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Age-related changes in normal human basement membrane
Mechanisms o f Ageing and Development, 19 (1982) 315-324
315
AGE-RELATED CHANGES IN NORMAL HUMAN BASEMENT MEMBRANE
YU-PING XI, E. GERDA NETTE, DONALD WEST KING and MOSHE ROSEN College o f Physicians and Surgeons, Department o f Pathology, New York, New York 10032 (U.S.A.)
(Received March 2, 1982)
SUMMARY Basement membranes were studied in the seminiferous tubules of the male, and capillaries of the pectoralis muscles in "normal" males and females. Age-related changes in basement membrane thickness were investigated on electromicrographs by quantitative morphometry. Specimens obtained from either autopsies or surgical procedures were divided into four different age groups ranging from age 16 to age 87 years. The results show a statistically significant increase in the basement membrane thickness correlated with age, in basement membranes associated with both mesenchymal (capillaries) and epithelial (testes) tissue. A comparison of capillary basement membrane in the pectoralis muscle and seminiferous tubule basement membrane in the male show a constant increase in thickness until the age of 60 years, following which only slight increases were noted. The basement membrane thickness in capillaries of the pectoralis muscle in females showed linear increases throughout the lifespan, although at a lower rate than in the male; at age 80 years, it had the same thickness as the male. A comparison of basement membrane thickness between the youngest and oldest groups showed an increase of approximately 50% in all tissues studied.
INTRODUCTION Basement membranes, associated with both epithelial and mesenchymal tissue, have been altered in many diseases as well as in the aging process [ 1 - 7 ] . Basement membranes are widely distributed throughout the body as extracellular components of blood vessels and muscle, and are important structures in the genitourinary, respiratory, gastrointestinal and endocrine systems. Although their function is not well understood, they may serve as a vascular mechanical barrier through which all cells and macromolecules entering tissue must pass, and provide a framework for tissue differentiation as well as regeneration following injury. Thickening of basement membrane has been proposed as an inhibitor to neoplastic invasion [8], is seen with arteriosclerosis [9], and is a prominent feature in many of the 0047-6374/82/0000-0000/$02.75
© Elsevier Sequoia/Printed in The Netherlands
316 collagen diseases [9, 10]. Many reports are available indicating that basement membranes thicken with age [1, 2, 4] particularly in association with maturity-onset diabetes [1,2, 11--19]. The latter is often suggested to be an age-related disease. The duration and/or severity of the diabetes has been correlated with the thickness of the muscle basement membrane [1]. This study quantitatively measures the increase in basement membrane thickness correlated with age in seminiferous tubules of the testes. Measurements of basement membranes in capillaries of the pectoralis muscles, in males and females, are also correlated with age.
METHOD AND MATERIALS The thickness of basement membranes was measured in tissues obtained from both autopsies and surgical specimens. The 111 specimens were received from surgical specimens or autopsies performed at the Columbia-Presbyterian Medical Center, St. Luke's Hospital, or from the New York Medical Examiner's Office (Table I). Autopsy specimens were mainly from post mortem examinations performed within 24 hours after death (Table II). Diabetes was ruled out both from the patients' personal histories and laboratory information obtained from patients' charts and autopsy protocols. The specimens were fixed overnight in 2.5% glutaraldehyde in cocadylate buffer at pH 7.4. After rinsing in the same buffer, the tissues were postfixed in 1% osmium tetroxide in cocadylate buffer for 2 hours, processed for embedding through an ascending series of ethanol and embedded in epon. Thin sections were cut with a Sorvall MT-1 ultramicrotome, stained with uranyl acetate and lead citrate [20], and examined and photographed on a Siemens Elmiskop 1 or 1A electron microscope. Ten to twelve basement membranes of capillaries from the pectoral muscle or seminiferous tubules were measured from each specimen and were averaged to obtain the mean basement membrane thickness. The micrographs were enlarged to a final magnification of 30 000 for capillaries and 15 000 for seminiferous tubules. Variations in magnification related to instability of the electron microscope were monitored by calibration plates. Morphometric measurements of basement membranes were employed as described by Underwood [21]. The estimate of basement membrane thickness was calculated as follows: After outlining the basement membrane, a midline between the inner and outer boundary of the basement membrane was drawn. A plastic template (square grid) of known size (D) was placed over the enlarged electron micrograph. To measure the mean thickness (T) two counts were made: (1) the number of intersections between the drawn midline of the basement membrane and the overlaying grid system (N~); (2) the number of nodal points of the grid falling inside the area of the basement membrane (NA). The mean thickness was calculated from the formula NA
4
NL
n
=--X--XDXM
317 TABLE I SOURCE AND NUMBER OF MEASURED SPECIMENS Capillaries in pectoralis
Post mortem Surgery Total
Testes
Male
Female
Total
31 10 41
40 0 40
18 12 30
89* 22 111
*89 Specimens were collected from 59 autopsies. 30 Specimens of testis and pectoralis came from the same case. TABLE II HOURS OF DEATH BEFORE AUTOPSY IN 59 CASES 12 hours
20
1 3 - 2 4 hours
> 24 hours
Unknown *
23
12
4
• Exact time of death unknown, but estimated time definitely more than 24 hours.
