Exp. Geront. Vol. 12. pp. 207-214. Pergamon Press 1977. Printed in Great Britain.
MORPHOMETRIC ANALYSIS OF ENDOCRINE PANCREAS IN OLD RATS C. R E M A C L E * , N . H A U S E R * , M . JEANJEAN~ a n d A. G O M M E R S ~ *Unit of Animal Morphology and 1"Unit of Experimental Gerontology, Universit6 de Louvain, Claude Bernard, PI. Croix du Sud, 5, B-1348 Louvain-la-Neuve, Belgium
(Received 11 December 1976) Abstract--The hypothesis of a degenereseence of Langerhans islets in senile animals has been tested by the morphometrical analysis of the pancreas of rats 6 and 24 months old. The method has allowed to show the presence in old animals of a higher number of islets per area unit; the distribution curve shows a shifting towards the larger sizes. If the surrounding connective tissue which thickens with age is not taken into account, the relative volumes occupied by the sinusoid capillaries and the A and B cells appear similar in the two age groups, as well as the nucleo--cytoplasmicratio of these cells and the granulation of the cells secreting insulin. So, the morphological aspect of Langerhans islets does not reveal a decrease of the insulin stores in the pancreas of old rats. NUMEROUS epidemiological studies have shown an increased prevalence of diabetic disease in h u m a n ageing; moreover, glucose tolerance tests performed on subjects without clinical diabetic signs have indicated abnormalities whose frequency increases with the age of the examined population. The relationship between the ageing and the carbohydrate metabolism bas yet to be clarified. Several mechanisms have been considered, for example the decreased consumption of blood glucose in humans due to a loss of protoplasm brought about by ageing (Silverstone et al., 1957), the resistance to insulin of hormone-sensible tissues (Cerasi and Luft, 1972) or a deficiency of old Langerhans islets (Zhukov, 1964; Crockford et al., 1966; Burch and O'Meallie, 1967; Hadju et al., 1968). The aim of this paper then is to attempt at verifying this last hypothesis by means of a morphometric analysis of the insular tissue taken from adult (6 months old) or aged rats (24 months old). For the previous experiments have in fact demonstrated that those animals, considered free from diabetic genes, showed a decrease in their glucose assimilation coefficient with ageing comparable to that observed in humans (Gommers, 1971 ; Gommers and de Gasparo, 1972). MATERIALS AND METHODS The male albino rats used in our experiment were bred in our colony over a span of 40 generations. They were kept under constant conditions of temperature and humidity, and artificiallightingfrom 7 a.m. to 7 p.m. The animals were placed 3 in a cage beginning at the age of 1.5 months, and observed until their death which generally occurred at about 2.5 years. The rats were allowed to drink water at will and were also fed ad libitum with a wholesome diet consisting of 20.8 % of digestible proteins, 5 % fat, 55 % carbohydrates, 3 % cellulose, 6 % minerals and 1 2 % moisture. They were weighed at regular intervals. They reached their m a x i m u m weight at about 20 months with an average of 350 g, with a dispersion ranging from 250 to 500 g. The aged animals, 24 months or more, showed a weight decrease. Ten fed 6 month-old rats and ten fed 24 month-old rats,were paired off according to similar weights and
decapitated. The pancreases were dissected and weighed immediately, then separated into two parts. The region of the head and that of the tail were treated separately in order to diminish the risk of error inherent to the different distribution of the islets of Langerhans between these two regions. The fixation of Bouin 207
208
c . REMACLE, N. HAUSER, M. JEANJEAN AND A. GOMMERS
Allen, dehydration in ethanol and impregnation in butanol were followed by immersion into paraffin. The pieces were placed at random in the insertion blocks. The serially sections, 6t~m thick, were stained according to the following techniques: 1. Masson's trichrome for the tracing of insular tissue. 2. Heidenhain's azan, to reveal the collagen fibres and the A and B cells. 3. Thionin paraldehyde-S naphtol yellow, paraldehyde fuchsin-S naphtol yellow, alcian blue-phloxin, following Gabe (1968) to differenciate between the A and B cells. 4. The DDD reaction following Barnett and Seligman (1954), with or without reduction of cystine groups, to check the B cells observed under the preceding methods. The morphometrical analysis was made upon the two largest sections which come from the head and the tail. Because of the random position of the pieces at the time of immersion into paraffin, the distribution of the pancreas on these two sections may be attributed to chance. A sheet of transparent plastic carrying a stereological test system made up of 312 straight segments which are limited by two points (Weibel, 1966; Briarty, 1975) was applied on the screen of a Reichert Visopan microscope. The counting of the distribution of points when a randomly selected section is scanned allows us to compute the areas or the relative volumes of the examined structures. According to the magnifications used, different parameters may be studied (Fig. 2). RESULTS T h e m e a n weight o f the a n i m a l s a n d t h a t o f the pancreases does n o t differ significantly in the two age groups. Similarly, the histological aspects o f the exocrine p a n c r e a s are c o m p a r a b l e . T h e s t u d y was c o n c e r n e d with the r a t i o o f insular tissue to the total p a n c r e a t i c tissue, a n d with the n u m b e r o f islets by unit a r e a (magnification 1), the d i s t r i b u t i o n of islets a c c o r d i n g to size categories (magnification 2), the relative volumes o f the different insular structures (magnification 3).
