in the vitamin D-deficient group (P<.05).

Ovariectomy decreased the torque value significantly in

the calcium- and vitamin D-deficient group (P<.05).

When estrogen was given to ovariectomied rats, torque

values were reduced significantly in the normally fed group

(P<.05) and raised significantly in the calcium-deficient

group (P<.05).
















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Deformation. Dietary deficiencies had no effect on

deformation of the rat femurs.

When estrogen was administered to intact rats in each

group, the normally fed group and the calcium- and vitamin

D-deficient group showed significant decreases in deforma-

tion values (P<.005). This effect can be interpreted to

mean a harder bone with less bending ability resulting from

estrogen administration.

Ovariectomy did not affect deformation of the femurs in

any group.

Fig IV.6 represents the femoral deformation values for

the rats in the study.


Bone Ash

Values for bone ash as % of dry, fat-free femoral bone

are listed as means SE in Table IV.5. Differences in bone

ash content between rats on deficient diets and those ade-

quately fed were statistically significant in all treatments

(P<.01), with highest values in the adequately fed group.

Decreases in skeletal ash weight of rats on calcium-defi-

cient diets have been previously observed (P<.001) (Rockoff

and Armstrong, 1970).

Estrogen administration did not alter bone ash in any

dietary group, however, ovariectomy decreased % ash content

in the calcium-deficient group. The effect of ovariectomy

and estrogen was an increase in bone ash in vitamin D-defi-

cient and calcium- and vitamin D-deficient groups (P<.05).
















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Other normally fed animals have shown no alteration in

ash content of bones after estrogen administration, but

oophorectomy caused a significant decrease (P<.05) in ash

content which was reversed by estrogen (P<.05) (Cruess and

Hong, 1979). Other significant decreases in ash weight as %

of femur dry weight have also been reported with oophorec-

tomy (P<.01), even though the rats also had a high body

weight gain (P<.05) (Lindgren and Lindholm, 1979). By cas-

trating male rats and maintaining a normal diet, a decrease

in % ash was observed after 3 to 6 months (Burkhart and

Beresford, 1978; Wink and Felts, 1980). It is probable that

a longer period than one month must be observed post ovari-

ectomy in order to detect significant differences in bone

ash in rats on a normal diet.


Mast Cells

Bone Marrow Mast Cells.

Means of mast cell counts in the distal metaphyseal and

diaphyseal portions of the femoral bone marrow are presented

in Table IV.6. Means for each group were obtained and

adjusted to an area of 1 mm2. The range of mast cell

counts is also given as counts per 1 mm2 surface area.

Appendix I contains photomicrographs of bone marrow mast

cells (Fig I.1-1.4). Figure IV.7 provides a visual repre-

sentation of bone marrow mast cell populations observed in

this study.


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Normal diet group. The mean number of mast cells per

mm2 in the bone marrow of these normal, untreated young

rats was 202.920.7. Belanger (1977) recorded mast cell

populations of 12316 per mm2 bone marrow in normally fed

rats.

Estrogen increased the count in normally fed, intact

rats (P<.10)(Fig. IV.7). Belanger (1977) found no change in

count when estrogen was used in rats on normal diets.

Ovariectomy produced a decrease in mast cell count

(P<.10). When estrogen was given to ovariectomized rats,

the count continued to remain below that of the untreated

rat (P<.10).

Calcium-deficient group. The calcium-deficient, un-

treated rats in this study showed no change in bone marrow

mast cell count from that of the adequately fed rats. This

finding was somewhat surprising. The well-known study by

Urist and McLean (1957) describes extensive increases in

calcium-deficient rat bone marrow. However, no statistical

evidence was reported. Their rats were weaned at three

weeks to a calcium-deficient diet, whereas the rats on this

study began the deficient diet at two months of age. Great-

est increases in mast cell counts in their rats were re-

ported as occurring after six to 15 weeks. The rats on the

present study were fed a calcium-deficient diet for only six

weeks. Rockoff and Armstrong (1970) also experienced marked

mast cell hyperplasia in calcium-deficient rats. However,


I








mean number per field and distribution of cells in calcium-

deficient rats did not vary from the normally fed rats in

the study of Rasmussen (1972). It may well be that age of

rat and duration of calcium deficiency play an important

role in mast cell population changes, if alterations do, in

fact, occur.

In contrast to normally fed animals, calcium-deficient

rats administered estrogen did not show altered counts.

However, calcium-deficient, estrogen treated rats did show

increased counts when compared to normally fed, estrogen

treated rats (P<.05).

Ovariectomy produced a significantly decreased mast

cell count in this group (P<.01).

Vitamin D-deficient group. A combined lack of protein,

phosphorus, and vitamin D produced no changes in bone marrow

mast cell count in the untreated animals. Estrogen signifi-

cantly increased the marrow count in the intact rats (P<.05)

and ovariectomy also increased the count (P<.10). Ovariec-

tomized rats also had an increased bone marrow mast cell

count when compared to ovariectomized, normally fed rats

(P<.05) indicating an effect of diet.

Contrary to the above finding, Rasmussen (1972) re-

ported marrow mast cells in a vitamin D-deficient group to

be higher than those of normally fed rats. The statistical

significance level was not given.

Calcium- and vitamin D-deficient group. Effect of diet

in untreated rats in this group was not observed. Rasmussen








(1972) reported significantly higher numbers of marrow mast

cells per field in a calcium- and vitamin D-deficient group

when compared with a normally fed group.

In the present study, when rats in this dietary group

were treated with estrogen, the mast cell count decreased

from that of the estrogen treated, normally fed group

(P<.05) and from the untreated rats in the same calcium- and

vitamin D-deficient group (P<.10) indicating an effect of

diet and treatment.

Ovariectomy also caused a significant decrease in mast

cell marrow count (P<.05).

It has been suggested that mast cell increases in bone

marrow are due to secondary hyperparathyroidism caused by

hypocalcemia (Rasmussen, 1972). As was mentioned previously

in the serum calcium section, dietary deficiencies did not

consistently cause hypocalcemia in the present study nor in

other studies. As stated, diet did not affect serum calcium

or bone marrow mast cell numbers in the present study.