where D is the size of the grid ( = 1 / 4 cm) and M is the magnification (/am on the specimen/cm on the image).
RESULTS The specimens of seminiferous tubules of the testes were divided into four age groups, as were the capillary specimens from the pectoral muscle: group 1 specimens, 1 0 - 2 9 years of age; group 2 specimens, 3 0 - 4 9 years; group 3 specimens, 5 0 - 6 9 years; and group 4 specimens, 7 0 - 8 9 years. Representative examples of basement membranes of capillaries in the pectoralis muscle as well as basement membrane in the testes are shown (Fig. 1). The average basement membrane thickness (BMT) of each specimen (seminiferous tubules) examined and the mean BMT in the four different age groups are shown in Table III. Comparison of the thickness of the basement membrane in the different age groups showed a statistically significant increase in BMT between groups 1 (age 1 0 - 2 9 ) , 2 (age 3 0 - 4 9 ) and 3 (age 5 0 - 6 9 ) , but no statistical difference could be detected between group 3 and group 4 (age 70--89). There was a sharp increase in BMT with increased age (Fig. 2), which reached a plateau at the age of 60 and showed only a slight increase between the ages of 60 and 80. When the average BMT for each specimen was plotted (Fig. 3A), it became apparent that the distribution of values for BMT in the under 40 years of age was small. After the age of 40 the BMT showed a marked increase and with much greater individual variation in distribution.
318
i
Fig. 1. A + B. Basement membrane (arrows) of capillaries of pectoralis muscle with red blood cells. (×30000). (A) Male, 16 years old, thickness of membrane 951 A. (B) Female, 53 years old, thickness of membrane 1752 A. C + D. Basement membrane (arrows) of seminiferous tubules in testes (× 15 000). (C) Male, 23 years old, thickness of membrane 1585 A. (D) Male, 58 years old, thickness of membrane 3775 A. The m e a n m e a s u r e m e n t s o f BMT o f capillaries in s t r i a t e d muscle ( p e c t o r a l i s ) in t h e d i f f e r e n t age g r o u p s are given in T a b l e IV for males a n d in T a b l e V for females. Comparing the BMT in t h e d i f f e r e n t age g r o u p s it was a p p a r e n t t h a t b o t h sexes s h o w e d a statisti-
319
TABLE III BASEMENT MEMBRANE THICKNESS* (A) OF SEMINIFEROUS TUBULES IN D I F F E R E N T AGE (YEARS) GROUPS
Group 1 (16-29)
Group 2 (30-49)
Group 3 (50-69)
Group 4 {70-89)
1329 1721 1738 1524 1675 1513 1621 1883
1168 1728 1819 1580 1693 1680 1628 1946 1937 2063 1864 2400 1834
2625 1956 2581 2116 2413 2979 2481 2271 2222
2140 2360 2897 2596 2206 2136 2244 2190 3281 2859 2382 2060 2446
± ± ± ± ± ± ± ±
123 201 220 176 188 107 142 195
1626 ± 170
± ± ± ± ± ± ± ± ± ± ± ± ±
176 184 161 187 375 196 436 154 287 208 314 354 230
± 273 ± 146 ± 363 ± 247 ± 324 +_456 ± 477 +_439 _+ 119
2405 ± 306
+_ 214 ± 304 _+419 ± 340 ± 251 ± 226 ± 269 +- 184 +- 319 ± 368 ± 195 ± 272 ± 342
*BMT is represented as mean ± S.D. p value between group 1 and group 2 < 0.05.
p value between group 2 and group 3 < 0.001.
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Fig. 2. Mean basement membrane thickness o f seminiferous tubules and capillaries in peetoralis muscle in males in different age groups. Bars represent the standard error o f the mean.
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Ago In Years Fig. 3. Distribution of individual basement membrane thickness. (A) Values of seminiferous tubules ot males o f different ages. (B) Values of capillaries in pectoralis in males o f different ages. (C) Values of capillaries in pectoralis in females of different ages.
