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Study of the basement membrane in experimental hyperplasia of the rat thyroid using the electron microscope… Mackenzie, Margaret June 1971

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A STUDY OF THE BASEMENT MEMBRANE IN EXPERIMENTAL HYPERPLASIA OF THE RAT THYROID USING THE ELECTRON MICROSCOPE AND STEREOLOGICAL ANALYSIS by MARGARET JUNE MACKENZIE B. S c , University of B r i t i s h Columbia, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s thesis as conforming to the required standard SHE UNIVERSITY OF BRITISH COLUMBIA September, 1971 \ In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and Study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thes.is for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada Date ABSTRACT Hyperplasia was induced by physical and chemical means. The r e s u l t i n g goitres were examined by electron microscopy, and the basement membranes were analyzed by st e r e o l o g l c a l methods. The basement membrane i n experi-mental c o l l o i d goitre was found to be s i g n i f i c a n t l y thinner than i t s c o n t r o l . A consideration of the course of the various goitres and of the chemical nature of basement membranes leads to the conclusion that the thinning of the basement membrane was the r e s u l t of stretching. i i i Table Of Contents Page Introduction 1 Materials and Methods 3 Results 7 Goitre Production 7 Light Microscopy 7 E l e c t r o n Microscopy 1 2 Stereologlcal Analysis 2 6 Discussion Jf7 Summary 6 3 Bibliography 61* iv L i s t Of Tables Table Page I. Condition of Experimental Animals and Sizes of Goitres. 8 II. Means, Standard Deviations, and Variances for F o l l i c l e Diameters. 13 III. Means, Variances, and 5% Confidence Intervals for Cell Heights. 22 IV. Cell Widths. 23 V. Measurements of Basement Membrane Widths. 2k VI. Relationship of t/T and T/D Ratios to DE (Portion of F o l l i c l e Examined). kj VII. Effect of Bias Modifications on t-tests on Parenchymatous Goitre and Colloid Goitre. k6 V L i s t Of Figures Figure Page 1 Typical thyroid f o l l i c l e s — hemithyroidectomy. 9 2 T y p i c a l thyroid f o l l i c l e s — parenchymatous g o i t r e . 10 3 Typical thyroid f o l l i c l e s — c o l l o i d g o i t r e . 11 k Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — hemithyroidectomy. Ik 5 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — sham-operated. 15 6 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — control f o r hemithyroidectomy. 16 7 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter —- parenchymatous goi t r e . 17 8 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — control f o r parenchymatous goitre . 18 9 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — parenchymatous goitre preceding c o l l o i d g o i t r e . 19 10 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — c o l l o i d g o i t r e . 20 11 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — control f o r c o l l o i d g o i t r e . 21 12 Frequency d i s t r i b u t i o n of means of basement membrane widths — hemithyroidectomy. 27 13 Frequency d i s t r i b u t i o n of means of basement membrane widths — control f o r hemithyroidectomy. 28 Ik Frequency d i s t r i b u t i o n of means of basement membrane widths — parenchymatous g o i t r e . 29 v i L i s t Of Figures, cont. Figure Page 15 Frequency d i s t r i b u t i o n of means of basement membrane widths — control f o r parenchymatous g o i t r e . 30 16 Frequency d i s t r i b u t i o n of means of basement membrane widths — c o l l o i d g o i t r e . 31 17 Frequency d i s t r i b u t i o n of means of basement membrane widths — control f o r c o l l o i d g o i t r e . 32 18 Ty p i c a l portion of thyroid c e l l showing basement membrane. 33 19 T y p i c a l portion of thyroid c e l l showing basement membrane. 3^ 20 Typical portion of thyroid c e l l showing basement membrane. 35 21 Diagram of the c y t o l o g l c a l o r i g i n of the symbols used i n the st e r e o l o g l c a l analysis. 38 22 Diagrammatic representation of the determination of the zone of ac c e p t a b i l i t y . kO 23 A graph of apparent thickness of basement membrane as a function of the i n t e r v a l over which measurements are made. 24 Bias l i m i t s f o r treatments. Acknowledgements My advisor, Alfred B. Acton, originally suggested the research topic. Prom him I learned also many things outside the f i e l d of c e l l biology, and he has proved to be one of the most Interesting persons I have met. Cyr i l V. Flnnegan, Nelly Auersperg, and Anthony Perks also offered much helpful criticism and advice. Maureen Douglas and Leslie Veto taught me the day-to-day practice of electron microscopy. Elizabeth Vizsolyi taught me how to handle my research animals, Dolores Lauriente wrote a computer program for frequency dis-tributions. Donald Guthrie helped with the preparation of the manuscript and fended off assorted demons. Special mention must be made of the contribution of Stephen Borden and Frederick Glick to this thesis and to the f i e l d of stereology. Inspired, no doubt, by the non-ichthyo-ecological nature of the subject matter, they good-humouredly provided the stereological analysis used in this study. I ask a l l these people to accept my thanks. Introduction The basement membrane i s the moderately electron-dense muco-protein sheet that underlies a l l e p i t h e l i a l t issues. Hlstochemical reactions Indicate i t s chemical composition. A po s i t i v e reaction with periodic a c i d -S c h l f f where the basement membrane i s very thick, and with periodic acid-silver-methenamine where i t i s thinner, demonstrate the carbohydrate moiety. Conjugated a n t i -body reactions demonstrate a protein component. The basement membrane i s important to developing and mature tis s u e s . For embryonic tissues, i t provides a framework, giving strength and orientation i n develop-ment (Menefee, 1957; Cohen, 1961); f o r mature tissues i t provides a framework on which conformational i n t e g r i t y may be maintained (Pease, 1958) . As the membrane l i e s between a tissue and the c i r c u l a t i o n , i t also acts as a f i l t e r , and considerable work has been done to determine the size of molecules that can penetrate the basement membrane (Gelke et al.5:1966a, 1966b) . In c e r t a i n pathological conditions, the basement membrane undergoes i n t e r e s t i n g changes. In experimental scurvy, basement membranes of synovial c a p i l l a r i e s become thin and tenuous ( F r i e d e r i c i et a l . , 1966) . In Hashimoto's t h y r o i d i t i s , the basement membrane changes from the thick and seemingly multi-layered structure i n the normal gland to a much thinner membrane i n the diseased gland (Irvine and Muir, 1963) . In nephrosis, the glomerular basement 2 membrane becomes very Irregular with large deposits of sim i l a r material scattered along i t s length (Alousi et a l . , 1 9 6 9 ) . Often, these basement membrane changes occur i n a condition that features a concomitant hyperplasia of the affected t i s s u e . The purpose of t h i s study was to induce an experimental hyperplasia i n some tissue i n a laboratory animal and then investigate possible changes i n the basement membrane of the alte r e d tissue. The thyroid was chosen because i t i s a tissue i n which hyper-p l a s i a can be e a s i l y induced. The thyroid i s under the control of the p i t u i t a r y hormone, thyrotropin (TSH). When thyroid hormone metabolism i s i n h i b i t e d , the l e v e l of TSH Increases, causing the gland to increase i n s i z e . This aspect of thyroid metabolism can be effected by sur g i c a l removal of part of the gland or by administration of chemicals that i n t e r f e r e with the gland's production of lodinated proteins (Turner, 1 9 7 1 ) . 