The rats given estrogen were made hypocalcemic in all

deficient diet groups, but did not exhibit mast cell

increases. The reverse was found with normally fed rats,

i.e., an increased mast cell count was observed in estrogen

treated rats with normal serum calcium levels. Hormone

treatment may have interfered with the theory mentioned

above, even though estrogen receptors are not known to

occur in bone.








A consistent decrease in marrow mast cell number was

observed in calcium- and vitamin D-depleted, estrogen defi-

cient rats (ovariectomized). With the addition of estrogen,

marrow mast cell numbers were returned to the normal range.

This finding clearly indicates the presence of hormonal

activity in bone.


Vaginal Tissue Mast Cells

The mean number of mast cells per mm2 vaginal tissue

in the different groups of rats is given in Table IV.5 and

is illustrated graphically in Fig. IV.8. Photomicrographs

of vaginal tissue mast cells observed in this study are con-

tained in Appendix I (Fig. I.5-1.8).

Normal diet group. Vaginal mast cells in the untreated

control group numbered 27.22.6 with a range of 14.5 to

39.3. Estrogen given to intact rats in this group did not

alter the count. However, ovariectomy did increase the

count significantly (P<.05). Estrogen given to ovariecto-

mized rats, however, did not return the count to a normal

range.

Estrogen given to intact mice increased vaginal mast

cells substantially (Westin and Odeblad, 1956) again sug-

gesting species difference; however, Johannson and Westin

(1959) report estrogen as suppressing true mast cell numbers

in mouse vaginal tissue.













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Calcium-deficient group. Calcium deficiency produced a

decrease in the vaginal tissue mast cell count at the P<.10

level, indicating a very weak, almost non-existent effect.

Calcium deficiency also produced a decrease (P<.01) in

estrogen treated rats. Likewise, the deficient diet showed

an effect of decreasing the mast cell count when these rats

were ovariectomized and given estrogen (compared with nor-

mally fed rats of similar treatment (P<.05).

Within the group, when compared with untreated rats,

ovariectomized rats exhibited an increased vaginal tissue

mast cell count (P<.01). With the addition of estrogen, the

cell count was reduced to the normal range of a calcium-

deficient animal.

Vitamin D-deficient group. Untreated rats in this

group had higher vaginal tissue mast cell counts than nor-

mally fed rats (P<.05). Treated rats did not display an

effect of diet deficiencies on mast cell numbers.

Estrogen did not alter mast cell counts; but lack of

estrogen caused a reduction in number (P<.10). These rats

were given a diet deficient in protein and phosphorus as

well as vitamin D; results would possibly be affected by the

lack of those nutrients.

Calcium- and vitamin D-deficient group. Diet had no

effect on mast cell count in this group.

Within the group, estrogen given to intact rats pro-

duced no changes in vaginal tissue mast cell counts. Ovari-

ectomy, however, caused an increase in cell numbers (P<.01).








Estrogen added to the ovariectomized rats maintained a count

higher than normal for this group (P<.10). Overall, mast

cell counts in rat vaginal tissue were much less variable

within groups than in bone marrow.

Summarizing the influence of diet and treatment on

vaginal tissue mast cell populations, one observes that

depletion of bone in the rat by dietary calcium deficiency,

as evidenced by densitometric bone analysis and bone ash

levels, has no effect on the vaginal tissue mast cell

number. This is a finding which has not been supported by

the literature since studies of vaginal tissue mast cells

do not exist.

It is important to examine the pattern of hormonal

effects. Estrogen given to intact rats did not affect mast

cells in the vaginal epithelium, an estrogen sensitive

tissue. But when the rats were deprived of estrogen by

ovariectomy, mast cells increased above normal, irrespective

of diet. When estrogen was added, the increased populations

were maintained above normal, but a hormonal effect of

reducing the numbers may be a possibility.

The vitamin D-deficient group does not fit this pat-

tern, most probably because of the effects of additional

dietary deficiencies. As an effect of this diet, however,

mast cells did increase in vaginal tissue. With ovariectomy

a decrease was observed.














CHAPTER V
CONCLUSIONS


Change (reduction) in mast cell numbers in rats made

osteopenic by dietary deficiencies of calcium and vitamin D

as evaluated by bone densitometry and bone ash content was

observed in vaginal tissue as an effect of a calcium-defi-

cient diet at the P<.10 level of significance. Because of

the weak evidence of mast cell reduction occurring, the

first hypothesis is not proven. This finding is not docu-

mented in the literature as studies concerning mast cell

populations in rat vaginal tissue do not exist.

Bone marrow mast cell populations did not vary as a

result of dietary deficiencies. That significant changes in

mast cell populations did not occur in the bone marrow was

surprising because other studies have indicated a substan-

tial increase in bone marrow mast cells with calcium defi-

ciency and osteoporosis (Urist and McLean, 1957; Frame and

Nixon, 1968; Rockoff and Armstrong, 1970; te Velde et al.,

1978).

Two possible explanations for lack of change in marrow

mast cell numbers are suggested. The rats in the present

study were at least two months old before being fed defi-

cient diets. It was desirable to have rats with estrous







cycles in order to observe the effect of removing the circu-

lating estrogen. The rats in the aforementioned studies

were weanlings.

In addition, the duration of dietary deficiencies may

have been too short to overcome the effect of age, even

though osteopenia was indicated. The rats in the present

study were kept on deficient diets for a period of five

weeks which approximated .05% of their life span. With

dietary deficiencies extended over a longer period of time,

significant alterations in marrow mast cell numbers may have

been seen.

The effects of exogenous estrogen were seen to reduce

bone marrow mast cell numbers in rats both calcium and vita-

min D deficient, which supports the second hypothesis of

this study, in part. An opposite effect of estrogen admini-

stration was seen in normally fed rats: the bone marrow

increased significantly in mast cells. This inconsistency

is not understood. However, these results strongly suggest

that bone is an estrogen sensitive tissue, even though

estrogen receptors have not been found in bone.

Bilateral ovariectomy significantly affects both

vaginal tissue and bone marrow with respect to mast cell

populations. Removal of estrogen production in the rat

reduces bone marrow mast cells in rats normally fed and also

in those deficient in calcium and vitamin D. An opposite

effect of estrogen removal is seen in the vaginal tissue.