1. Magnification 1 : 1 m m = 8 /zm T h e coefficients o f c o r r e l a t i o n between the weight o f p a n c r e a s a n d the p r o p o r t i o n of insular tissue are n o t significantly different either at 6 m o n t h s ( r = 0.28) o r at 24 m o n t h s (r = - 0.49). T h e percentage o f insular tissue in the t o t a l p a n c r e a t i c v o l u m e shows a significant increase o f a b o u t 50 ~o in senile rats as c o m p a r e d to y o u n g rats (Table 1). The n u m b e r o f islets reaching a d i a m e t e r l a r g e r t h a n 4 0 / z m - - s e n s i t i v e n e s s o f this first t e s t - - c a l c u l a t e d b y unit o f a r e a (1-4 m m ~) is significantly higher in o l d rats. T h e increase o f the n u m b e r o f islets thus plays a role in the increase o f the v o l u m e o f insular tissue. TABLE 1. EFFECT OF AGEING ON THE PROPORTION OF INSULAR TISSUE IN THE PANCREAS AND ON THE NUMBER OF ISLETS BY UNIT OF AREA
Insular volume/pancreas N. islets/1.4 mm ~
6 months (n=10)
24 months (n=10)
0.71 + 0.18* 1.35 + 0'36
1.07 + 0.21 1.72 + 0'31
p < 0.01 p < 0'05
*Mean + S.D., p: probability of significance.
2. Magnification 2 : 1 m m = 2 / ~ m This magnification allows us to t a k e into a c c o u n t the smallest islets c o n t a i n i n g even one single e n d o c r i n e cell. T h e surface areas o f 1000 islets (100 p e r a n i m a l ) in the pancreases o f 10 rats o f 6 m o n t h s a n d 10 o f 24 m o n t h s were m e a s u r e d . T h e y are d i s t r i b u t e d in classes o f size within intervals o f 2.212/z z which is a surface a r e a c o r r e s p o n d i n g to 25 points o f the
MORPHOMETRIC ANALYSISOF ENDOCRINEPANCREAS IN OLD RATS
209
stereological sheet at this magnification. The distribution of the number of islets by categories of size is transformed into a percentage of the total area of the measured insular tissue. These percentages are cumulated (Fig. 1) and the Kolmogorov-Smirnov test is applied. The two distributions are significantly different (N1/2Dmm=2"9; ~= 1.95; for a=0.001). A shifting of the distribution towards the large sizes was observed in old rats (Fig. 1). Thus, the increase of the volume of insular tissue in senescent rats is due to a larger number o f islets and to an increase in size. This last can be roughly estimated at 11 ~o by comparing the total insular areas measured in the two age groups. %S t00
AAA "o •
• *AAA~A • •
e
e
•
• t:
:O:..''"
°
O o
t
• 6 rnon~ h~ o24 rnonths
i
~
~
~,
~
~
/Y
~'3 ,o'.L,~
1~. I. Cumulative distribution curves of the Langerhans islets in young (6 months) and old (24 months) rats. %S~: percent of the total area.
3. Magnification 3:1 m m = 0 . 8 t~m 50 islets with a diameter larger than 80 tz are randomly taken, in the pancreases of old and young rats. The relative volumes of the following structures were computed: the surrounding connective tissue, the sinusoid capillaries, and concerning the A and B cells, the nuclei, the cytoplasms with or without secretion granules. The comparison of these volumes, in terms of percentages of the total volume of the islet (Table 2) shows that: 1. the surrounding connective tissue is thicker in old rats (p < 0.01), 2. the volume occupied by the blood sinusoids is not at all modified, 3. the relative volumes of the total of A cells and the total of B cells are diminished
(p < 0.05). TABLE 2. COMPOSITION OF THE ISLETS, IN PERCENTAGE OF THE TOTAL VOLUME
6 months (n=50)
24 months (n=50)
Connective tissue SinusoIds
11.4 + 3.3* 17.4 + 4"4
19.6 + 6.4 16"2 + 4.9
p < 0.01 N.S.