TABLE IV BASEMENT MEMBRANE THICKNESS* (A) OF CAPILLARIES IN PECTORALIS IN D I F F E R E N ] AGE (YEARS) GROUPS IN MALES
Group 1 (16-29)
Group 2 (30-49)
Group 3 (50-69)
Group 4 (70-89)
920 944 967 994 938 1219 1048 1028 1081 1208 1017
1213 1128 1206 1117 1232 1181 1087 1186 1203 1356 1367 1512 1232
1641 1788 1601 1778 1433 1639 1424 1363 1560
1400 1453 1956 1447 1745 1470 1671 1615
-+ 87 +_ 147 +_ 81 -+ 148 _+ 101 _+ 112 _+ 138 _+ 108 + 99 -+ 116 _+ 116
1022 _+ 102
± 117 -+ 69 _+ 201 -+ 57 _+ 119 +_ 97 _+ 166 + 139 _+ 115 +_ 263 ~ 97 +_ 169 _+ 122
*BMT is represented as mean +_ S.D. p value between group 1 and group 2 < 0.001. p value between group 2 and group 3 < 0.001.
± 361 _+ 147 ~ 247 _+ 230 ~ 156 _+ 303 +_ 119 _+ 194 _+ 284
1582 +_ 152
+- 127 _+ 121 +- 205 ± 318 -+ 205 ± 146 ± 214 -+ 128
1595 -+ 191
321 TABLE V BASEMENT MEMBRANE THICKNESS* (A) OF CAPILLARIES IN PECTORALIS IN DIFFERENT AGE (YEARS) GROUPS IN FEMALES Group 1 (16-29)
Group 2 (30-49)
Group 3 (50- 69)
Group 4 (70-89)
1046 _+136 1029 _+86 1043 ± 75 998 ± 101
1144 ± 153 1145 ± 116 1122 ± 159 1086 ± 233 1053 +_160 1360 ± 224 1138 ± 95 1509 ± 172 1355 ± 119 1174 ± 142
1631 ± 201 1684 _+267 1620 _+175 1318 ± 150
1029 _+22
1209 +_148
1623 ± 161 1315 ± 137 1503 +_168 1405 _+93 1493 +_167 1312 ± 89 1253 ± 205 1410 ± 185 1230 ± 123 1555 ± 236 1267 ± 185 1268 ± 100 1386 ± 132
1563 ± 166
*BMT is represented as mean ± S,D. p value between group 1 and group 2 < 0.05. p value between group 2 and group 3 < 0.01. p value between group 3 and group 4 < 0.05. cally significant increase between age groups 1, 2, and 3. Only the females showed a significant increase between groups 3 and 4. A comparison of the mean BMT in the same age groups in males and females (Tables IV and V) showed that the BMT was similar in both sexes in group 1 (1022 vs. 1029), group 2 (1232 vs. 1209), and group 4 (1595 vs. 1563); only group 3 (1582 vs. 1386) showed a statistical difference in the BMT. The capillary BMT in males showed little increase between group 3 (age 5 0 - 6 9 ) and group 4 (age 7 0 - 8 9 ) and the results were similar to that of the seminiferous tubules. As seen in Fig. 4, the basement membrane in males steadily increased before the age of 60 and reached a plateau with only a slight increase after the age of 60. In contrast, in females a linear increase in capillary basement membranes was observed throughout the different age groups, reaching, by the age of 80, the same thickness as seen in males (Fig. 4). There appears to be a close clustering of values within a fairly narrow range before the age of 40. After 40, individual variation becomes a much more prominent feature (Fig. 3B and C). This also correlates with findings in the testes (Fig. 3A) previously described.
DISCUSSION This study included some autopsy specimens from post mortems performed more than 24 hours after death. Although these specimens were not suitable for electronmicroscopic studies of fine intracellular details, the morphology of the basement membranes remained intact [22].
322 1700
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Fig. 4. Mean basement membrane thickness of capillaries in pectoralis muscle in males and females in different age groups. Bar represents the standard error of the mean.