Both observations support the hypothesis that ovariectomy








alters mast cell populations in bone marrow and vaginal

tissue of calcium- and vitamin D-deficient rats.

In absolute terms, ovariectomized rats resupplied with

estrogen demonstrated increases in vaginal tissue mast cells

and a decrease in marrow mast cells. These are changes

similar to those stated as an effect of ovariectomy alone.

However, upon observing Figs. IV.7 and IV.8, one can detect

that replacement of estrogen may be causing a reversal of

mast cell population change due to ovariectomy. Statistical

analyses between ovariectomized groups with and without

estrogen will be performed in the near future to determine

whether added estrogen changes mast cell populations in

ovariectomized rats.

It is important to consider again the possibility that

both age of the rat when begun on a deficient diet and

duration of feeding a deficient diet may greatly influence

mast cell populations in the two tissues examined in this

study.

Ovariectomy, the procedure which removes the endogenous

gonadal hormone supply has known consequences relating to

bone loss in the human female. Whether mast cell changes

similar to those in the rat occur in the human is not known.

Considering the results of the present study, a quantitative

investigation of mast cell populations in vaginal tissue of

ovariectomized women may provide information useful in the

study of osteopenia and bone resorption.





54


It has also become apparent that qualitative investiga-

tion of the mast cell populations, including histologic bone

evaluations, needs to be considered from data obtained in

the present study. The need for further examination of

correlations between bone densitometric measurements, bone

ash, serum calcium, biomechanical tests and the mast cell

populations is also immediate. Statistical analysis is

scheduled for these parameters.


































APPENDIX A

SIGMA XI GRANT-IN-AID OF RESEARCH




















rS, 1586 ,I




SIGMA XI
THE SCIENTIFIC RESEARCH SOCIETY OF NORTH AMERICA
For the Encouragement of Scientific Research





Grants-in-Aid of Research




Grants-in-Aid of Research are supported by voluntary contributions to 'he Researi
Program from the membership of SIGMA XI. Awards are normally made in amounts rngin.
from 5100 (or less) to a maximum of S 1.000.

Research awards may be made to support scientific investigation in any field. Each
award is made payable to the individual recipient. No part of a irant may be used for
the payment of any indirect costs to the recipient's institution-al, of the funds must be
expended directly in support of the proposed investigation. All equipment purchased shall
be the property of the institution. Grants normally are rot made for expenses of publication.
salary or tuition, travel to meetings. or usual and routine institutional obligations. Priority is
usually given to applicants who are in an early stage of their scientific careers.

The Committee on Awards meets on or about the first of March. June. and December
of each year and applicants are notified of the Committee's Jecisions within si.\ weeks.
In order to be considered, applications must be received by February I for the .'la-ch
meeting. May 1 for the June meetiiin, and November 1 for the December eetring at Sigma
Xi National Headquarters. 345 Whitney Avenue. New Haven. Connec::cut 06511. Attention:
Committee on Awards.








Franklyr. S. VL.n licuHoe. C:;









APPLICANT: Please filU i0n these thee item only.


APPLICANT: Tesar Rogene E.
APPLICANT: ............................... .......
LAST NAME FIRST NAME MIDDLE NAME
FIELD: ..Nutrition and Osteoporosis
............ .........................


TITLE OF STUDY: .The RelatiO.n h~Between
Epithelial and Connective Tissue Mast Cell
Populations in the Female Rat
....... .................... ...................


FOR COMMITTEE USE ONLY
Date Received.........
Amount Requested:
......................
Action:
Not Granted
Granted:
Full ($ )
Partial ($ )
Conditional ($ )
Date of mailing award:


Date of receipt of
final report:


COMMENTS AND RECOMMENDATIONS

COMMITTEE ACTION









DATE OF MEETING




ADDITIONAL REMARKS













Please type or print all information

APPLICATION FOR GRANT-IN-AID OF RESEARCH


ame Tesar Rogene E.
LAST NAME FIRST NAME MIDDLE NAME
Addess 6916 N.W. 20th Place, Gainesville, Florida 32605

..............................................Ae42

Peent iion and int on Graduate Assistant, Department of
Obstetrics and Gynecology, University of Florida


Degrees, it un coneing them, daetu B.Sc. (Home Economics).
Kansas State University, 1962; B.Sc. (Food Science) University of
Florida, 1977; M.Ag. (Human Nutrition) University of Florida, 1979;
Ph.D. (in progress) University of Florida

MembeAzhip in SIGMA XI ..........onmember...................................

Please attach a. ti.t o6 titezS o6 articlu published dwZing the tast
jive yeau, with names o6 peAiodicaZc and dates: Lis;t of tit.es
appended ...........
Tt.e. o ppod on: The Relationship Between Epithelial
Title of proposed investigation: .....................................
and Connective Tissue Mast Cell Populations in the Female Rat

Proposed investigation, desucibed in non-technica language:
Previous studies suggest a relationship between bone marrow mast cell
(MC) activity and local bone loss. There is also evidence that changes
in skin MC activity may be indicative of bone loss. The proposed study
is designed: 1) to determine whether a correlation exists in female
rats between MC activity in bone marrow and vaginal tissue and 2) to
examine the effects of calcium- and vitamin D-deficient diets, exogenous
estrogens and removal of the ovaries on this relationship.
The following hypotheses will be tested: 1) dietary calcium and
vitamin D deficiencies produce an increase in bone marrow MCs and a
decrease in vaginal epithelial MCs in female rats; 2) administration
of exogenous estrogens alters the bone and vaginal tissue MC activity
in the osteoporotic female rat; and 3) removal of the ovaries (removal
of primary source of endogenous estrogens) produces changes in bone and
vaginal tissue MC activity in the female rat.
Should the correlation be shown to exist, a similar correlation in
the pre-osteoporotic and osteoporotic human female could be suggested.





59



At present there is no universally applicable, non-invasive method for
evaluation of bone resorption and formation. Evaluation of MC activity
in vaginal tissue may prove useful as a non-invasive means of detecting
increased bone resorption (indicative of osteoporosis associated with
endogenous or exogenous excess of corticosteroids, hyperthyroidism,
hyperparathyroidism and osteomalacia). In a similar manner, the method
could be used to assess treatment efficacy.