3 Materials and Methods Male rats of the Wistar s t r a i n from the colony main* tained In the U. B. C. vivarium were used i n t h i s study. The animals were kept In separate pans i n mesh-covered racks. Water and food (U. B. C. r a t i o n #66) were fed ad l i b i t u m . Induction of Hyperplasia Hyperplasia was induced by physical (surgical) and by chemical (goitrogenic) means. Physical — Hemithyroldectomy. The animals were anesthe-ti z e d with sodium pentobarbital at 40 mg / kg of body weight, given i . p . The thyroid was exposed by central i n c i s i o n , the r i g h t lobe gripped with forceps, the tissue eased,off the trachea with a needle, and the isthmus out with s c i s s o r s . The i n c i s i o n was closed with s u r g i c a l (000) s i l k and smeared with an a n t i b i o t i c ointment. The remaining lobe was taken f o r microscopy eight days l a t e r . Sham operations were performed to test the e f f e c t of the s u r g i c a l procedure on the thyroid. The same procedure was followed, but no tissue was removed. These animals were maintained under the same conditions as the animals with hemi-thyroidectomies. Both lobes of the sham-operated thyroids were taken f o r microscopy eight days l a t e r * Controls (no treatment at a l l ) were established to check the e f f e c t s of f i x a t i o n , embedding, and seasonal v a r i a t i o n on the thyroid. The controls were maintained under laboratory conditions f o r eight 4 days p r i o r to c o l l e c t i o n of tissue f o r microscopy. Chemical — Parenchymatous Goitre. Parenchymatous goitre was induced by feeding 5% potassium perchlorate (KClOij,) mixed with ground feed (U. B. C. rat i o n #66) . This mixture was allowed ad l i b i t u m . Tissue f o r microscopy was taken a f t e r 30 days of t h i s treatment. Estimated average dose was 0 .6 g KClOij. / day. Control animals were given ground feed with no additives f o r 30 days, a f t e r which tissue was taken f o r microscopy. Chemical — C o l l o i d Goitre. C d l l o i d goitre was induced by f i r s t causing parenchymatous goitre (20 mg of 6-n-p r o p y l t h i o u r a c i l (PTU) i n o l i v e o i l , sub-outaneously, per day f o r 30 days) and then leaving the animals un-treated f o r 14 days. Some animals were s a c r i f i c e d with-out a recovery period to check that PTU had induced a parenchymatous g o i t r e . Tissue f o r microscopy was taken at these times ( a f t e r 44 days f o r c o l l o i d and a f t e r 30 days f o r parenchymatous). Tissue from untreated animals was taken at 44 days and at 30 days. Fix a t i o n and Embedding The animals were anesthetized with an overdose of sodium pentobarbital (80 mg / kg body wt.), i . p . The thyroid was exposed and flooded with buffered 6% g l u t a r -aldehyde (pH 7.1*). The portion of the trachea with the gland adhering was removed to a p e t r i d i s h containing more f i x a t i v e . The thyroid was dissected into cubes 3 1 mm and placed i n v i a l s containing f r e s h glutaraldehyde. 5 The tissues, except f o r the tissue from the parenchymatous goitre and i t s control, were then post-fixed i n osmium tetroxlde (pH 7 . R a f t e r which they were dehydrated i n graded ethanols and propylene oxide, and f i n a l l y embedded i n Epon 812. Microscopy Thick sections (1 u) f o r l i g h t microscopy were cut manually on an LKB microtome, a f f i x e d to glass s l i d e s by heating i n a Bunsen flame, and stained with 1% methylene blue i n sodium borate. The s l i d e s were examined and photographed on a Zeiss microscope. Measurements of f o l l i c l e diameter and c e l l width were made on p r i n t s with a f i n a l magnification of 710 X. Thin sections (less than 500 A) f o r electron microscopy were cut on an LKB microtome, mounted on neoprene-coated copper grids (Kushida and P u j l t a , 1964), and stained with uranyl acetate and lead c i t r a t e . The sections were ex-amined i n a Hitachi HU-11A electron microscope with accelerating voltage of 50 kV. Micrographs were taken at an i n i t i a l magnification of 19 .000 X. Measurements were made on p r i n t s enlarged to 53*000 X. Light Microscopy A l l measurements were made on p r i n t s with a f i n a l magnification of 710 X. Only those f o l l i c l e s that could be considered as having been sectioned near the middle of the f o l l i c l e were measured. To be near the middle, the lumen must be v i s i b l e , and the c i r c l e of c e l l s must be a monolayer. This second c r i t e r i o n would be met by 6 a s i n g l e row of n u c l e i and no changes i n c o l o u r or t e x t u r e of the cytoplasm t h a t might I n d i c a t e t h a t two c e l l s had been s e c t i o n e d through a t an a n g l e . Frequency d i s t r i -b u t i o n s of f o l l i c l e diameter were p l o t t e d . C e l l width (the d i s t a n c e between l a t e r a l c e l l membranes as judged by c o l o u r and t e x t u r e d i f f e r e n c e s ) was measured a t the narrowest p a r t of the c e l l i n each case. S e v e r a l c e l l s i n s e v e r a l d i f f e r e n t f o l l i c l e s were measured and the means computed. C e l l h e i g h t (the d i s t a n c e from the base of the c e l l t o the apex) was measured a t f o u r p o i n t s on s u i t a b l e f o l l i c l e s , a t the p o i n t s of maximum and minimum f o l l i c l e d iameter. Means were computed. E l e c t r o n Microscopy Measurements of basement membrane t h i c k n e s s were made on p r i n t s w i t h a f i n a l m a g n i f i c a t i o n of 53iOOO X. M a g n i f i c a t i o n on the microscope was v e r i f i e d by comparison w i t h standard specimens. Twenty micrographs r e p r e s e n t i n g twenty d i f f e r e n t f o l l i c l e s were used. Measurements were taken a t 1-micron i n t e r v a l s p e r p e n d i c u l a r to the b a s a l c e l l membrane w i t h a measuring m a g n i f i e r t h a t f u r t h e r m a g n i f i e d the membrane 8 X. Measurements were made to the n e a r e s t 0.05 mm. Means were c a l c u l a t e d f o r each f o l l i c l e , converted to angstroms, and ranked. Frequency d i s t r i b u t i o n s were p l o t t e d . Means, standard d e v i a t i o n s , medians, and i n t e r - q u a r t i l e ranges (IQR) were c a l c u l a t e d . T - t e s t s were made to t e s t f o r d i f f e r e n c e s between the means of means f o r each c o n d i t i o n and i t s c o n t r o l . Results 7 Goitre Production The usual method of judging the extent of hyperplasia i n experimental goitre p r i o r to h i s t o l o g i c a l examination i s weighing the excised glands and comparing the weights with weights of control glands. This procedure was considered incompatible with s a t i s f a c t o r y f i x a t i o n f o r electron microscopy. Therefore, the goitres were judged subjectively before excision and were ranked. The severity of the goitre i s described by a series of plus signs. Size and colour of gland were both used as in d i c a t o r s . The most severe goitre was chosen f o r electron microscopical examination. In that instance where two goitr e s appeared equally severe, the gland selected was from the animal that seemed healthier, on the basis of body weight, weight gained, food intake, and behaviour. These data are sum-marized i n Table I. The gland selected f o r electron microscopy i s marked by an a s t e r i s k . Micrographs of t y p i c a l portions of the various tissues are shown i n Pigs. 1 - 3 * Light Microscopy A l l l i g h t microscopy was done on the same tissue that was chosen f o r electron microscopy. A l l measurements f o r f o l l i c l e diameter were taken along the longest axis of the f o l l i c l e and were made on only those f o l l i c l e s that met the c r i t e r i a previously described. No s i g n i f i c a n t differences between experimental 8 Table I_. Condition of Experimental Animals and Sizes of Goitres. Experiment F i n a l Wt. Food Behaviour Goitre size body wt. gain intake (subjective) (subjective) (g) (g) (g/day) Hemithyroidectomy 190 33 18.5 normal ++ 180 22 n ++ 170 15 II + Sham operated 190 40 20.5 normal 0 Control 240 50 25.5 good 0 * 184 30 II 0 Parenchymatous 150 75 nervous ++++ •tt-116 41 II +++ 90 15 nervous & staggering +++ Control 235 160 good 0 * 208 133 n 0 242 167 II 0 C o l l o i d Goitre Parenchymatous 182 6 12.0 l i s t l e s s +++ (15 days) 121 3 9.1 II +++ Pa ren chyma t ou s 160 53 11.4 l i s t l e s s +++ (30 days) 16.4 224 55 II +++ C o l l o i d 263 120 15.8 improved ++++ • 309 127 19.3 ti +++ Control 333 60 22.4 good 0 * 1 T y p i c a l thyroid f o l l i c l e s — hemithyroidectomy. a. Hemithyroidectomy b. Sham-operated c. Control A l k a l i n e methylene blue. 710X 10 F i g . 2 T y p i c a l thyroid f o l l i c l e s — parenchymatous g o i t r e . a. Parenchymatous goitre b. Control A l k a l i n e methylene blue. 71OX Note: increased c e l l height i n parenchymatous goitre (a) 11 F i g . 3 Ty p i c a l thyroid f o l l i c l e s — c o l l o i d g o i t r e . a. Parenchymatous goitre — 30 days "b. C o l l o i d goitre c. Control A l k a l i n e methylene blue. 710X Note increased c e l l height i n parenchymatous goitre (a) and lower (cuboidal) c e l l height i n c o l l o i d goitre (b) and control tissues were found, nor was there any d i f f e r -ence between test tissues (the sham operations f o r hemi-thyroidectomy and parenchymatous goitre f o r c o l l o i d goitre) and t h e i r controls. The f o l l i c l e diameter means, v a r i -ances, and standard deviations are summarized i n Table I I . Frequency d i s t r i b u t i o n s of f o l l i c l e diameters with a class size of 0.01 mm are shown i n F i g s . 