A cells B cells
14.4+ 7.3 56.9 + 8.7
11.4 + 6.7 52"7+ 8-0
p < 0.05 p < 0"05
i
*Mean_+S.D., p: probability of significance. However, if the surrounding connective tissue is not taken into account, any significant difference disappears between young and old rats (Table 3).
210
c. REMACLE, N. HAUSER, M. JEANJEAN AND A. GOMMERS TABLE 3. COMPOSITION OF THE ISLETS, WITHOUT THEIR SURROUNDING CONNECTIVE TISSUE
Sinusolds A cells B cells
6 months (n=50)
24 months (n=50)
19.6+ 5"0" 16.3+ 8.2 64.3 + 9.9
20.2 _.+6.1 14.1 + 8.3 65'3 _ 8.9
N.S. N.S. N.S.
*Mean + S.D. The nucleo-cytoplasmic ratios of the A and B cells are not modified (Table 4). The cytoplasmic granulation of B cells is estimated by the percentage of cytoplasm volume containing secretion granules, compared with the total volume of cytoplasm. This cytoplasmic granulation is not modified in old rats (Table 4). TABLE 4. CELL PARAMETERS
6 months (n=50)* A cells: nucleo--cytoplasmicratio B ceils: nucleo-cytoplasmic ratio B cells: cytoplasmic granulation
24 months (n=50)
1.2 _+0.6f
1.4__0.6
N.S.
0-4_+0"1
0'5 _+0-1
N.S.
74.3 _+10.9
76.8 + 9.4
N.S.
*These cell parameters are calculated from global data gathered from each of the 50 islets measured. i'Mean__ S.D. DISCUSSION Numerous methods pertaining to the estimation of the absolute or relative volume of the Langerhans islets in the pancreases have been proposed. This diversity is explained by the difficulty of quantifying the observations on the insular system. The most elaborate of these methods (Hellman, 1959a, b,c; Hellman et al., 1964) comes from a mathematical development o f Wicksell (1926) concerning the estimation of a real volumetric distribution of spheric or ellipsoid corpuscules dispersed in a solid, based upon the apparent distribution observed in sections in this solid. Wicksell's formulae have been applied to Langerhans islets by Tejning (1947) and Hellman (1959a, b,c). They allow us to obtain an estimation of the insular tissue volume and the volumetric distribution of the islets. The morphometrical technique used in this work is very different. It does not oblige the confection of serial sections, as the calculations are made from a section randomly taken among those of a large area in two regions of the pancreas. With magnification 1, we obtain the proportion of insular tissue in the pancreas, and the number of islets of a diameter more than 40 ~m by unit of area. As compared to Hellman's method, we lose the advantage of an absolute volume calculation, but we are compensated by a greater rapidity in the calculations and at the same time, avoid the mathematical transformations. The results obtained are suitable for the estimation of a difference of relative volumes between two groups of animals. The magnification 2 permits the determination of the distribution of size of the islets by direct measuring. The high sensitivity of the method is evident in the fact that, at the lower limit, 'islets' formed by a single endocrine cell are included in the sample. With appropriate cytologic stains, at magnification 3, the quanti-
FIG. 2. Weibel's test on a section in a Langerhans islet of an old rat. A: A-cell, B: B-cell, S: sinusoid Aldehyde Fuchsin-naphtol yellow. Fl~. 3. Small islets in pancreas of an old rat (arrows). Trichrome stain. FiG. 4. The largest islet of the sample (old rat). Trichrome stain.