60


Locations wheAe problem wii be studied: Department of Animal
Resources, J. Hillis Miller Health Center, University of Florida;
i...............................................................
Department of Animal Science, College of Agriculture, University of
***********...........................................................
Florida.
Nature o6 assistance desired and amount o6 grant needed, itemized:
Purchase of 128 Sprague-Dawley female rats $544.00
......... o ......................
Feed and bedding for above rats for 5 wk. period 394.25
..................................................................
TOTAL $938.25
...........................................................
****..........................* .................................



Institutiona support 6or study o problem: Remaining necessary support
to carry out entire research project: anesthesia, estrogen, stains
.. ...........................................................
and chemicals, microscope slides and use of all equipment.
...........................................................

Previous gtrant-s received fom SIGMA XI and other: .none..............



OtheA applications pending: .none

o ...........................................................

Attach a list giving name od each assistant oa co-wodkeA, i6 any,
engaged in the. investigation:
List attached X Number o6 co-workker 7
Names and addrAesse o0 at least two specialists* in thki6 ield who
will be ASKED BY THE APPLICANT to send toSigma Xi National
Headquattea statements indicating (7) the importance o6 the proposed
investigation and (2) the qualijications o6 the investigator.
Morris Notelovitz, M.D., Ph.D., Dept. of Obstetrics and Gynecology,
Box J-294 JHMHC, College of Medicine, U of F, Gainesville, FL 32610
..........................................................
J.P. Feaster, Ph.D., Dept. of Animal Science, 20 Nutrition Lab, IFAS

College of Agriculture, U of F, Gainesville, FL 32610


*I1 applicant is a degree candidate, one must be that facuLty or
research sta6j membeA su.peAvising hi research.


Applicant's Signatuwe .--

Date October 28, 1980









Title: The Relationship Between Epithelial and Connective Tissue Mast
Cell Populations in the Female Rat

Investigator: Rogene Tesar





Assistants and Co-workers Engaged in Investigation:


Morris Notelovitz, M.D., Ph.D.
J.P. Feaster, Ph.D.
A.F. Moreland, D.V.M.
Laboratory Technician of Dr. Moreland
Marsha Ware
Lynda McKenzie, R.N.
Cindy Soroski


Advisor Endocrine Functions
Advisor Dietary
Laboratory Assistance
Laboratory Assistance
Laboratory Assistance
Laboratory Assistance
Statistical Assistance















The University of Florida College of Medicine

Department of Obstetrics and Gynecology


iIdf-/ ?November 26, 1980




BOX J-294. JHMHC
GAINESVILLE. FLORIDA 32610
TELEPHONE: 904-392-2893

Committee on Awards
Sigma Xi
National Headquarters
345 Whitney Avenue
New Haven, CT 06511

RE: Application by Rogene Tesar for grant to investigate
the relationship between epithelial and connective
tissue mast cell population in the female rat.

Sirs,

The above application relates to the potential role
that mast cell activity may have in the development of
osteoporosis, a condition that affects some 25% of meno-
pausal women and about 75% of women who have undergone a
surgical menopause. Osteoporosis is a significant disease
since it is not only associated with pathologic fractures
and potential invalidism, but can frequently result in the
premature death of elderly women. One of the problems in
the clinical management of this condition is the difficulty
of its diagnosis and measurement of its response to treat-
ment. Mrs. Tesar's research could provide the answer to
this problem.

.I have been acquainted with Mrs. Tesar for approxi-
mately two years and am currently an advisor to her for
her Ph.D. requirements. She is a highly competent
research worker, and I am confident that she will be able
to accomplish all the goals of her research project. I
have no hesitation in supporting her request.

Warm kind regards. ince

\ urs sincrely ,*


Morris Note ovitz, M.D.
(RAND), Ph.D., F.R.C.O.G.,
F.A.C.O.G.
Director of the Center for
Climacteric Studies
MN:mlh


EQUAL. EMPLOYMENT OPPORTUNITIES FFIRMATIVE ACTION EMPLOYER






63
















SIGMA X., THE SCIENTIFIC RESEARCH SOCIETY

345 WHITNEY AVENUE
OFFICE OF THE NEW HAVEN. CONNECTICUT 06511
COMMITTEE ON AWARDS 20 April 1981 (203 624.93





Ms. Rogene E. Tesar
6916 N.W. 20th Place
Gainesville, FL 32605

Dear Ms. Tesar:

I am happy to inform you that at a recent meeting of the Committee on Awards a
Grant-in-Aid of Research of $250.00 was given you to further the work in your
application: The Relationship Between Epithelial and Connective Tissue Mast
Cell Populations in the Female Rat. Please complete the enclosed acceptance
form so that we may write and forward your check.

This award is one of eight made possible this year from the income of a
specific gift to the Research Fund by Mrs. Daisy Yen Wu in memory of her
husband, Dr. Hsien Wu.

It is understood that in accepting this award you will at the close of the
academic year (1981-82) submit a report of the work done to the Committee on
Awards, Sigma Xi, The Scientific Research Society, 345 Whitney Avenue, New
Haven, Conn. 06511. This should be a short one or two-page summary of the work
accomplished with your Sigma Xi grant.

It is further understood that all published reports of your work will contain a
statement that the research was aided by a Grant-in-Aid of Research from Sigma
Xi, The Scientific Research Society. Also, any equipment purchased with the
funds which have been made available is to be considered the property of the
institution where the research is being carried on. It is also to be under-
stood that no indirect costs are to be paid to your institution from this
grant.

It is a great pleasure to express the Committee's hope for your continued
success in scientific research.

Sincerely yours,

'7. i. V'1 1_^A*-

Franklyn B. Van Houten
Chairman


FVH/ia
Enclosure











Date April 27, 1981


Committee on Awards
Sigma Xi, The Scientific Research Society
345 Whitney Avenue
New Haven, CT 06511

Gentlemen:

I have received your letter stating that a grant has been
awarded to me by the Committee on Awards.