4 - 1 1 . The c e l l heights give a good estimate of the severity of the g o i t r e s . These measurements are summarized i n Table I I I . There i s a d e f i n i t e increase i n c e l l height (from ouboidal c e l l s to columnar c e l l s ) f o r the CIO4F (parenchymatous) goitre and f o r the parenchymatous goitre preceding c o l l o i d g o i t r e . The c e l l height i n c o l l o i d goitre can be seen to be s i m i l a r to i t s cont r o l . Hemi-thyroidectomy d i d not make any s i g n i f i c a n t difference i n c e l l height i n the remaining lobe. The sham operations had no e f f e c t on c e l l height. C e l l widths were obtained f o r the s i x tissues to be examined by electron microscopy. These measurements are summarized i n Table IV. E l e c t r o n Microscopy Measurements of basement membrane thickness were obtained on 20 micrographs representing 20 d i f f e r e n t f o l l i c l e s . A mean basement membrane width was obtained from each micrograph. Table V summarizes these measure-ments and presents a mean of means f o r each t i s s u e . Also shown i n Table V are the r e s u l t s of t - t e s t s done to 13 Table I I . Means, Standard Deviations, and Variances f o r F o l l i c l e Diameters. Treatment No. Mean Variance Standard (cm) deviation Hemlthyroidectomy Sham 111 Rt. Lobe 65 Control 57 Parenchymatous Goitre ClO^F 70 Control 148 C o l l o i d Goitre Parenchymatous 139 C o l l o i d 431 Control 75 0.0442 0.0606 0.0593 O.0368 0.0434 0.0502 0.0392 0.0535 0.0002 0.0003 0.0003 0.0001 0.0003 0.0003 0.0002 0.0003 0.0149 0.0179 0.0198 0.0104 0.0186 0.0185 0.0164 0.0199 14 F i g . 4 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — hemithyroidectomy. 40-0+ cn LLI 35.0.. UJ tz ZD U) < UJ 30.0. U. a Ul • 25-0-1 UJ LY LY D U U O EO-O.: U. a z LU Z) a Ul 2 10.0. LL Ul u LY UJ •_ 5-0.. 0*0. 0-00 0«0E O.04 O.OG O.OB 0*10 0-1E DIAMETER OF F O L L I C L E S (MM) 0-14 15 Pig. 5 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — sham-operated. 40.0. CD Z UJ z UJ u CL UJ CL 35.0.. UJ CL • < UJ 30-0. L L • UJ ( J 25-OL UJ CL CL ZD U u D E0.0.. U. • >15.0j z UJ a UJ K 10*0. 5 . 0 . . o.o. + + o.oo O'OP. 0*04 O'OG O'OB 0-10 0-1S DIAMETER OF F O L L I C L E S (MM) 0-14 16 F i g . 6 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — control f o r hemithyroidectomy. 40.0. Ul Id 35.0.. LU CL ID i n < Ly 30.0. LL a LU U 25*0. Ld CL CL Z) u u a so-o.. u. a > 15.01 z UJ ZD a UJ ix 10.0. z u CL Ul CL 5.O.. 0.0. 0*00 • OE 0«04 0.06 O.OB 0-10 0»1E DIAMETER OF F O L L I C L E S (MM) H 0-14 17 Pig. 7 Percent frequency d i s t r i b u t i o n of f o l l i c l e  diameter — parenchymatous g o i t r e . 8 * o PERCENT FREQUENCY OF OCCURRENCE OF MEASUREMENTS & e s n. 8 M Ul 6 p p o • a M > r n H m o O? "n o n r m o LO ,1 o 18 F i g . 8 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter ~ Control f o r parenchymatous g o i t r e . 40.0. in H Z LU 35-OL. LU LY Z3 < LU 30*0. LL • LU U z LU LY LY ZD L J U a LL a >-u z LU ZD a LU LY LL I — Z LU U LY LU • L E5-0.. EO-0.. 15.0.. 10.0.. 5-0.. 0'4 1 1 1 1 1 1 1 I I 1 1 1 C-00 0-OE 0.04 0«0B 0-08 0-10 0-1H DIAMETER OF F O L L I C L E S (MM) 0«14 19 Pig. 9 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — parenchymatous goitre preceding c o l l o i d goitre 40.0. U l 35-0.. UJ LY ZI in < UJ 30.0. LL a UJ U 25'Cq z U l LY LY ZJ u u a 20-0.. LL a > 15.QJ s Z] a rx i o « o . u . LY •_ 5.0.. O-QJ 1 i—\ 1 1 1 1 -I 1 1 I 1 1 h 0.00 O.OE 0*04 O'OF O'OB 0-10 0-1S 0*14 DIAMETER OF F O L L I C L E S CMM) 20 F i g . 10 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — c o l l o i d g o i t r e . 40.0. 01 LU 35.0.. LU LY Z) LO < LU 30-0. LL • LU U 25-a LU LY LY Z l U U Q EO-O.. LL a > 15.QI z LU Zl a LU LY 10 «0. LL Z LU U LY LU CL 5.0.. 0.0. 0*00 O.OE 0-04 O.OB COB 0.10 0-1E r 0*14 DIAMETER DF F O L L I C L E S (MM) 21 F i g . 11 Percent frequency d i s t r i b u t i o n of f o l l i c l e diameter — control f o r c o l l o i d g o i t r e . 40. Q in I— | o S . O LU LY Z l i n < LU 30-0. L L a LU U S5.Q LU LY LY n u u a LU u LY LU •L E0-0.. U. a > 15.0. z LU Z) a LU LY 10.0. r - 5.O.. 0-0. 0-00 —I— •02 0-04 O'OG O.OB 0-10 0«1E DIAMETER OF F O L L I C L E S (MM) 0.14 22 Table I I I , Means, Variances, and Confidence Intervals f o r C e l l Heights. Treatment X , S 5% C. I. (mm) Hemlthyroidectomy Sham 0.0053^3 0.002874 0.004889 0.005797 Right Lobe 0.00697^9 0.0048748 0.0063455 0.0076044 Control 0.006571 0.003635 0.005921 0.007221 Parenchymatous Goitre ClOjjF 0.009789 0.007770 0.0087599 0.018196 Control 0.004503 0.003125 0.004103 0.004903 C o l l o i d Goitre Parenchymatous 0.011516 0.009486 0.016868 0.012346 C o l l o i d 0.004797 0.002577 0.0043488 0.005244 Control 0.0066325 0.004986 0.005916 0.0073^9 23 Table IV. C e l l Widths. Treatment Mean C e l l Width + Standard Deviation (u) Hemithyroidectomy 4.4 0.8 Control 6.4 1.1 Parenchymatous Goitre 4.2 1.0 Control 7.2 1.2 C o l l o i d Goitre 7.4 1.1 Control 4.9 0.9 Table V. Measurements of Basement Membrane Widths. Ranked means and t- t e s t s f o r difference between means of means. Hemi thyroidectomy 304.2? 395.04 307.69 400.15 349.78 425.31 370.32 427.56 371.09 427.79 375.18 436.5$ ' 377.35 443.39 381.78 447.52 388.17 1*51.57 388.45 474.97 Mean = 397.20 Variance = 2076.679 Control 298.69 417.86 305.66 422.90 319.70 445.01 350.57 445.75 360.20 446.54 375.26 451.88 392.45 479.^1 399.09 1+99.99 405.65 503.42 415.09 567.48 Mean = 415.13 Variance = 4868.311 P r o b a b i l i t y = 0.31723 Difference between means of means i s not s i g n i f i c a n t . C o l l o i d Goitre Control 262.73 3^6.55 332.76 424.03 279.68 352.42 338.05 432.69 283.87 358.57 359.97 433.95 285.30 366.40 372.23 441.54 291.02 373.58 376.02 444.62 309.19 380.78 381.44 448.11 314.68 400.94 383.99 464.74 317.21 436.36 399.71 488.78 329.63 437.29 402.48 496.46 331.27 479.76 419.44 575.47 Mean = 346.89 Mean = 420.82 Variance = 3^53.773 Variance = 3401.060 P r o b a b i l i t y = 0.00028 Difference between means of means i s highly s i g n i f i c a n t . Table V. cont Parenchymatous Goitre Control 350.48 356.13 376.60 387.69 394.50 436.79 438.67 440.25 444.12 475.47 486.49 486.59 489.44s, 503.88 504.22 540.56 553.39 578.44 637.09 653.23 Mean = 476.70 Variance = 7350.061 383.41 425.53 443.84 445.71 447.83 451.92 464.95 472.32 473.69 499.99 512.26 520.83 538.78 547.16 553.06 561.69 561.89 578.01 581.43 596.91 Mean = 503.06 Variance = 3665.678 P r o b a b i l i t y = 0.26842 Difference between means of means i s not s i g n i f i c a n t . test f o r differences between the means of means f o r each experimental tissue and i t s control. Frequency d i s t r i b u t i o n s of the means were plotted and are shown i n Fi g s . 12 - 17. The class size of 100 A i s that generally used i n t h i s type of work. No difference i n the basement membrane width between the hemithyroidectomy and i t s control was found, nor was there any difference i n parenchymatous (ClOjjF) g o i t r e . A highly s i g n i f i c a n t difference was found f o r c o l l o i d goitre (p a 0.00028). No other change i n ult r a s t r u c t u r e was found; the basement membrane had remained a continuous and f a i r l y uniform sheath throughout. Electron micrographs of representative conditions are shown i n Fi g s . 18 - 20. Stereologlcal Analysis The basement membrane that i s considered i n t h i s study i s e s s e n t i a l l y the coating of a sphere. The most accurate measurement of t h i s basement membrane would be made on a section that cut through the exact middle of the f o l l i c l e . However, f i n d i n g the exact middle would require s e r i a l sections and such an approach would be impractical. Therefore, random sections were used i n t h i s study. Most of these sections w i l l not pass through the middle of the f o l l i c l e , and the true width of the basement membrane w i l l be masked not only by er r o r but also by bias, the e f f e c t of sectioning away from the middle of the f o l l i c l e . I t i s e s s e n t i a l to get an estimate of bias i n order to make an accurate inference of the true width of the basement membrane. 27 F i g . 12 Frequency d i s t r i b u t i o n of means of basement membrane widths — hemlthyroldeotomy. 12*Qi. i l . Q . a * a . 8-0.. b 7 . a i a 5.Q. b 4 W 3'QI 1*Q. 0.Q 4- 4- 4-200*0 300*0 400*0 500*0 600*0 700*0 800*0 THICKNESS OF BASEMENT MEMBRANE WIDTH (A) 28 F i g . 13 Frequency d i s t r i b u t i o n of means of basement  membrane widths — control f o r  hemi thyrolde ctomy. 12'GU. 11*0.. 