In this study we have quantitated BMT in seminiferous tubules and skeletal muscle (pectoralis) capillaries in different age groups by morphometry. A comparison of basement membranes derived from specimens obtained more than 24 hours after death with those obtained less than 24 hours was impossible, because the 16 cases were scattered in the different age and sex groups making a statistical analysis impossible. Our aim was twofold: (1) to correlate the change in BMT with age in epithelial and mesenchymal associated basement membranes, and (2) to compare the change with age in males and females. We chose the seminiferous tubules of the testis for the epithelial associated basement membrane. The testes are under the influence of hormonal control which decreases with age and are well known to show an increase in BMT with increased age [8]. The attempt to correlate the testis with a female reproductive organ failed, because the basement membranes of the endometrium, fallopian tubes and ovarian follicles were found to show with age either a multilaminar, a diffusely fragmented, or too thin a structure to be measured by the method used. The mesenchymal tissue selected for quantitative analysis for both sexes was the capillary of skeletal muscle. Unlike lung, kidney, liver and other organs, the pectoralis muscle is relatively immune from primary neoplasia and inflammation and in contrast to the reproductive organs is not under as prominent an endocrine influence. Any changes noted in the basement membranes of the capillaries selected could be most likely ascribed to the aging process per se, rather than to a specific disease process. An increase in BMT in diabetes has been reported by many investigators [1, 2, 1 1 - 1 9 ] . In this study, diabetes was ruled out by either the patients' histories or from laboratory results from patients' charts and autopsy protocols. Cases from the New York Medical Examiner's Office, in which insufficient information was available, involved mostly young people dying accidental deaths. Since the average BMT in these cases was found to be in the normal range for that particular age group, it was assumed that longstanding or severe diabetes was not present.
323 The morphometric method described [21] made it possible to measure the BMT of capillaries and the seminiferous tubules by the same method, since it does not require the total outline of the basement membrane for the measurement as do methods described by Siperstein et al. [13] and Williamson e t al. [11]. The error due to tangential sectioning is also minimized since the average BMT is derived by considering both the area and the length of the basement membrane. Although most of the basement membranes in capillaries and seminiferous tubules are homogeneous in structure, we occasionally observed, as described by Kilo et al. [ 1 ], diffuse, segmental or focal asymmetrical basement membrane thickening in different age groups. These findings are rather critical in morphometric studies. Thickening of the basement membrane may not involve the whole length or circumference of the vessels or seminiferous tubules. By measuring the most representative portion of the capillaries and seminiferous tubules, we obtained the best average BMT for all age groups. Our large number of random measurements in each specimen provided increased validity for our findings. The basement membrane width increased with age both in seminiferous tubules and capillaries. The data confirm the results obtained by other investigators who also observed an increase, with age, in BMT in animal and human tissue [ 1 - 7 ] . However, Deckert e t al. [ 19], while measuring basement membrane thickening in muscle capillaries in long-standing, insulin-dependent diabetics, found no correlation between BMT and age in the non-diabetic control group (ages 30-59). Our data clearly show an increase in capillary BMT in the 3 0 - 5 9 age range in males and females (Fig. 4). Comparison of the BMT from the pectoralis muscle in males and females shows that the total increase in BMT from the youngest to the oldest age group is very similar. Examination of the thickened basement membrane in males and females (Fig. 4) showed that, in males, the increase is faster than in females until it reaches a plateau at age 60. In females the increase seems to be a nonstop, continuous process throughout the female's life span as measured in these studies. There is a remarkable similarity in the increase in BMT whether the basement membrane is associated with mesenchymal or epithelial tissue or whether the surrounding cells are under direct hormonal influence (testis vs. muscle). A direct comparison of basement membrane from the two tissues in males (Fig. 2) shows a parallel increase until the age of 60 and only a slight increase in both organs after age 60. The increase in BMT from the youngest samples (age 10-29) to the oldest age groups (age 7 0 - 8 9 ) was 50.4% in seminiferous tubules, 54.4% in capillaries of the pectoralis muscle in males, and 51.9% in females--a remarkable similarity. The distribution of individual values of BMT seen in Fig. 3 for seminiferous tubules in males and capillaries in males and females showed clustering in the under 40 years of age but a wide variation over the age of 40. These findings cannot be explained in terms of strict chronological age [23]. Aging appears as a biologically independent process, rather than a strictly chronologically dependent process, with considerable variation in both males and females, especially in the middle and older age groups.