I. (X ) I accept the award in the amount of $250.

() I cannot accept the award because


)I shall let you know by whether
or not I can accept it.

II. The name of the President or Chancellor or Chief
Executive Officer of my institution is:

President: Robert Q. Marston

III. The name of the Head or Chairman of my Department
is:
Animal Science: H.D. Wallace, Chairman
Center for Climacteric Studies:
M. Notelovitz, M.D., Ph.D.

IV. Will you please have Sigma Xi, the Scientific
Research Society, forward to me a check made
payable for the amount of the award. I under-
stand that it will be sent to my institutional
address only, and I have made arrangements for
its being forwarded if necessary.

NAME (Please Print): Rogene Tesar, R.D., M.Ag.

INSTITUTIONAL
ADDRESS ONLY: Center for Climacteric Studies
Univ. of Fla., 901 N.W. 20 P1.
Suite B-l Gainesville, FL 32601



Signature





















SIGMA XI, THE SCIENTIFIC RESEARCH SOCIETY

345 WHITNEY AVENUE
OFFICE OF THE NEW HAVEN, CONNECTI(UTokW I
EXECUTIVE DIRECTOR 10 June 1981 i20Ml 624-~W












Ms. Rogene E. Tesar
University of Florida
Center for Climacteric Studies
901 N.W. 20th Place
Suite B-1
Gainesville, FL 32601

Dear Ms. Tesar:

Enclosed please find our check in the amount of $250.00,
which represents the Grant-in-Aid of Research award made
to you by the Sigma Xi Committee on Grants-in-Aid at their
March meeting.

This award is one of eight made possible this year from
the income of a specific gift to the Research Fund by Mrs.
Daisy Yen Wu in memory of her husband, Dr. Hsien Wu.

Upon completion of your research a report of your findings
should be forwarded to the Committee on Grants-in-Aid, 345
Whitney Avenue, New Haven, CT 06511.

May I take this opportunity to wish you every success with
your research.

Sincerely,



Thomas T. Holme
Executive Director

TTH/ia
Enclosure


cc: Office of the President
Department Chairman











































APPENDIX B


LABORATORY ANIMAL USE


L











All University Cornitcee on the Care and Use
of Laboratory Animals

In order to comply with DHEW policy and all federal, state and local rules
and regulations concerning the care, treatment and use of laboratory animals,
tne following information is necessary for grants to be processed by the
Division of Sponsored Research. Instructional programs and research projects
supported internally using laboratory animals must also complete this question-
naire and return to the Comrnittee completing the appropriate parts.


1. Principal investigator: Rogene E. Tesar
Department Obstetrics and Gynecology
College College of Medicine


2. Proposal submitted to:


Sigma Xi
Name


Starting date: December 15, 1980


Telephone 392-3184 Date Nov. 17, 1980


345 Whitney Ave., New Haven,
Aaoress Conn. 06511

; conclusion: August 30, 1981


4. Proposal title:
The Relationships Between Epithelial and Connective Tissue Mast Cell
Populations in the Female Rat

5. Animal species: Rat
Strains Sprague-Dawley
Numbers 137
Sex Female
Age 3 weeks and 2 months
Size 50 gram and 120 gram

6. Is the animal-model appropriate?
Explain Yes. The female rat has been used in many experiments involving
the mast cell. The metabolism of the rat closely relates to that of the human
and will produce results due to the treatment given which can be implicated
as occurring in the human.
7. Abstract of animal use:
(Use continuation sheet if needed)
137 rats will be utilized to observe differences in population and activity of the
mast cell in bone and vaginal tissue. Of these rats, 12 will be utilized for a
preliminary trial to define whether mature rats or weanlings are to b used for
treatment (a) castration. Treatments are as follows: a) castration (7 rats)
at beginning of study b) estroeen (LV.) (67 rats) 3 X week for 5 weeks. c) normal
diet (32 rats). d) calcium deficient diet (32 rats) e) vitamin D deficient diet
(32 rats), f) calcium and vitamin D deficient diet t32 rats), (continued)
8. Care and location of animals:
The rats will be maintained at the Animal Resources Department, JHMHC, University
of Florida in individual cages with daily care and feeding.

9. Is the study designed to avoid inflicting needless pain and/or suffering
including the appropriate use of tranquilizers, analgesics and/or
anesthetics? If not, explain: Yes;
Anesthesia will be used for surgery purposes.


10. Method(s) of euthanasia to be used:
At the end of a maximum 5 week feeding
decapitation


Pri6 rpal Investigator
Rogene Tesar, M.A., R.D.


period, the rats will be killed by



/Decartment Chairman
Eduard G. Friedrich, Jr., M.D.






68





All University Committee on the Care and Use
of Laboratory Animals
Continuation Sheet



7. h) photon absorptiometry of the femur and tibia during the 5 week diet period.
Castration (removal of ovaries) will be performed under anesthetization. At
the termination of the 5 week diet period the rats will be killed by decapitation.
-Bone and vaginal tissue specimens will be obtained for histological purposes.












University of Florida
All-University Committee for
The Care and Use of
Laboratory Animals





1. Acknowledge receipt of your form for the care and use
of laboratory animals entitled:

"The Relationship Between Epithelial and Connective
Tissue Mast Cell Populations in the Female Rat"
submitted 11/17/80.