9*0.. 8'Q. 7*0.. 6-Q. 5.Q. 3»Qi 1*Q 0*0. + + 800*0 300*0 400*0 500*0 600*0 700*0 800*0 THICKNESS OF BASEMENT MEMBRANE WIDTH (A) 29 F i g . 14 Frequency d i s t r i b u t i o n of means of basement membrane widths — parenchymatous g o i t r e . 12*04. 11*0.. P* 10*Q. 9*0.. 8.Q. 6-Q. 5*0.. I 300*0 300*0 400*0 500*0 600*0 700*0 800*0 THICKNESS OF BASEMENT MEMBRANE WIDTH (A) 30 F i g . 15 Frequency d i s t r i b u t i o n of means of basement membrane widths — control f o r parenchymatous goitre i i * a . Jfl ±o*a. 9.Q. e*a. b ™ . 6 * 0 . 5*Q. 3*Q. e*a. i . a . o*oJ + •0 300*0 400*0 500*0 600*0 700*0 600*0 THICKNESS OF BASEMENT MEMBRANE WIDTH (A) 31 F i g . 16 Frequency d i s t r i b u t i o n of means of basement membrane widths — c o l l o i d g o i t r e . lE'OU. li*Ol P* lD*Qi 9 * O L B-Ol 6»0l 5 « Q b 4" 3*01 S.Ql 1'Oi I I I 1 I I aOO'O 300*0 400*0 900*0 600*0 700*0 600*0 THICKNESS OF BASEMENT MEMBRANE WIDTH (A) 32 F i g . 17 Frequency d i s t r i b u t i o n of means of basement membrane widths — control f o r c o l l o i d goitre l£*Oi. ll'Q. P» 1D*Q. 9.Q. B»Q. b *< G * O . 5*Q. 3*01 s*a. 1*Q. O*Q + + •0 300-0 400*0 500*0 600*0 700*0 800*0 THICKNESS OF BASEMENT MEMBRANE WIDTH (A) 33 F i g . 18 Ty p i c a l portion of thyroid c e l l showing basement  membrane. a. hemithyroidectomy b. control f o r hemithyroidectomy epi = thyroid epithelium bm = e p i t h e l i a l basement membrane end = endothelium 34 F i g . 19 T y p i c a l portion of thyroid c e l l showing basement membrane. a. parenchymatous goitre b. control f o r parenchymatous goitre epi = thyroid epithelium bm = e p i t h e l i a l basement membrane end = endothelium 35 F i g . 20 T y p i c a l portion of thyroid c e l l showing basement  membrane. a. c o l l o i d goitre b. control f o r c o l l o i d goitre epi = thyroid epithelium bm = e p i t h e l i a l basement membrane end = endothelium ct = connective tissue c e l l The bias can be calculated knowing the measured values of basement membrane thickness and the size of thyroid f o l l i c l e s . An equation which w i l l predict the e f f e c t of bias can be derived as follows: T i s the r e a l thickness of basement membrane. t i s the measured thickness — the best possible To f i n d the average value of t, the following data are needed: the several values of t that were found, the frequency with which the values were found, and the range over which the values were found. Thus, to f i n d the average value of t, E{t}, the function of the frequency of the occurrence of an event (the frequency of being able to f i n d t ) , f ( x ) , i s multi p l i e d by the function of which the average i s being found, t ( x ) , and then the product i s integrated over a l l possible values of x ( i . e . a l l possible places where t can be found). This gives the equation: However: t h i s i s a uniform d i s t r i b u t i o n and x has equal chances of occurring over i t s range. Therefore: f(x) i s a constant and i s equal to l/length where x occurs. Therefore: f(x) may be withdrawn from the i n t e g r a l and estimate since average values of measurements must be used. (1) t(x) integrated over the range where t(x) may be found. fx Equation (1) becomes: E ft] = -v-y I t(x) dx JQ (2) Prom Pig. 21, i t can be seen that x = D - T, so: • r l 1 f D " T E(t|= V D - T / d x (3) 3 J 0 Now: t = d n - d, (See F i g . 21) and d Q 2 = D 2- X 2 2 2 2 d i = (D - T) - X* Therefore: t = (D 2 - X 2 ) * - ((D - T ) 2 - X 2 ) * (4) Substituting the value of t from equation (4) into equation (3), and putting D - T = DT, equation (4) becomes E(t}= VDT j ^ l V 2 - X 2 ) * dx - ^  D T 9 2 * (DT^ - X r dx 0 (5) This equation can be integrated and s i m p l i f i e d : w/f\ V ™ X(D 2- X 2 ) ^ ^  D 2 . -1 X X(DT 2- X 2 ) ^ E(.t^= /DT 2 + 2~ s l n D 2 2 IDT - -g— s i n •1 X_ " T D T Jo 2 ^ DT (D 2- DT 2)* 4 D 2 s i n ' 1 g£ - DT 2 + f -1 ( D 2 - D f 2 ) i + D£ s i n - l DT _ D T r £ (D 2 - D 2 + 2(D)(T) - T 2 ) * + . . . Diagram of the oytol o g l c a l o r i g i n of the symbols  used i n the stereologioal a n a l y s i s . radius of f o l l i c l e true width of basement membrane radius of thyroid f o l l i c l e ( c e l l s only) apparent thickness of basement membrane estimate of D i n the section where t i s found estimate of D-T i n the section where t i s found any portion of f o l l i c l e used i n analysis zone of a c c e p t a b i l i t y 39 E (t) _ 1 ,2D . v l ^ D f -IDT D T t . T *" 2 ^ T " ' DT(T) s l n D " T 2 Now, t h i s equation allows f o r t to be measured anywhere i n the f o l l i c l e . However, certain portions of the f o l l i c l e that give l e s s accurate measurements can be eliminated by inspection. The nearer the measurements are to the centre of the f o l l i c l e , the more c l o s e l y w i l l the measure-ments approach the r e a l value, and as a r e s u l t , the le s s w i l l be the error. A l l sections that are obviously not near the centre are eliminated. Such sections would be those that, while showing the lumen, show also at l e a s t one transection of a l a t e r a l cellsmembrane. Only those sections showing a perfect monolayer would be acceptable. If t h i s "zone of a c c e p t a b i l i t y " i s designated as D - E, or DE, (Fig. 22) an equation predicting bias i s obtained: _ 1 X(D 2- X 2 ) ^ D 2 4 -1 X X(DT 2- X 2 ) ^ E ( t ) D E = 5E 2 + 2~ s i n D 2 DT 2 . -1 X~1 D E - — s i n m j Q 1 , 2 2 -1 DE , 2 ZA = •gjjjgr DE(D - X ) + D s i n - DE (DT - DE ) - D ^ s i n ' 1 ^ (?) If the graph of t h i s equation i s plotted with various portions of the radius of the f o l l i c l e as D - E, the graph shown i n F i g . 23 i s obtained. Equation (7) can also be used to gauge the e f f e c t 40 P i g . 22 Diagrammatic r e p r e s e n t a t i o n of the determina-t i o n of the zone of a c c e p t a b i l i t y . D = r a d i u s of f o l l i c l e ( c f F i g . 21) \ 41 F i g . 2 3 A graph of apparent thickness of basement  membrane as a function of the Interval over which measurements are made. CO 1.4+ 1.34 24.5% X 1-24 15.9%' X 10.8% X 1.14 7.3 %X 4.7 %X 0.2% 0.7% X - I -0.2 .6%X 1-2.9%X — r — 0.8 0.1 0.3 —I— 0.4 — f -05 — l — 0.6 —I-0 7 0.9 MEASUREMENT INTERVAL (PORTION OF RADIUS) (DE) of f o l l i c l e size on the accuracy of measurements. While f o l l i c l e size i s variable (Jackson, 1931), the methods of sampling f o l l i c l e size emphasize t h i s v a r i a b i l i t y . We need to be sure that the v a r i a b i l i t y does not i n -validate the measurements by changing the bias through the range of f o l l i c l e s i z e s . But, i f a given value of DE and a range of values f o r D are substituted into equation (7), a range of values i s obtained f o r t/T, which i s the r a t i o of measured thickness to true thick-ness. These values are summarized i n Table VI. I t can be seen that the t/T r a t i o s do not change as the T/D r a t i o s ( r a t i o s of basement membrane thickness to f o l l i c l e radius) ohange u n t i l DE becomes quite substantial, and then the t/T r a t i o s change only s l i g h t l y . The e f f e c t of bias f o r each of the 6 tissues can be estimated using c e l l widths and f o l l i c l e diameters. The c e l l widths give an idea of the size of the "zone of a c c e p t a b i l i t y " . No section was acceptable that showed a transection of a l a t e r a l c e l l membrane, so the zone of a c c e p t a b i l i t y i s lim i t e d by the c e l l widths ( i . e . i t i s one c e l l wide). The zone w i l l , of course, be d i f f e r e n t i n d i f f e r e n t t i s s u e s . In F i g . 24 zones of a c c e p t a b i l i t y f o r the 6 tissues are shown as per cent from centre. These zones were calculated by d i v i d i n g the mean c e l l width by one-half the mean f o l l i c l e diameter t one-half f o l l i c l e diameter standard deviation. The brackets i n Fi g . 24 enclose the range of the possible extremes of Table VI. Relationship of t/T and T/D Ratios to DE (Portion of F o l l i c l e Examined) DE T/D (x 1 0 " 3 ) t/T (x 1 0 " 1 ) Q.l 1.000 0.1002 3.162 0.1002 0 .2 1.000 0.1007 3.162 0.1007 0.3 1.000 0.1016 3.162 0.1016 0.4 1.000 0.1029 3.162 0.1029 0.5 11000 0.1047 3.162 0.1047 0.6 1.000 0.1073 3.162 0.1073 0.7 1.000 0.1108 3.162 0.1108 0.8 1.000 0.1159 3.162 0.1160 0.9 1.000 0.1245 3.162 0.1246 0.95 1 .000: 0.1320 3.162 0.1322 0.985 1.000 0.1421 3.162 0.1426 . 