MORPHOMETRIC ANALYSIS OF ENDOCRINE PANCREAS IN OLD RATS
213
fication of internal composition of the islets and of cell parameters such as the nucleocytoplasmic ratio and the cytoplasmic granulation is obtained. One of the eventual causes of the glucose tolerance decrease in rats as in aged humans, is the degeneration of the insular tissue through ageing. Important insular tissue alterations have been seen on autopsy specimens from old people by Houcke et al. (1964) and Zhukov (1964). Besides, Hajdu and Rona (1967) and Hajdu et al. 0968) drew attention to the very frequent occurrence of modifications in Langerhans islets, such as fibrosis and hyperplasia, in male rats pancreases of 48, 52 and 82 weeks. These lesions affect more than 50 % of the animals studied. Lewis and Wexler (1974) describe a very marked hyperplasia of the islets in virgin male rats 15-18 months old, on the other hand, breeding male animals ranging from 6-8 months considered by these same authors to be affected by premature ageing, show an atrophy and a marked fibrosis of the insular system. The observations of these different researchers are not based upon a quantitative analysis, except for Hellman et al. (1964) who studied the growth curves of the insular tissue in rats from birth to the age of 16 months only. In our results, the senile rats of 24 months present a greater relative volume of insular tissue than do rats 6 months old. The greater number of islets and their larger size simultaneously influence this increase. The distribution curve shows a slight shift towards the larger sizes. The only significant difference in the structure of the examined islets is an increased thickness of their surrounding connective tissue. The relative volumes occupied by sinusoid capillaries and the A and B cells, the nucleocytoplasmic ratios of these cells, and the cytoplasmic granulation of the B cells are the same in the two age groups. The pictures of insular degenerescence arc rare in the rats examined. Some islets degenerate in t o t o and though infrequent may be observed in young and old rats alike. These degenerated islets have been eliminated from the calculations, which only considered healthy tissue. On the other hand, in apparently normal islets some cells degenerate; their nucleus is pycnotic. The proportion of dead cells is negligible in the two age groups. The walls of the sinusoid capillaries seen through a photon microscope are perfectly normal in old rats and no intrainsular fibrosis was observed. At a preliminary histological examination, the B cells seem rather similar in young and old rats, but the A cells were less stainable in old rats. The calculations show that their volume occupied within the islets is not modified. A cytoplasmic granulation index for A cells has not been calculated because of a very high individual variation, especially in senile rats; moreover, the staining techniques for the displaying of the granules in A cells are more variable than for the B cells. To conclude, the morphological aspects of Langerhans islets taking into particular account the insulin stores, certainly do not demonstrate a decrease of their functional capacity. On the contrary, this insulin store will be increased in the pancreas of ageing rats. This observation does not exclude the possibility of a slow or abnormal response of the B cells to glucose or other stimuli of hormonal secretion. REFERENCES BARNETT,J. R. and SEUGMAN,A. M. (1954)J. natn. Cancer Inst. 14, 769. BIUARTY,L. G. (1975) Sci. Progr. 62, 1. BtrRCH, G. E. and O~MEALLIE,L. P. (1967) Am. J. reed. Sci. 254, 602.
214
c. REMACLE,N. HAUSER,M. JEANJEANAND A. GOMMERS
CERASI,E. and Ltwr, R. (1972) In Handbook of Physiology, Section 7: Endocrinology, 1 Endocrine Pancreas (Edited by D. F. STERNERand N. FREINKEL).American Physiological Society, Washington, DC. CROCKFORD,P. M., HARnECK,R. J. and WILLIAMS,R. H. (1966) Lancet i, 465. GABE, M. (1968) Techniques Histologiques. Masson, Paris. GOMMERS,A. (1971) Gerontologia 17, 228. GOMMERS,A. and DE GASPARO,M. (1972) Gerontologia 18, 176. ~u, A., HERR, F. and RONA, G. (1968) Diabetologia 4, 44. HINDU, A. and RONA, G. (1967) Diabetes 16, 108. HELLMAN,B. (1959a) Acta Endocr. 31, 91. HELLMAN,B. (1959b) Acta Endocr. 32, 63. HELLMAN,B. (1959c) Acta Endocr. 32, 78. HELLMAN,B., PETERSON,B. and HELLERSTROM,C. (1964) In The Structure and Metabolism of the Pancreatic Islets (Edited by S. E. BROLIN,B. HELLMANand H. KNUTSON).Pergamon Press, Oxford. HOUCKE,E., HOUCKE,M. and LEBLOIS,J. (1964) Presse med. 24, 1887. LEwis, B. K. and WEXLER,B. C. (1974) J. Geront. 29, 139. SrLVERSTONE, F. A., BRANOFONBRENER,R. M., SHOCK, M. W. and YIENGST, M. J. (1957) J. clin. Invest. 36, 504. TEJNIN~, S. (1947) Acta med. scand, suppl. 198, 1. WEmEL, E. R., KISTLER,G. S. and SCHERLE,W. (1966) J. cell. Biol. 30, 23. WlCKSELL,S. D. (1926) Biometrika 17, 84. ZmSKOV,N. A. (1964) Soy. Med. 28, 597.