2. Review Results

Approval: XX
Disapproval:
Incomplete-please provide




3. For questions, please call: Dr. Halliwell at (904)
392-4751.















APPENDIX C
EXPERIMENTAL ANIMAL BODY WEIGHTS

TABLE C.1
NORMAL DIET


Sample
ID OW 1W 2W 3W 4W 5W


192
175
184
180
185
183
186
197
195
189
185

212
195
177
184
177
183
183
197
191

184
201
194
206
196
200
171
180
209
194


197
184
198
186
198
183
187
204
195
196
196

205
198
175
195
182
187
202
191
196

190
209
203
204
209
209
188
191
214
199


195
189
202
187
198
189
195
237
227
251
240

248
253
222
259
206
197
199
215
206

190
254
225
230
228
242
201
231
241
226


191
197
208
190
201
196
197
231
259
299
264

291
282
257
298
217
205
228
209
214

208
242
234
238
247
267
217
250
267
245


195
200
213
192
204
199
197
234
276
321
282

308
297
270
325
219
209
231
214
204

204
241
244
255
251
275
226
256
273
252


196
204
212
193
209
200
198
237
280
332
294

315
309
278
337
217
206
230
216
206

209
245
250
264
255
285
228
263
275
259


Weights expressed in grams








TABLE C.2
-Ca DIET


Sample
ID OW 1W 2W 3W 4W 5W 6W

1 161 179 176 179 177 169 171 176 176 175 181
2 187 204 197 197 196 195 196 202 196 195 205
3 181 192 189 190 197 193 198 196 198 197 195
4 183 202 201 197 196 196 193 199 199 208 209
5 175 175 176 187 190 188 196 200 201 202 200
6 175 187 185 185 184 181 188 188 195 195 197
7 177 194 192 182 173 185 187 189 188 186 190
8 183 198 205 210 210 210 215 213 214 212 215
la 211 211 211 215 216 222
2a 197 201 213 212 213 211
3a 182 187 187 194 200 194
4a 188 196 203 203 209 211
9 181 191 210 225 239 247 255 261 275 274 290
10 167 174 190 205 220 226 236 244 250 259 265
11 190 204 222 248 262 275 286 295 301 305 310
12 204 210 233 264 282 295 310 320 328 336 342
13 170 178 203 221 238 242 253 259 268 275 285
14 168 184 200 228 247 257 270 276 279 290 294
15 194 212 226 241 255 260 262 263 258 265 265
16 185 199 217 242 258 274 291 299 303 310 317
5a 191 192 251 275 292 298
6a 177 177 214 253 269 278
7a 186 196 243 283 295 306
8a 183 191 239 275 291 299
17 184 207 192 195 194 200 203 213 208 215 218
18 196 195 201 205 205 209 215 224 223 233 228
19 187 201 204 215 219 229 233 233 234 242 235
20 202 217 213 220 229 236 233 239 239 234 244
21 172
22 167 186 184 192 190 191 191 201 203 207 207
23 183 198 197 206 207 215 230 215 215 214 204
24 200 216 217 225 219 230 216 235 232 234 220
25 181 189 189 199 199 202 205 210 209 210 210
26 184 198 210 220 225 228 239 246 246 245 249
27 187 195 203 214 219 224 230 233 236 242 241
28 181 188 194 201 206 210 212 216 219 222 223
29 173 190 203 208 216 217 223 228 228 235 235
30 177 198 211 223 225 228 234 239 242 245 247
31 185 204 210 219 226 231 242 247 239 242 245
32 191 204 213 221 228 232 241 248 242 245 249


Weights expressed in grams








TABLE C.3
-D DIET


Sample 2
ID Ow 1W2W 3W 4W


144
157
146
158
146
151
154
154
156
175
161
153
203
184
173
177
202
134
136
142
148
161
139
152
133
154
149
155
150
175
187
184


161
166
173
181
165
173
171
180
180
190
173
165
192
183
166
176
209
145
151
165
174
189
157
177
149
176
172
181
169
171
180
183


165
167
171
179
166
177
169
175
187
193
174
172
196
184
164
179
199
146
157
169
177
190
158
181
158
182
171
184
174
174
179
185


164
167
167
186
165
171
168
172
207
208
189
182
213
203
179
203
196
149
158
169
181
189
163
183
168
188
185
203
182
182
195
191


172
170
174
196
176
175
173
171
223
224
204
200
231
210
201
220
210
161
170
173
195
203
169
190
173
201
197
210
191
193
211
191


168
176
174
198
180
174
177
172
232
234
214
216
250
219
213
230
218
163
176
177
193
206
172
194
188
210
208
217
199
197
219
203


Weights expressed in grams








TABLE C.4
-Ca, -D DIET


Sample 5
ID i W 1W 2W 3W 4W 5W


204
204
199
176
194
187
218
184
205
171
212
191
187
197
212
197
196
196
221
195
198
201
210
200
211

217
205
214
194
194
207


217
215
207
184
202
192
225
185
205
183
194
200
198
214
223
208
205
209
229
197
210
212
212
201
224

225
213
229
201
203
208


211
210
199
180
190
190
216
186
234
214
258
238
223
239
224
204
209
224
234
209
209
216
215
202
248

237
220
243
219
221
242


223
224
213
187
197
201
230
194
269
251
303
278
262
283
240
224
246
214
244
224
212
230
234
220
246

243
231
257
225
229
225


229
225
224
194
207
203
239
196
294
272
329
305
280
306
248
228
222
252
258
234
200
232
245
229
268

259
259
270
253
241
268


228
217
231
196
206
202
242
197
315
287
343
318
300
323
258
242
177
253
246
215
190
231
234
198
268

266
257
277
247
247
280


Weights expressed


in grams














APPENDIX D
COMPOSITION OF EXPERIMENTAL DIETS


AIN-76 Semipurified Diet (Ca:P


1.14:1 )*


Casein 20.0%
DL-Methionine 0.3%
Cornstarch 15.0%
Sucrose 50.0%
Fiber 5.0%
Corn Oil 5.0%
AIN Mineral mix 3.5%
Calcium Phosphate, Dibasic (CaHPO4)
Sodium Chloride (NaCl)
Potassium Citrate, Monohydrate
(HOC(COOK)CH4COOK)4 H20
Potassium Sulfate (K2SO4)
Magnesium Oxide (MgO)
Manganous Carbonate (43-48% Mn)
Ferric Citrate (16-17% Fe)
Zinc Carbonate (70% ZnO)
Cupric Carbonate (53-55% Cu)
Potassium lodate (KIO3)
Sodium Selenite (Na2SeO3 5H20)
Chromium Postassium Sulfate (CrK
(SO4)2 12H20)
Sucrose, finely powdered
All Vitamin mix 1.0%
Thiamine HC1
Riboflavin
Pyridoxine HC1
Nicotinic Acid
D-Calcium Pantothenate
Folic Acid
(Vit. E), Pre-mix
Cholecalciferol (Vit. D3)
Menaquinone (Vit. K)
Sucrose, finely powdered
Choline bitrate 0.2%


g/kg Mixture
500.0
74.0


220.0
52.0
24.0
3.5
6.0
1.6
0.3
0.01
0.01

0.55
118.0
per kg Mixture
600 mg
600 mg
700 mg
3 mg
1.6 mg
200 myl Acetate
20 g
2.5 mg
5.0 mg
972.9 g