24 Bias l i m i t s f o r treatments. CONTROL s= control f o r hemithyroidectomy RLOBE = hemithyroidectomy COLLCON = control f o r c o l l o i d goitre COLLOID = c o l l o i d goitre CONF = control f o r parenchymatous goitre CLOjjF = parenchymatous goitre RLOBE COLLCON COLLOID 24.5%. X CONF "V CL04F J 15.9 %X 10.8% X 7.3°/«< 4.7 %X 0.2% * 0.7% X 1.6% X 2.9%X 0.1 1 0.2 1 0.3 1 0.4 1 0.5 1 0.6 1 07 1 0.8 I 0.9 MEASUREMENT INTERVAL (PORTION OF RADIUS) (DE) the zones of a c c e p t a b i l i t y . I t must be remembered that not a l l measurements were taken at the worst possible bias, but were randomly d i s t r i b u t e d and had equal chances of occurring anywhere from the centre up to the l i m i t s of worst possible bias. However, i n order to test the surety of the t - t e s t probabil-i t i e s , the bias l i m i t s can be used to modify the means of basement membrane widths. I f , when the means are modified and the t - t e s t * i s repeated, the p r o b a b i l i t i e s s t i l l stand, then the v a l i d i t y of the t - t e s t i s enhanced. The t-tests f o r modified means f o r c o l l o i d goitre and CIO4F goitre are shown i n Table VII. E s s e n t i a l l y what has been done i s that the means have been divided by a fa c t o r that accounts f o r bias. This f a c t o r i s obtained (from F i g . 24) by taking the inverse of the sum of the amount of bias calculated to be present at the extreme l i m i t of the zone of a c c e p t a b i l i t y and the e f f e c t of no bias (100$). For c o l l o i d g o i t r e , the fa c t o r i s 0.93» and i t i s applied to the mean f o r the experimental t i s s u e . As can be seen i n Table VII, t h i s has the e f f e c t of pushing the means f o r experimental and control closer together, but the t- t e s t shows that there i s s t i l l a s i g n i f i c a n t d i f f e r e n c e . For ClO^F goitre the fa c t o r i s 0.98, and i t i s applied to the mean f o r the control tissue. Applied t h i s way, the fact o r tends to push the means far t h e r apart. A t - t e s t done a f t e r t h i s modification shows that there i s s t i l l no s i g n i f i c a n t difference. Table VII* E f f e c t of Bias Modifications on t-t e s t s  on Parenchymatous Goitre and C o l l o i d Goitre, Parenchymatous Goitre 2 x s  476.701 7350.061 P r o b a b i l i t y = 0.26842 476.701 7350.061 P r o b a b i l i t y = 0.12943 C o l l o i d Goitre  2 x s  346.886 3453.773 Pr o b a b i l i t y = 0.00028 (Adjusted by fa c t o r = 0.93) 372.995 3993.229 P r o b a b i l i t y = 0.01737 Control 503.060 3665.678 ; Not s i g n i f i c a n t (Adjusted by fact o r = 0. 513.326 3816.797 ; Not s i g n i f i c a n t C o l l o i d Control  2 x s  420.823 3401.060 ; Highly s i g n i f i c a n t 420.823 3^01.060 ; S i g n i f i c a n t Discussion The purpose of t h i s investigation was to study the ultrast r u c t u r e of the "basement membrane i n an experiment-a l l y Induced hyperplasia. From Table I i t can be seen that the treatments did Indeed cause the thyroid gland to enlarge by what i s considered to be hyperplasia (Voitkevich, 1964; Turner, 1973.) • When the basement membrane was measured and corrections f o r bias made i n each of the enlarged glands and then compared to the basement membrane i n the control f o r each experimental condition, i t was found that: 1. There was no change i n basement membrane width i n hemi thyroidectomy..^: 2. There was no change l n basement membrane width i n parenchymatous g o i t r e . 3. There was a change i n basement membrane width i n c o l l o i d goitre — the basement membrane was s i g n i f i c a n t l y thinner when compared with i t s c ontrol. As F r i e d e r i c i (1966) , A l o u s i et a l . (1969), and Irvine and Muir (1963) have observed, a change i n the thickness of basement membrane i s one of the most s t r i k i n g features of certain pathological states. Thus, basement membrane thickness i s considered to be one of the more important aspects of the pathological conditions induced i n t h i s study. However, merely observing a thinning or a thickening i s not s u f f i c i e n t to evaluate a pathological state. Stereological methods are required i f an accurate estimate of "basement membrane thickness i s to be obtained. Several ways to measure basement membranes have already been devised. iWaiserhouse and Squier (1969) have been interested i n the basement membrane of oral epithelium and, thus, i n the best way to measure the basement membrane when i t i s present as an undulating sheet. Their measure-ments, when plotted as a histogram, show a decidedly skewed d i s t r i b u t i o n which, according to these two workers, indicates that most of t h e i r measurements are too large and that the median and I. Q. R. ( i n t e r - q u a r t i l e range) are, therefore, the best estimates of thickness and error respectively. The d i f f i c u l t y here l i e s i n the lack of clues as to orientation; there i s no way of knowing i f the measurement axis i s exactly perpendicular to the basement membrane. Other researchers have been studying the cylinders that are c a p i l l a r y basement membranes, e s p e c i a l l y i n muscles and the glomeruli. Osawa et a l . ( I 9 6 6 7 ) , working on the glomerulus, measured from the c e l l membrane of the endothelial c e l l s to that of the e p i t h e l i a l foot processes, excluding a l l s i t e s that were "unsuitable", s i t e s where the c e l l membrane was not sharp, i n d i c a t i n g tangential sectioning, s i t e s bordering mesangial cytoplasm, or areas of to r t u o s i t y . Means and standard deviations were used as estimates of thickness and error. Jorgensen and Bentzon (1968) devised a d i f f e r e n t method to study glomerular basement membrane, measuring only i n the peripheral portions and eliminating those areas where one c e l l ' s membranes were not sharp. They measured the area of selected portions of basement membranes and related the area to the length of the portions (measured at the midpoints of the basement membrane) to f i n d the thickness. The thicknesses were expressed as logarithms to give a normal d i s t r i b u t i o n , with the geometric mean as an estimate of thickness and three d i f f e r e n t variances to estimate error. Basement membrane of muscle c a p i l l a r i e s i s of intere s t to workers i n the f i e l d of diabetes research. Vracko and Strandness (1967), using a planimeter, took the area occupied by the basement membrane and related i t to the area of the whole ve s s e l . This method eliminated several d i f f i c u l t i e s such as choosing a s i t e at which to measure and an angle at which to measure. I t also eliminated v a r i a t i o n due to functional shrinkage and distension of the vessel, or differences i n c a l i b e r of c a p i l l a r y , and permitted sections to be taken at an angle. Siperstein et a l . ( 1968) , also working on muscle c a p i l l a r y , used a grid to choose measurement s i t e s . They eliminated those s i t e s that crossed pericytes and in s i s t e d on a minimum of 10 measurements per c a p i l l a r y . Their estimate of thickness and error i s expressed as the mean and standard error. When the thickness of thyroid "basement membrane i s to be discussed, a certain amount of d i f f i c u l t y i s encount-ered. In cross-section, the basement membrane appears as a c i r c l e concentric with a c i r c l e of thyroid c e l l s , not unlike those surrounding c a p i l l a r i e s , and i t would seem easy enough to just measure across i t . However, the thyroid f o l l i c l e i s a sphere and a random section through the sphere may not pass through the best region to give the best estimate of basement membrane thickness. The id e a l section from a thyroid f o l l i c l e would cut through the exact middle. This would give the thinnest and most accurate measurement of basement membrane thickness. Finding the middle of the f o l l i c l e would require s e r i a l sections and such a systematic approach would be labor-ious, expensive of time, and yet not very accurate. Therefore, random sections must be used, and these sections w i l l probably not be through the centre. The true width of the basement membrane w i l l be masked by error plus bias, and i t i s e s s e n t i a l to get an estimate of these s t a t i s t i c s . The estimate of bias was calculated as outlined previously and the bias values plotted on the grapte i n F i g . 23. This graph shows, f o r example, that i f a l l measurements are made within k0% of the distance from the centre of the f o l l i c l e to the edge, the measured thickness of the basement membrane i s affected by a maximum bias of le s s than % . By employing cert a i n c r i t e r i a , a l l sections obviously not near the centre can be eliminated (see "Results" section). There w i l l remain a zone of accept-a b i l i t y straddling the middle of the f o l l i c l e , that can be used to estimate worst possible bias l i m i t s f o r each of the t i s s u e s . The bias l i m i t s are shown i n Pig. 2k superimposed on the graph from F i g . 