MORPHOMETRIC ANALYSIS OF ENDOCRINE PANCREAS IN OLD RATS C. R E M A C L E * , N . H A U S E R * , M . JEANJEAN~ a n d A. G O M M E R S ~ *Unit of Animal Morphology and 1"Unit of Experimental Gerontology, Universit6 de Louvain, Claude Bernard, PI. Croix du Sud, 5, B-1348 Louvain-la-Neuve, Belgium
(Received 11 December 1976) Abstract--The hypothesis of a degenereseence of Langerhans islets in senile animals has been tested by the morphometrical analysis of the pancreas of rats 6 and 24 months old. The method has allowed to show the presence in old animals of a higher number of islets per area unit; the distribution curve shows a shifting towards the larger sizes. If the surrounding connective tissue which thickens with age is not taken into account, the relative volumes occupied by the sinusoid capillaries and the A and B cells appear similar in the two age groups, as well as the nucleo--cytoplasmicratio of these cells and the granulation of the cells secreting insulin. So, the morphological aspect of Langerhans islets does not reveal a decrease of the insulin stores in the pancreas of old rats. NUMEROUS epidemiological studies have shown an increased prevalence of diabetic disease in h u m a n ageing; moreover, glucose tolerance tests performed on subjects without clinical diabetic signs have indicated abnormalities whose frequency increases with the age of the examined population. The relationship between the ageing and the carbohydrate metabolism bas yet to be clarified. Several mechanisms have been considered, for example the decreased consumption of blood glucose in humans due to a loss of protoplasm brought about by ageing (Silverstone et al., 1957), the resistance to insulin of hormone-sensible tissues (Cerasi and Luft, 1972) or a deficiency of old Langerhans islets (Zhukov, 1964; Crockford et al., 1966; Burch and O'Meallie, 1967; Hadju et al., 1968). The aim of this paper then is to attempt at verifying this last hypothesis by means of a morphometric analysis of the insular tissue taken from adult (6 months old) or aged rats (24 months old). For the previous experiments have in fact demonstrated that those animals, considered free from diabetic genes, showed a decrease in their glucose assimilation coefficient with ageing comparable to that observed in humans (Gommers, 1971 ; Gommers and de Gasparo, 1972). MATERIALS AND METHODS The male albino rats used in our experiment were bred in our colony over a span of 40 generations. They were kept under constant conditions of temperature and humidity, and artificiallightingfrom 7 a.m. to 7 p.m. The animals were placed 3 in a cage beginning at the age of 1.5 months, and observed until their death which generally occurred at about 2.5 years. The rats were allowed to drink water at will and were also fed ad libitum with a wholesome diet consisting of 20.8 % of digestible proteins, 5 % fat, 55 % carbohydrates, 3 % cellulose, 6 % minerals and 1 2 % moisture. They were weighed at regular intervals. They reached their m a x i m u m weight at about 20 months with an average of 350 g, with a dispersion ranging from 250 to 500 g. The aged animals, 24 months or more, showed a weight decrease. Ten fed 6 month-old rats and ten fed 24 month-old rats,were paired off according to similar weights and
decapitated. The pancreases were dissected and weighed immediately, then separated into two parts. The region of the head and that of the tail were treated separately in order to diminish the risk of error inherent to the different distribution of the islets of Langerhans between these two regions. The fixation of Bouin 207
208
c . REMACLE, N. HAUSER, M. JEANJEAN AND A. GOMMERS
Allen, dehydration in ethanol and impregnation in butanol were followed by immersion into paraffin. The pieces were placed at random in the insertion blocks. The serially sections, 6t~m thick, were stained according to the following techniques: 1. Masson's trichrome for the tracing of insular tissue. 2. Heidenhain's azan, to reveal the collagen fibres and the A and B cells. 3. Thionin paraldehyde-S naphtol yellow, paraldehyde fuchsin-S naphtol yellow, alcian blue-phloxin, following Gabe (1968) to differenciate between the A and B cells. 4. The DDD reaction following Barnett and Seligman (1954), with or without reduction of cystine groups, to check the B cells observed under the preceding methods. The morphometrical analysis was made upon the two largest sections which come from the head and the tail. Because of the random position of the pieces at the time of immersion into paraffin, the distribution of the pancreas on these two sections may be attributed to chance. A sheet of transparent plastic carrying a stereological test system made up of 312 straight segments which are limited by two points (Weibel, 1966; Briarty, 1975) was applied on the screen of a Reichert Visopan microscope. The counting of the distribution of points when a randomly selected section is scanned allows us to compute the areas or the relative volumes of the examined structures. According to the magnifications used, different parameters may be studied (Fig. 2). RESULTS T h e m e a n weight o f the a n i m a l s a n d t h a t o f the pancreases does n o t differ significantly in the two age groups. Similarly, the histological aspects o f the exocrine p a n c r e a s are c o m p a r a b l e . T h e s t u d y was c o n c e r n e d with the r a t i o o f insular tissue to the total p a n c r e a t i c tissue, a n d with the n u m b e r o f islets by unit a r e a (magnification 1), the d i s t r i b u t i o n of islets a c c o r d i n g to size categories (magnification 2), the relative volumes o f the different insular structures (magnification 3).