*Obtained from Nutritional Biochemicals, Cleveland, OH









Calcium Deficient Diet (Ca:P .07:1)*


Casein (purified) 24.0%
Sucrose 68.0%
Corn Oil 5.0%
Calcium Free Salt Mixture 3.0%
Dipotassium Phosphate
Monosodium Phosphate
Magnesium Sulfate
Sodium Chloride
Ferric Citrate
Potassium Iodine
Manganese Sulfate
Zinc Chloride
Copper Sulfate
Plus Special ICN Vitamin Diet Fortification

Vitamin A Concentrate (200,000 units/gm)
Vitamin D Concentrate (400,000 units/gm)
Alpha Tocopherol
Ascorbic Acid
Inositol
Choline Chloride
Menadione
-Aminobenzoic Acid
Niacin
Riboflavin
Pyridoxine Hydrochloride
Thiamine Hydrochloride
Calcium Pantothenate

Biotin
Folic Acid
Vitamin B-12


52.873%
10.313%
8.188%
23.125%
4.500%
0.130%
0.741%
0.080%
0.050%
Mixture:
gm/kg
.1
.006
.1
1.0
.1
1.7
.05
.1
.1
.02
.02
.02
.007
mgs/kg
.4
2.0
.03


Rachitogenic U.S.P. No. 2 Diet
(Ca:P 4.23:1)*


Ground Gluten
Ground Whole Yellow Maize
Calcium Carbonate
Sodium Chloride


20%
76%
3%
1%


Custom Vitamin D and Calcium Deficient Diet
(Ca:P .07:1)*

Based on the Calcium-Deficient Diet
with omission of Vitamin D Concentrate


* Obtained from Nutritional Biochemicals, Cleveland, OH
** Formulated and obtained from Nutritional Biochemicals,
Cleveland, OH














APPENDIX E
SERUM CALCIUM ANALYSIS


Sample Collection and Procedurel

Blood was obtained at decapitation of the laboratory

rats by exsanguanation. The blood was collected in tubes,

allowed to clot and centrifuged. Serum was drawn off using

Pasteur glass pipettes, transferred to clean tubes, labeled,

and frozen for future determination.

A protein-free filtrate was required for analysis of

calcium. After thawing the serum samples at room temperature

for one half-hour, precipitation of serum protein was accom-

plished as follows:

(a) 9 ml 10% (w/v) trichloracetic acid (TCA) were deliv-

ered into labeled test tubes.

(b) 1 ml serum from each well-mixed sample was pipetted

into the TCA.

(c) the solutions were mixed on a vortex mixer, allowed

to stand for 10 minutes, and centrifuged 10 minutes

at 2,500 rpm. This filtrate represented a 1410

dilution of the serum samples.

(d) 1 ml of the supernatant was diluted to 5 ml with

1% lanthanum. The dilution factor was 10 x 5 = 50.



1Fick et al., 1979








This procedure provided the appropriate serum calcium

concentration for the reading of absorbance by the Perkin-

Elmer 306 atomic absorption spectrophotometer (AAS), which

has a linear working range of 7 ppm for calcium. Confirma-

tion of the above is shown by the following calculation:


(ppm calcium expected
calcium concentration = for original sample)ample)
sample dilution

calcium concentration =(100 ppm) x (1 ml)
50 ml

calcium concentration = 2 ppm.

Calcium standards of 0, 2, 3, 4, 5, and 7 vg/ml were

prepared in 100 ml volumetric flasks. The 1,000 ppm stock

standard calcium solution was first diluted to 100 ppm. Each

standard was made to contain 18 ml of 10% TCA to match the

final dilution of serum and 16 ml of 5% lanthanum. Table E.1

lists the concentration and absorbance of the standards, as

read by AAS.


TABLE E.1
SERUM CALCIUM STANDARDS

Standard Readout Calculated
pg/ml Absorbance (A) Slope (a)

0 .000 .000
2 .074 .037
3 .120 .040
4 .164 .041
5 .184 .037
7 .268 .038

Calcium in the sample solutions was then measured for

absorbance by AAS and concentration was calculated as mg/100

ml serum as follows:








sample ppm = absorbancee) x (dilution factors)
(slope) x (sample weight)

This equation is derived from Beer's Law, which states that

A = abc

where

A = absorbance (optical density)

a = absorptivity or slope of the standard

b = length of the light path (always constant)

c = concentration

The absorbance reading was obtained from the machine.

The slope was determined as an average of the slopes of

the standards (a = A/c from Beer's Law). The slope used

in calculations for samples was .0386, obtained from

Table E.1.

Tables E.2 through E.5 contain serum calcium concen-

tration values of the laboratory rats.








TABLE E.2
SERUM CALCIUM ANALYSIS DATA


Rat Diet Normal
Total Dilution 50


Date


2/16/82


Technician R. Tesar


Sample Readout
ID Absorbance I ppm mg/100 ml


.100
.099
.096
.097
.097
.102
.102
.082
.103
.067
.096

.084

.088
.092
.082
.088
.091
.092
.098

.088
.093
.062
.074
.092
.070
.068
.068
.073
.087


129.5
128.2
124.4
125.6
125.6
132.1
132.1
106.2
133.4
86.8
124.4

108.8

114.0
119.2
106.2
114.0
117.9
119.2
126.9

114.0
120.5
80.3
95.9
119.2
90.7
88.1
88.1
94.6
112.7


13.0
12.8
12.4
12.6
12.6
13.2
13.2
10.6
13.3
8.7
12.4

10.9

11.4
11.9
10.6
11.4
11.8
11.9
12.7

11.4
12.1
8.0
9.6
11.9
9.1
8.8
8.8
9.5
11.3








TABLE E.3
SERUM CALCIUM ANALYSIS DATA


Rat Diet -Ca
Total Dilution 50


Date


2/16/82


Technician


R. Tesar


Sample Readout
ID Absorbance jppm mg/100 ml


.074
.064
.068
.060
.074
.074
.064
.074
.069
.071
.064
.064
.082
.082
.076
.093
.076
.073
.076
.095
.074
.056
.084
.082
.077
.072
.071
.071