23. I t can be seen that i n only two cases did the bias l i m i t s exceed 3% ( c o l l o i d goitre and parenchymatous goitre c o n t r o l ) , and that these were l i m i t e d to a maximum bias of l e s s than 8%. Therefore, the manner of sampling ensures small bias. Since there was considerable v a r i a t i o n i n f o l l i c l e size within each treatment, i t was necessary to ascertain the e f f e c t of f o l l i c l e size on bias. Table VI summarizes the r a t i o s of measured thickness to true thickness of the basement membrane f o r various values of DE, the portion of the f o l l i c l e studied. The t/T r a t i o s f o r large f o l l i c l e remain the same as the t/T r a t i o s f o r small f o l l i c l e s u n t i l DE exceeds 80% ( i . e . u n t i l the section taken i s f a r from the centre). F i g . 2k shows that DE never exceeds 6.5% and, therefore, bias i s independent of f o l l i c l e size i n t h i s study and the basement membrane may be confidently measured on any f o l l i c l e . T-tests were used to test f o r differences between treatment means. The bias can be used to strengthen the t - t e s t s . Since the strengthened t - t e s t s provided the same res u l t s as the o r i g i n a l tests, confidence i n the accuracy of the measurements i s reinforced. (See Table VII) The mean was considered an accurate estimate of "base-ment membrane width, even though the d i s t r i b u t i o n was truncated at both ends. At one end, the d i s t r i b u t i o n stops because the basement membrane does not become i n f i n i t e l y t h i n . At the other end, the d i s t r i b u t i o n does not continue outside the zone of a c c e p t a b i l i t y . Within these l i m i t s , the d i s t r i b u t i o n can be considered normal, Influenced by the sampling method and by the shape of the thyroid f o l l i c l e i n the region of examination. The major f i n d i n g i n t h i s study was that one of the treatments resulted in,a thinner basement membrane. An examination of the phy s i o l o g i c a l changes associated with each treatment i s necessary to evaluate the significance of t h i s f i n d i n g . The thyroid i s a bilobed, endocrine gland that l i e s on the trachea just below the larynx. The gland synthesizes iodine-containing hormones, p r i n c i p a l l y thyroxine and triiodothyronine, and they are produced at a rate i n f l u -enced by the p i t u i t a r y hormone, thyrotropin (TSH). As the thyroid hormone l e v e l drops i n the peripheral c i r c u -l a t i o n , TSH l e v e l s increase and act on the thyroid, stimulating both rate of release and rate of synthesis of the hormones. If peripheral hypothyroidism cannot be a l l e v i a t e d by the normal cybernetic mechanisms, the thyroid responds by becoming hyperplastic (Astwood, 1970). These changes are observed In both n a t u r a l l y occurring and experimentally induced g o i t r e s . The removal of one lobe of the thyroid causes the gland to regenerate. This growth i s i n response to reduced thyroid hormone l e v e l s and i s not merely an e f f e c t of trauma (Voitkevich, 1964) . The thyroid fragment shows an increased iodine-trapping a b i l i t y and an increased rate of radio-iodine turnover (Reichlin, 1958) . The growth of the remain-131 ing tissue i s f a i r l y rapid and by eight days, the I -/ uptake i s 90$ of normal (Logothetopoulos and Donlach, 1955)• These observations r e s u l t from increased TSH l e v e l s since sham-operated animals (Reichlin, 1958) and thyroxine treated animals (Voitkevich, 1964) do not react i n the same manner as hemi-thyroidectomized animals. Removal of more than one-half of the gland leads to a more severe hypothyroidism, and the stress on the thyroid fragment leads to c e l l u l a r hypertrophy and hyperplasia. But, i f only h a l f of the gland i s removed, new f o l l i c l e s are formed and c e l l heights approximate normal c e l l heights (Voitkevich, 1964). In t h i s study, no s i g n i f i c a n t d i f f e r -ence was found between c e l l heights of control and experi-mental tissue, nor was there any difference i n f o i l i c l e diameters. There was an increase i n size of the remaining lobe, so i t i s considered that the number of f o l l i c l e s must have increased. However, the animals' requirements f o r thyroid hormone would not cause the gland to increase i n size i n excess of "normal". Nor i s there any pathological condition present, and the hormone metabolism of the gland i t s e l f has not been a l t e r e d . The response to increased TSH l e v e l s i s mild and, thus, no a l t e r a t i o n s i n u l t r a -structure would be expected. Indeed, there was no s i g n i f i -cant difference between the widths of the basement membranes f o r the experimental and control tissues. Interference with hormone metabolism has a much more s t r i k i n g e f f e c t on the thyroid than does p a r t i a l thyroid-ectomy. If the dosage of a blocking agent i s s u f f i c i e n t , the gland w i l l synthesize no hormone at a l l , the peripheral hypothyroidism w i l l be severe, and the TSH l e v e l s w i l l be high. A marked hyperplasia should r e s u l t . The perchlorate ion, usually administered as a sodium or potassium s a l t , i s a hydrated mono-valent anion s i m i l a r i n size to iodide, and i t acts on the thyroid by i n t e r f e r i n g with the uptake of iodide ion. I t has the e f f e c t , then, of ^starving" the thyroid f o r iodine, a form of competitive i n h i b i t i o n which can be neutralized by the administration of excess iodide (Astwood, 1 9 7 0 ) . I t s use as a drug to combat hyperthyroidism i s much c u r t a i l e d by the prevalance of side-effects (skin and g a s t r i c i r r i t a t i o n s , and lymph-adenopathy). In t h i s study, a r e l a t i v e l y large dose of perchlorate ion was fed, and, as expected, the gland greatly increased i n s i z e . The gross pathological changes included a more uniform d i s t r i b u t i o n of f o l l i c l e size than the controls. C e l l heights increased d r a s t i c a l l y , causing the lumen to a l l but disappear. However, no s i g n i f i c a n t change i n basement membrane thickness was found, and bias corrections d i d not produce s i g n i f i c a n t differences. The lack of s i g n i f i c a n t differences i s almost c e r t a i n l y due to the large variance associated with the treatment (7350.061 as compared to 3665.678 f o r the controls), since there was a considerable difference i n the means. This variance i s probably not the r e s u l t of side-effects of ClOj^F. I r r i t a t i o n caused by the ingestion of the drug may well stimulate the secretion of ACTH, which i n t e r f e r e s with the production of TSH (D'Angelo, 1953; Carriere and I s l e r , 1959). hut i f TSH secretion i s hampered, the e f f e c t i s not l i k e l y to be observed i n some thyroid f o l l i c l e s and not i n others. The variance i n basement membrane widths i s more l i k e l y to be influenced by the manner i n which the number of f o l l i c l e s i s increased. The thinner basement membranes might be found surrounding newly-budded f o l l i c l e s while the thicker ones may be found on older f o l l i c l e s i n a dormant stage. The tissue examined here i s s t i l l under stimulation and i s s t i l l undergoing hyperplasia, and i t i s not l i k e l y that a l l f o l l i c l e s present are i n the same stage of growth. P r o p y l t h i o u r a c i l i s an a n t i - t h y r o i d agent that i n h i b i t s the formation of thyroid hormone by Interfering with the binding of iodine into an organic form. Propyl-t h i o u r a c i l produces a parenchymatous goitre s i m i l a r to that produced by the perchlorate ion. The mechanism of action of p r o p y l t h i o u r a c i l would seem to be competitive i n h i b i t i o n of a peroxidase system that oxidizes the iodide ion.and e f f e c t s the coupling of mono- and d i - iodotyrosines to tri-iodothyronine and thyroxine (Astwood, 1970; Morris and Hager, 1966; Maloof and Soodak, 1964; Bjousksten, 1966). A PTU-induced parenchymatous goitre can become trans-formed into a c o l l o i d goitre ( F o l l i s , 1959) hy removing the anti - t h y r o i d agent. In t h i s study the administration of PTU induced a parenchymatous goitre as Judged by c e l l heights (Table I I I ) . The removal of PTU resulted i n a lowering of c e l l heights. The formation of c o l l o i d goitre from experimental parenchymatous goitre may be brought about i n the following