1. Magnification 1 : 1 m m = 8 /zm T h e coefficients o f c o r r e l a t i o n between the weight o f p a n c r e a s a n d the p r o p o r t i o n of insular tissue are n o t significantly different either at 6 m o n t h s ( r = 0.28) o r at 24 m o n t h s (r = - 0.49). T h e percentage o f insular tissue in the t o t a l p a n c r e a t i c v o l u m e shows a significant increase o f a b o u t 50 ~o in senile rats as c o m p a r e d to y o u n g rats (Table 1). The n u m b e r o f islets reaching a d i a m e t e r l a r g e r t h a n 4 0 / z m - - s e n s i t i v e n e s s o f this first t e s t - - c a l c u l a t e d b y unit o f a r e a (1-4 m m ~) is significantly higher in o l d rats. T h e increase o f the n u m b e r o f islets thus plays a role in the increase o f the v o l u m e o f insular tissue. TABLE 1. EFFECT OF AGEING ON THE PROPORTION OF INSULAR TISSUE IN THE PANCREAS AND ON THE NUMBER OF ISLETS BY UNIT OF AREA
Insular volume/pancreas N. islets/1.4 mm ~
6 months (n=10)
24 months (n=10)
0.71 + 0.18* 1.35 + 0'36
1.07 + 0.21 1.72 + 0'31
p < 0.01 p < 0'05
*Mean + S.D., p: probability of significance.
2. Magnification 2 : 1 m m = 2 / ~ m This magnification allows us to t a k e into a c c o u n t the smallest islets c o n t a i n i n g even one single e n d o c r i n e cell. T h e surface areas o f 1000 islets (100 p e r a n i m a l ) in the pancreases o f 10 rats o f 6 m o n t h s a n d 10 o f 24 m o n t h s were m e a s u r e d . T h e y are d i s t r i b u t e d in classes o f size within intervals o f 2.212/z z which is a surface a r e a c o r r e s p o n d i n g to 25 points o f the
MORPHOMETRIC ANALYSISOF ENDOCRINEPANCREAS IN OLD RATS
209
stereological sheet at this magnification. The distribution of the number of islets by categories of size is transformed into a percentage of the total area of the measured insular tissue. These percentages are cumulated (Fig. 1) and the Kolmogorov-Smirnov test is applied. The two distributions are significantly different (N1/2Dmm=2"9; ~= 1.95; for a=0.001). A shifting of the distribution towards the large sizes was observed in old rats (Fig. 1). Thus, the increase of the volume of insular tissue in senescent rats is due to a larger number o f islets and to an increase in size. This last can be roughly estimated at 11 ~o by comparing the total insular areas measured in the two age groups. %S t00
AAA "o •
• *AAA~A • •
e
e
•
• t:
:O:..''"
°
O o
t
• 6 rnon~ h~ o24 rnonths
i
~
~
~,
~
~
/Y
~'3 ,o'.L,~
1~. I. Cumulative distribution curves of the Langerhans islets in young (6 months) and old (24 months) rats. %S~: percent of the total area.
3. Magnification 3:1 m m = 0 . 8 t~m 50 islets with a diameter larger than 80 tz are randomly taken, in the pancreases of old and young rats. The relative volumes of the following structures were computed: the surrounding connective tissue, the sinusoid capillaries, and concerning the A and B cells, the nuclei, the cytoplasms with or without secretion granules. The comparison of these volumes, in terms of percentages of the total volume of the islet (Table 2) shows that: 1. the surrounding connective tissue is thicker in old rats (p < 0.01), 2. the volume occupied by the blood sinusoids is not at all modified, 3. the relative volumes of the total of A cells and the total of B cells are diminished
(p < 0.05). TABLE 2. COMPOSITION OF THE ISLETS, IN PERCENTAGE OF THE TOTAL VOLUME
6 months (n=50)
24 months (n=50)
Connective tissue SinusoIds
11.4 + 3.3* 17.4 + 4"4
19.6 + 6.4 16"2 + 4.9
p < 0.01 N.S.