.071
.068
.078
.071
.073
.065
.070
.073
.070
.070
.068


95.9
82.9
88.1
77.7
95.9
95.9
82.9
95.9
89.4
92.0
82.9
84.2
106.2
106.2
98.4
120.5
98.4
94.6
98.4
123.1
95.9
72.5
108.8
106.2
99.7
93.3
92.0
92.0

92.0
88.1
101.0
92.0
94.6
84.2
90.7
94.6
90.7
90.7
88.1


9.6
8.3
8.8
7.8
9.6
9.6
8.3
9.6
8.9
9.2
8.3
8.4
10.6
10.6
9.8
12.1
9.8
9.5
9.8
12.3
9.6
7.3
10.9
10.6
9.9
9.3
9.2
9.2

9.2
8.8
10.1
9.2
9.5
8.4
9.1
9.5
9.1
9.1
8.8








TABLE E.4
SERUM CALCIUM ANALYSIS DATA


Rat Diet -D
Total Dilution 50


Date 2/
Technician


16/82
R. Tesar


Sample Readout
ID Absorbance I ppm mg/100 ml


.080
.059
.065
.065
.067
.077
.070
.068
.057
.047
.078
.076
.082
.078
.079
.090
.079
.077
.066
.064
.073
.073
.061
.065
.082
.068
.079
.067
.064
.081
.072
.066


103.6
76.4
84.2
84.2
86.8
99.7
90.7
88.1
73.8
60.9
101.0
98.4
106.2
101.0
102.3
116.6
102.3
99.7
85.5
82.9
94.6
94.6
79.0
84.2
106.2
88.1
102.3
86.8
82.9
104.9
93.3
85.5


10.4
7.6
8.4
8.4
8.7
10.0
9.1
8.8
7.4
6.1
10.1
9.8
10.6
10.1
10.2
11.7
10.2
10.0
8.6
8.3
9.5
9.5
7.9
8.4
10.6
8.8
10.2
8.7
8.3
10.5
9.3
8.6









TABLE E.5
SERUM CALCIUM ANALYSIS DATA


Rat Diet -Ca, -D
Total Dilution 50


Date


2/16/82


Technician


R. Tesar


Sample Readout
ID Absorbance I ppm mg/100 ml


1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32


.059
.070
.071
.058
.057
.053
.072
.057
.053
.063
.079
.062
.057
.069
.065
.074
.031
.067
.043
.043
.031
.060
.036
.025
.055

.038
.023
.031
.017
.023
.048


76.4
90.7
92.0
75.1
73.8
68.7
93.3
73.8
68.7
81.6
102.3
80.3
73.8
89.4
84.2
95.9
40.2
86.8
55.7
55.7
40.2
77.7
46.6
32.4
71.2

49.2
29.8
40.2
22.0
29.8
62.2


7.6
9.1
9.2
7.5
7.4
6.9
9.3
7.4
6.9
8.2
10.2
8.0
7.4
8.9
8.4
9.6
4.0
8.7
5.6
5.6
4.0
7.8
4.7
3.2
7.1

4.9
3.0
4.0
2.2
3.0
6.2














APPENDIX F
DENSITOMETRIC BONE ANALYSIS


Instrumentation and Procedure

A Norland Digital Bone Densitometer Model 278 (Norland

Instruments, Ft. Atkinson, WI) was used to measure bone

mineral content, bone width, and bone length in the excised,

cleaned rat femur by direct photon absorptiometry. This

technique measures the attenuation of a beam of gamma radi-

ation by calcified tissue and is based on the concept that

the mass of bone mineral present is directly proportional

to the attenuation by bone (Sanchez et al., 1980).

The densitometer consisted of a scanner module and a

computer module (Fig. F.1). The scanner transported a

highly collimated beam of monoenergetic gamma rays from a
125
radioactive sealed source of I25 across the bone being

measured. A 1/16" diameter detector collimator and a thresh-

old setting of 85% were used to enhance accuracy. The compu-

ter module calculated the bone mineral content (BMC) and

bone width (BW) values from the resulting absorption curve.

These values and the bone profiles were displayed on the

computer module screen. See Fig. F.2.

BMC and BW measurements were made at six distinct scan

sites of the femoral bone, beginning at the edge of the








lesser trochanter proximall end) and progressing to the

widening of the distal end. Scans were made perpendicular

to the bone axis.

The quantity measurement of BMC is a linear mass den-

sity of g/cm length of bone, an average linear density over

the approximately 4 mm wide scan path. In terms of another

explanation, BMC is the grams of mineral which would be

obtained if a 1 cm thick crosswise slice were cut out of

the bone and this slice were heated in a crucible to burn

away all non-mineral material (Norland Corp., 1980).

BW represents the distance in cm from one longitudinal

edge of the bone to the other.

The value of BMC/BW, calculated by the computer module,
2
provides a measurement of linear bone density in cm The

entire depth of the bone, i.e., the distance from top sur-

face to bottom surface of a bone lying flat, is included in

this measurement. The BMC, BW, and BMC/BW values are re-

corded in Tables F.1 through F.4.

Bone lengths were measured by placing each of the bones

in a longitudinal position along the scan path of the scanner

module. Data on these lengths are recorded in Table F.5.
























Fig. F.1 The Norland Digital Bone Densitometer,
Model 278, with computer module at left. The densitometer
scanner module transports a highly collimated beam of
monenergetic gamma rays from a radioactive sealed source
(125I) across the bone to be measured. The computer
module calculates the values from the resulting absorption
curve and displays the results on the screen. A calibra-
tion standard is shown on the scanner deck.
























Fig. F.2 Printout display of rat femur profile.
Measurement results are BMC (bone mineral content-mass)
expressed in gr ms per cm, BW (bone width) in cm, BMC/BW
in grams per cm .

















_'_-.----- -




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