manner. A l l Tyanused iodine has been excreted previously. Once the block i s removed, there i s an immediate demand f o r a l l a vailable iodine, but there i s not s u f f i c i e n t iodine to s a t i s f y the requirements. TSH l e v e l s remain high and a l l iodine present i s u t i l i z e d . TSH continues to stimulate growth of the thyroid, causing c o l l o i d accumulation i n the lumen (Greer,et a l . , 1967; Ryan, 1968) . The period follow-ing goitrogen withdrawal i s marked by a rebound i n I ^ l uptake and altered mono- / d i - iodotyrosine and t r i -iodothyronine / thyroxine r a t i o s (Studer and Greer, 1967) . A l l features return to normal a f t e r a prolonged recovery period. The course of c o l l o i d goitre i s considerably d i f f e r -ent from that of parenchymatous g o i t r e . The growth of the thyroid i n parenchymatous goitre i s gradual while that of c o l l o i d goitre Is almost explosive. I t has already been shown that regeneration and the growth of the gland i n parenchymatous goitre do not s i g n i f i c a n t l y a l t e r the width of the basement membrane. I f basement membranes are produced p r i n c i p a l l y by t h e i r associated tissues, i t would be reasonable to assume that the rate of growth of the gland i n c o l l o i d goitre i s responsible f o r the basement membrane change. Considerable evidence has accumulated that e p i t h e l i a l tissues are the prime contributors to the basement membrane that underlie and support them. This evidence has been collected with such varied techniques as conjugated a n t i -bodies, s i l v e r n i t r a t e stains, and t r i t i a t e d glucose. (Pierce et a l . , 1962, 1963; Pierce et a l . , 1964; Mukerjee et a l . , 1965; Kurtz and Feldman, 1962; Nadol and Gibbons, 1970). Any a l t e r a t i o n or renewal of thyroid basement membrane would be the major " r e s p o n s i b i l i t y " of the appropriate synthetic mechanisms i n the thyroid gland. The change i n basement membrane thickness i n c o l l o i d goitre could be due to a f a i l u r e of the gland to synthesize a d d i t i o n a l basement membrane material when the gland was growing most rapidly, the rapid growth causing the base-ment membrane to stretch. If stretching i s a tenable hypothesis, then examination of the chemical nature of the basement membrane must show a composition or structure that would permit stretching to occur. The chemical composition of basement membranes has been studied by Mukerjee et a l . (1965) and by Misra and Berman (1966, 1968). Protein i s the major constituent (about 80$) and the carbohydrates are primarily reducing sugars and amine sugars. Diseased glomerular basement membrane has an increased cholesterol content and the glycoproteins are much altered, probably through the linkages of s i a l i c acid to other constituents with the glycoprotein. However, the chemical changes were not enough to explain a l l the changes i n kidney physiology. Misra and Berman (1968) speculate that the disease a l s o brings about changes i n the arrangements of constituents. Certainly, i n experimental nephrosis, there are changes i n both electrophoretic mobility and the d i f f r a c t i o n patterns of glomerular basement membrane, probably due to loss of phospholipid and increased ordering within the c r y s t a l l a t t i c e (Kalant et a l . , 1966). Gang et a l . (1970) confirm the loss of phospholipids i n experimental n e p h r i t i s , t h i s l o s s coinciding with the onset of albuminuria and the permeability of the glomerular basement membrane to lanthanum hydroxide, a small, electron-dense p a r t i c l e . The basement membrane would seem to be f a i r l y l a b i l e . F r i e d e r i c i (1965) observed that c a p i l l a r y basement membrane would repair a gap formed by the extrusion and budding off of a projection of endothelium. Whole lymphocytes have been found within the boundaries of basement membrane of normal thyroid, and yet no breaks were observed i n the basement membrane (Irvine and Muir, 1963; Toujas et G u e l f i , 1969) . The d i f f e r e n t f i l t r a t i o n properties of glomerular basement membrane of diseased kidney are thought to be due to a less cross-linked structure (Huang et a l . , 196?; Gang et a l . , 1970; Parquhar and Palade, i 9 6 0 ; Misra and Kalant, 1966) . Larger molecules can pass through the basement membrane i n nephrosis and n e p h r i t i s ( f e r r i t i n -Parquhar and Palade, I960; l a b e l l e d glucose and urea -Gelke et a l . , 1966a, 1966b; lanthanum hydroxide - Gang et a l . , 1970) . These f i l t r a t i o n changes are not attributed s o l e l y to chemical changes (Misra and Kalant, 1966; Gang et a l . , 1970) . P r i e d e r i c i et a l . (1966), i n studies on experimental scurvy, found a much thinner basement membrane surrounding affected c a p i l l a r i e s . Affected c a p i l l a r i e s are characterized by increased c e l l s i z e , and increased synthetic and secre-tory apparatus. These authors cannot explain why the c a p i l l -ary c e l l s are active and yet there i s a reduction i n basement membrane material, when the c a p i l l a r y presumably produces mush of i t s own basement membrane. In scurvy, the size of the c e l l s i s much increased, a l l but o b l i t e r a t i n g the lumen. F r i e d e r i c i et a l . (1966) give no measurements, but i t i s possible that the increased c e l l size also i n -creases the circumference of the c a p i l l a r i e s . The base-ment membrane would thus be stretched. Another instance of a thinner basement membrane i s found i n Hashimoto's t h y r o i d i t i s (Irvine and Mulr, 1963). This i s not the r e s u l t of growth rate, as Hashimoto's t h y r o i d i t i s develops over several years. The basement menfe brane i n normal human thyroid i s a very unusual structure, much reduplicated and multilayered. I t s l i k e i s not seen i n other tissues. The reduplication and multilayeredness i s not a f i x a t i o n a r t i f a c t , else Irvine and Muir would not have found i t only i n normal tissue, nor can I t be repro-duced by deliberate poor f i x a t i o n (Mackenzie, 1966). There are no micrographs available showing how the base-ment membrane may change from what Is found i n normal thyroid to the thinner, single-layered basement membrane in Hashimoto's t h y r o i d i t i s . Hashimoto's t h y r o i d i t i s i s one of several n a t u r a l l y occurring thyroid disorders i n humans. From only two of these disorders can there be recovery. One i s the goitre due to diet a r y iodine deficiency, which i s corrected by the addition of iodine to the d i e t . The other i s adolescent idiopathic c o l l o i d g o i t r e . This goitre occurs at the onset of puberty and spontaneously regresses l a t e r i n adolescence (Nilsson, 1966) . Many goitres can be mimicked i n laboratory animals, but when the experimental stress on the thyroid i s removed, the animals recover. In humans, a high incidence of thyroid disorders within f a m i l i e s Indicates that there i s a genetic predisposition toward g o i t r e s . Inborn errors i n structure or metabolism w i l l not be observed u n t i l there Is some stress on the thyroid (Nilsson, 1966; Fraser, 1969). Experimental animals are probably able to recover from induced thyroid disorders because a l l such errors would have been selected against and there would be no predis-p o s i t i o n remaining l n the stock. When goitre i s induced, the l a b i l i t y of the basement membrane would prevent t o t a l i disorganization during hyperplasia. Maintenance of gland i n t e g r i t y would a i d recovery when the stress was removed. U l t r a s t r u c t u r a l changes i n the basement membrane, other than reduced thickness, were not observed i n t h i s series of experimentally induced hyperplasias. Those reports that described thickening or s t r u c t u r a l changes dealt with n a t u r a l l y occurring pathological conditions f o r which genetic predisposition, also, was often evident. I t i s l i k e l y that these authors were describing syndromes, and the changes they^observed were not due d i r e c t l y to to a single causative f a c t o r . Indeed, Slperstein et a l . (1968) examined animal models f o r thickening of basement membrane,inamuscle c a p i l l a r i e s . Chinese hamster with spontaneous non-diabetic hyperglycemia did not show any Increase i n thickness of basement membrane, such as i s found i n diabetes. I t i s , therefore, u n l i k e l y that interrupting only one phase of thyroid metabolism would Induce spectacular change i n the structure of the basement membrane. While c o r r e l a -tion does not necessarily imply a cause and e f f e c t r e l a t i o n ship, the close c o r r e l a t i o n between rate of growth and basement membrane change make the hypothesis of basement membrane stretching a very tenable one. This hypothesis i s also consistent with the changes found i n perchlorate goitre where rate of growth i s more v a r i a b l e . 