A cells B cells
14.4+ 7.3 56.9 + 8.7
11.4 + 6.7 52"7+ 8-0
p < 0.05 p < 0"05
i
*Mean_+S.D., p: probability of significance. However, if the surrounding connective tissue is not taken into account, any significant difference disappears between young and old rats (Table 3).
210
c. REMACLE, N. HAUSER, M. JEANJEAN AND A. GOMMERS TABLE 3. COMPOSITION OF THE ISLETS, WITHOUT THEIR SURROUNDING CONNECTIVE TISSUE
Sinusolds A cells B cells
6 months (n=50)
24 months (n=50)
19.6+ 5"0" 16.3+ 8.2 64.3 + 9.9
20.2 _.+6.1 14.1 + 8.3 65'3 _ 8.9
N.S. N.S. N.S.
*Mean + S.D. The nucleo-cytoplasmic ratios of the A and B cells are not modified (Table 4). The cytoplasmic granulation of B cells is estimated by the percentage of cytoplasm volume containing secretion granules, compared with the total volume of cytoplasm. This cytoplasmic granulation is not modified in old rats (Table 4). TABLE 4. CELL PARAMETERS
6 months (n=50)* A cells: nucleo--cytoplasmicratio B ceils: nucleo-cytoplasmic ratio B cells: cytoplasmic granulation
24 months (n=50)
1.2 _+0.6f
1.4__0.6
N.S.
0-4_+0"1
0'5 _+0-1
N.S.
74.3 _+10.9
76.8 + 9.4
N.S.
*These cell parameters are calculated from global data gathered from each of the 50 islets measured. i'Mean__ S.D. DISCUSSION Numerous methods pertaining to the estimation of the absolute or relative volume of the Langerhans islets in the pancreases have been proposed. This diversity is explained by the difficulty of quantifying the observations on the insular system. The most elaborate of these methods (Hellman, 1959a, b,c; Hellman et al., 1964) comes from a mathematical development o f Wicksell (1926) concerning the estimation of a real volumetric distribution of spheric or ellipsoid corpuscules dispersed in a solid, based upon the apparent distribution observed in sections in this solid. Wicksell's formulae have been applied to Langerhans islets by Tejning (1947) and Hellman (1959a, b,c). They allow us to obtain an estimation of the insular tissue volume and the volumetric distribution of the islets. The morphometrical technique used in this work is very different. It does not oblige the confection of serial sections, as the calculations are made from a section randomly taken among those of a large area in two regions of the pancreas. With magnification 1, we obtain the proportion of insular tissue in the pancreas, and the number of islets of a diameter more than 40 ~m by unit of area. As compared to Hellman's method, we lose the advantage of an absolute volume calculation, but we are compensated by a greater rapidity in the calculations and at the same time, avoid the mathematical transformations. The results obtained are suitable for the estimation of a difference of relative volumes between two groups of animals. The magnification 2 permits the determination of the distribution of size of the islets by direct measuring. The high sensitivity of the method is evident in the fact that, at the lower limit, 'islets' formed by a single endocrine cell are included in the sample. With appropriate cytologic stains, at magnification 3, the quanti-
FIG. 2. Weibel's test on a section in a Langerhans islet of an old rat. A: A-cell, B: B-cell, S: sinusoid Aldehyde Fuchsin-naphtol yellow. Fl~. 3. Small islets in pancreas of an old rat (arrows). Trichrome stain. FiG. 4. The largest islet of the sample (old rat). Trichrome stain.