63 Summary 1. Hyperplasia; was induced experimentally i n r a t thyroid to study the e f f e c t of hyperplasia on the basement membrane. 2. Hyperplasia was induced by hemithyroidectomy, by feeding perchlorate ion (parenchymatous g o i t r e ) , and by i n j e c t i n g p r o p y l t h i o u r a c i l and allowing a recovery period ( c o l l o i d g o i t r e ) . 3 . The enlarged thyroids were examined by electron microscopy and the basement membrane width subjected to s t e r e o l o g i c a l a n a l y s i s . 4. There was no s i g n i f i c a n t change i n basement membrane ultrast r u c t u r e i n hemithyroidectomy or i n parenchyma-tous g o i t r e . A s i g n i f i c a n t l y thinner basement membrane was found i n c o l l o i d g o i t r e . 5. The c o r r e l a t i o n between f a s t growth rate and thinner basement membrane i n c o l l o i d goitre suggest that the basement membrane had been stretched. E x i s t i n g knowledge of structure and chemical composition of basement membrane lends support to t h i s conclusion. Bibliography A l o u s i , M. A., R. S. Post, and W. Heymann. I 9 6 9 . Experimental autoimmune nephrosis i n r a t s . Amer. J . Pathol. 54: 35-45. Astwood, E. B. 1970. Thyroid and a n t i t h y r o i d drugs. p. 1466-1500. In Goodman, L. S. and A. Gilman,(eds.) The pharmacological basis of therapeutics. 4th ed. Macmillan, New York. Bjorksten, P. 1966. Do thionamide antit h y r o i d compounds act as free r a d i c a l scavengers? Biophys. Biochim. Acta 127: 265-268. Carriere, R. and H. I s l e r . 1959. E f f e c t of frequent housing changes and of muscle exercise on the thyroid gland of mice. Endocrinol. 64: 414-418. Cohen, A. I. I 9 6 I . Electron microscope observations on developing mouse eye. 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Kalant. 1970. Nephro-toxic serum n e p h r i t i s I I : chemical, morphologic, and functional correlates of glomerular basement membrane at the onset of proteinuria. Lab. Invest. 23: 150-157. Gang, N. F., E. Trachtenburg, J . j&llerhand, N. Kalant, and W. Mautner. 1970. Nephrotoxic serum n e p h r i t i s I I I : 65 c o r r e l a t i o n of proteinuria, excretion of the glomerular basement membrane-like protein, and changes i n the ultrastructure of the glomerular basement membrane as vi s u a l i z e d with lanthanum. Lab. Invest. 23: 436-441. Gelke, D., P. V. Bruchhausen, and G. Fuch. 1966. The size of the pore equivalents i n is o l a t e d basement membrane of the r a t kidney. (Transl. from German). Pflugers Arch. Gesamte Physiol. Mens Tiere 289: 180-190. Gekle, D., F. V. Bruchhausen, and G. Fuchs. 1966. Pore equivalents r a d i i of the is o l a t e d basement membrane of r a t kidney following the action of amino nucleoside. (Transl. from German). Pflugers Arch. Gesamte Physiol. Mens Tiere 290: 250-257. Greer, M. A., H. Studer, and J . W. Kendall. I 9 6 7 . Studies on the pathogenesis of c o l l o i d g o i t r e . Endocrinol. 81: 623-632. Huang, F., L. Hutton, and N. Kalant. I 9 6 9 . Molecular sieving by basement membrane. Nature 216: 8788. Irvine, W. J . and A. R. Muir. 1963* An electron microscope study of Hashimoto's t h y r o i d i t i s . Quart. J . Exp. Phys i o l . 48: 13-26. Jackson, J . L. 1931• The size and shape of the human thyroid f o l l i c l e i n health and disease. Anat. Rec. 48: 219-237. Jorgensen, F. and M. W. Bentzon. 1968. The ultrastructure of the normal human glomerulus. Lab. Invest. 18: 42-48. Kalant, N., R. P. Misra, R. Manley, and J . Wilson. 1966. Glomerular basement membrane i n experimental nephrosis: X-ray d i f f r a c t i o n and electrophoretic studies. Nephron 3 : 167-172. Kurtz, S. M. and J . B. Feldman. 1962. Experimental studies on the formation of the glomerular basement membrane. J . U l t r a s t r u c . Res. 6: 19-27. Kushida, H. and K. F u j i t a . 1964. A method to mount thin sections d i r e c t l y on supporting gr i d s . J . E l e c t r o n Microscop. 13: 27-28. Logothetopoulos, J . H. and I. Doniach. 1955* Compensatory hypertrophy of the rat thyroid a f t e r p a r t i a l thyroid-ectomy. B r i t . J . Exp. Pathol. 36: 617-627. 66 Mackenzie, M. J . 1966. A study of delayed f i x a t i o n on the u l t r a s t r u c t u r e of the basement membrane of the rat thyroid. B. Sc. thesis, U. B. C. Maloof, F. and M. Soodak. 1964. Competition between several a n t i t h y r o i d compounds and iodide f o r a common oxidizing enzyme system i n thyroid t i s s u e . J . C l i n . Invest. 43: 1292. Menefee, M. G. 1957. Some fi n e structure changes occurring i n the epidermis of embryo mice during d i f f e r e n t i a t i o n . J . U l trastrue. Res. 1: 4 9 - 6 1 . Misra, R. P. and L. B. Berman. 1966. Studies on glomerular basement membrane I. I s o l a t i o n and chemical analysis of normal glomerular basement membrane. Proc. Soc. Exp. B i o l . Med. 122: 705-710. Misra, R. P., and L. B. Berman. 1968. Studies on glomerular basement membrane. I I . I s o l a t i o n and chemical analysis of diseased glomerular basement membrane. Lab. Invest. 18: 131-138. Misra, R. P. and N. Kalant. 1966. Glomerular basement membrane i n experimental nephrosis: chemical composi-tio n . Nephron 3: 84-102. Morris, D. R. and L. P. Hager. I966. Mechanism of the i n h i b i t i o n of enzymatic halogenation by a n t i t h y r o i d agents. J . B i o l . Chem. 241: 3582-3589. Mukerje, H., J . S r i Ram, and G. B. Pierce. I 9 6 5 . Basement membranes V. Chemical composition of neo-p l a s t i c basement membrane mucoprotein. Amer. J . Pathol. 46: 49-57. Nadol, J . B. and J . R. Gibbons. 1970. Autoradiographic evidence f o r the e p i t h e l i a l o r i g i n of glucose-rich components of the basement membrane (basal lamina) and basement lamella i n the skin of Fundulus h e t e r o c l l t u s . Z. Z e l l f o r c V w l 0 6 : 398-549. Nilsson, L. R. 1966. Adolescent c o l l o i d g o i t r e . Acta Pediat. Scand. 55: 4 9 - 6 3 . Osawa, G., P. Kimmelstiel, V. S e l l i n g . 1966. Thickness of glomerular basement membranes. Amer. J . C l i n . Pathol. 45: 7 - 2 0 . Pease, D. C. 1958. The basement membrane: substratum of h i s t o l o g i c a l order and complexity. 4th Internat. Conf. on El e c t r o n Microscop. Springer Verlag. i 9 6 0 . Pierce, G. B., A. R. Midgley, J . S r i Ram, and J . D. Feldman. 1962. P a r i e t a l yolk sac carcinoma: clue to the h i s t o -genesis of Reichert's membrane of the mouse embryo. Amer. J . Pathol, 41: 549-566. Pierce, G. B., A. R. Midgley, and J . S r i Ram. 1963. Histogenesis of basement membranes. J.;Exp. Med. 117: 339-348. Pierce, G. B., T. F. Beals, J . S r i Ram, and A. R. Midgley. 1964. Basement membranes IV. E p i t h e l i a l o r i g i n and immunological cross reactions. Amer. J . Pathol. 45: 929-942. Re l c h l i n , S. 1958. Thyroid iodine metabolism following p a r t i a l thyroidectomy i n the r a t . Endocrinol. 62: 463-473. Ryan, R. J . , C. Faiman, W. E. Mayberry, and J . G. G r e s l i n . 1968. E f f e c t of iodide on recovery of function of rat thyroid gland a f t e r administration of a n t i t h y r o i d agents Endocrinol. 83: 452-460.::. Siperstein, M. D., R. H. Unger, and L. L. Madison. 1968. Studies of muscle c a p i l l a r y basement membranes i n normal subjects, d i a b e t i c , and prediabetic patients. J . C l i n . Invest. 47: 1973 - 1 9 9 9 . Studer, H. and M. A. Greer. 1967. Thyroid function during the rebound phase following the discontinuation of a n t i t h y r o i d drugs. Endocrinol. 80: 52-60. Toujas, L. et J . G u e l f l . I 9 6 9 . Sur 1'ultrastructure de l a glande thyroide humaine. Z. Z e l l f o r s c h . 94: 118-128. Turner, C. D. 1971. General endocrinology. 5#h ed. Saunders, Philadelphia. Voitkevlch, A. A. 1964. The phenomenon of regeneration and hypertrophy i n the thyroid gland following i t s injury. B u l l . Exp. B i o l . Med. 57: 8 9 - 9 3 . Vracko, R. and D. E. Strandness. I967. Basal lamina of abdominal s k e l e t a l muscle c a p i l l a r i e s i n d i a b e t i c s and nondiabetics. C i r c u l a t i o n 35: 690 - 7 0 0 . Waterhouse, J . P. and C. A. Squier. 1969. Measurements from electron micrographs of organelle size i n r e l a t i o n to t h e i r shape: a refinement applied to the epidermal melanosome and basal lamella. J . Mlcroscop. 89: 195-204. 

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