MORPHOMETRIC ANALYSIS OF ENDOCRINE PANCREAS IN OLD RATS
213
fication of internal composition of the islets and of cell parameters such as the nucleocytoplasmic ratio and the cytoplasmic granulation is obtained. One of the eventual causes of the glucose tolerance decrease in rats as in aged humans, is the degeneration of the insular tissue through ageing. Important insular tissue alterations have been seen on autopsy specimens from old people by Houcke et al. (1964) and Zhukov (1964). Besides, Hajdu and Rona (1967) and Hajdu et al. 0968) drew attention to the very frequent occurrence of modifications in Langerhans islets, such as fibrosis and hyperplasia, in male rats pancreases of 48, 52 and 82 weeks. These lesions affect more than 50 % of the animals studied. Lewis and Wexler (1974) describe a very marked hyperplasia of the islets in virgin male rats 15-18 months old, on the other hand, breeding male animals ranging from 6-8 months considered by these same authors to be affected by premature ageing, show an atrophy and a marked fibrosis of the insular system. The observations of these different researchers are not based upon a quantitative analysis, except for Hellman et al. (1964) who studied the growth curves of the insular tissue in rats from birth to the age of 16 months only. In our results, the senile rats of 24 months present a greater relative volume of insular tissue than do rats 6 months old. The greater number of islets and their larger size simultaneously influence this increase. The distribution curve shows a slight shift towards the larger sizes. The only significant difference in the structure of the examined islets is an increased thickness of their surrounding connective tissue. The relative volumes occupied by sinusoid capillaries and the A and B cells, the nucleocytoplasmic ratios of these cells, and the cytoplasmic granulation of the B cells are the same in the two age groups. The pictures of insular degenerescence arc rare in the rats examined. Some islets degenerate in t o t o and though infrequent may be observed in young and old rats alike. These degenerated islets have been eliminated from the calculations, which only considered healthy tissue. On the other hand, in apparently normal islets some cells degenerate; their nucleus is pycnotic. The proportion of dead cells is negligible in the two age groups. The walls of the sinusoid capillaries seen through a photon microscope are perfectly normal in old rats and no intrainsular fibrosis was observed. At a preliminary histological examination, the B cells seem rather similar in young and old rats, but the A cells were less stainable in old rats. The calculations show that their volume occupied within the islets is not modified. A cytoplasmic granulation index for A cells has not been calculated because of a very high individual variation, especially in senile rats; moreover, the staining techniques for the displaying of the granules in A cells are more variable than for the B cells. To conclude, the morphological aspects of Langerhans islets taking into particular account the insulin stores, certainly do not demonstrate a decrease of their functional capacity. On the contrary, this insulin store will be increased in the pancreas of ageing rats. This observation does not exclude the possibility of a slow or abnormal response of the B cells to glucose or other stimuli of hormonal secretion. REFERENCES BARNETT,J. R. and SEUGMAN,A. M. (1954)J. natn. Cancer Inst. 14, 769. BIUARTY,L. G. (1975) Sci. Progr. 62, 1. BtrRCH, G. E. and O~MEALLIE,L. P. (1967) Am. J. reed. Sci. 254, 602.
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c. REMACLE,N. HAUSER,M. JEANJEANAND A. GOMMERS
CERASI,E. and Ltwr, R. (1972) In Handbook of Physiology, Section 7: Endocrinology, 1 Endocrine Pancreas (Edited by D. F. STERNERand N. FREINKEL).American Physiological Society, Washington, DC. CROCKFORD,P. M., HARnECK,R. J. and WILLIAMS,R. H. (1966) Lancet i, 465. GABE, M. (1968) Techniques Histologiques. Masson, Paris. GOMMERS,A. (1971) Gerontologia 17, 228. GOMMERS,A. and DE GASPARO,M. (1972) Gerontologia 18, 176. ~u, A., HERR, F. and RONA, G. (1968) Diabetologia 4, 44. HINDU, A. and RONA, G. (1967) Diabetes 16, 108. HELLMAN,B. (1959a) Acta Endocr. 31, 91. HELLMAN,B. (1959b) Acta Endocr. 32, 63. HELLMAN,B. (1959c) Acta Endocr. 32, 78. HELLMAN,B., PETERSON,B. and HELLERSTROM,C. (1964) In The Structure and Metabolism of the Pancreatic Islets (Edited by S. E. BROLIN,B. HELLMANand H. KNUTSON).Pergamon Press, Oxford. HOUCKE,E., HOUCKE,M. and LEBLOIS,J. (1964) Presse med. 24, 1887. LEwis, B. K. and WEXLER,B. C. (1974) J. Geront. 29, 139. SrLVERSTONE, F. A., BRANOFONBRENER,R. M., SHOCK, M. W. and YIENGST, M. J. (1957) J. clin. Invest. 36, 504. TEJNIN~, S. (1947) Acta med. scand, suppl. 198, 1. WEmEL, E. R., KISTLER,G. S. and SCHERLE,W. (1966) J. cell. Biol. 30, 23. WlCKSELL,S. D. (1926) Biometrika 17, 84. ZmSKOV,N. A. (1964) Soy. Med. 28, 597.