324 REFERENCES 1 C. Kilo, N. Vogler and J, R, Williamson, Muscle capillary basement membrane Changes related to aging and to diabetes mellitus. Diabetes, 21 (1972) 881-905. 2 J. M. B. Bloodworth, Jr., R. L. Engerman, R. A. Camerini-Davalos and K. L. Powers, Variations in capillary basement membrane width produced by aging and diabetes mellitus. Adv. Metab. Disord. Suppl., 1 (1970) 279-295. 3 W. Andrew and D. Pruett, Senile changes in the kidneys of Wistar Institute rats. Am. J. Anat., 100 (1957) 5 1 - 7 9 . 4 C. T. Ashworth, R. R. Erdmann and N. J. Arnold, Age changes in the renal basement membrane in rats. Am. J. Pathol., 36 (1960) 165-179. 5 S. Yagihashi and N. Kaseda, Age-related changes of glomerular basement membrane in normal rats. Tohoku J. Exp. Med., 126 (1978) 2 7 - 4 9 . 6 S. Yagihashi, Quantitative estimation of age-related thickening of glomerular basement membrane in normal rats. Tohoku J. Exp. Med., 126 (1978) 8 5 - 9 3 . 7 R. Vracko, Effects of aging and diabetes on basal lamina thickness of six cell types. In N. A. Kefalides (ed.), Biology and Chemistry of Basement Membranes, Academic Press, New York, 1978, pp. 4 8 3 - 4 9 3 . 8 D. W. King, C. M. Fenoglio and J. H. Lefkowitch, General Pathology. Principles and Dynamics, Lea & Fegiber, 1982, in the press. 9 S. L. Robbins and R. S. Cotran, Pathologic Basis of Diseases, W. B. Saunders, Philadelphia, London, Toronto, 1979, pp. 302 and 1169. 10 M. G. Farquhar, R. L. Vernier and R. A. Good, An electron microscope study of the glomerulus in nephrosis, glomerulonephritis and lupus erythematosus. J. Exp. Med., 106 (1957) 649-660. 11 J. R. Williamson, N. J. Vogler and C. Kilo, Estimation of vascular basement membrane thickness. Diabetes, 18 (1969) 5 6 7 - 5 7 8 . 12 S. A. Bencosme, R. D. West, J. W. Kerr and D. L. Wilson, Diabetic capillary angiopathy in human skeletal muscles. Am. J. Med., 40 (1966) 6 7 - 7 7 . 13 M. D. Siperstein, R. H. Unger and L. L. Madison, Studies of muscle capillary basement membranes in normal subjects, diabetic, and prediabetic patients. J. Clin lnvest., 47 (1968) 1973-1999. 14 R. Vracko, Skeletal muscle capillaries in diabetics: A quantitative analysis. Circulation, 41 (1970) 271-283. 15 J. R. Williamson and C. Kilo, Current status of capillary basement membrane disease in diabetes mellitus. Diabetes, 26 (1977) 6 5 - 7 5 . 16 T. S. Danowski, R. C. Khuranae, A. R. Gonzalez and E. R. Fisher, Capillary basement membrane thickness and the pseudodiabetes of myopathy. Am. J. Med., 51 (1971) 757-766. 17 V. W. Fischer, H. B. Barner and M. L. Leskiw, Capillary basal laminar thickness in diabetic human my0cardium. Diabetes, 28 (1979) 7 1 3 - 7 1 9 . 18 G. E. Peterson and P. H. Fursham, Variation in thickness of the capillary basement membrane in single muscles of diabetic subjects. Diabetes, 28 (1979) 548-551. 19 T. Deckert, J. Egerberg, C. Frimodf-M¢ller, E. Sander and A. Svejgaard, Basement membrane thickness, insulin antibodies and HLA-antigens in long standing insulin dependent diabetics with and without severe retinopathy. Diabetologia, 17 (1979) 9 1 - 9 6 . 20 E. S. Reynolds, The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 1 7 (1963) 208-213. 21 E. E. Underwood, Quantitative Stereology, Addison-Wesley, Massachusetts, 1970, pp. 23-45. 22 J. M. B. Bloodworth, Jr., Diabetic microangiography. Diabetes, 12 (1963) 99-114. 23 D. A. Hall, Theories o f Ageing in the Ageing of Connective Tissue, Academic Press, New York, 1976, pp. 1-12.
315
AGE-RELATED CHANGES IN NORMAL HUMAN BASEMENT MEMBRANE
YU-PING XI, E. GERDA NETTE, DONALD WEST KING and MOSHE ROSEN College o f Physicians and Surgeons, Department o f Pathology, New York, New York 10032 (U.S.A.)
(Received March 2, 1982)
SUMMARY Basement membranes were studied in the seminiferous tubules of the male, and capillaries of the pectoralis muscles in "normal" males and females. Age-related changes in basement membrane thickness were investigated on electromicrographs by quantitative morphometry. Specimens obtained from either autopsies or surgical procedures were divided into four different age groups ranging from age 16 to age 87 years. The results show a statistically significant increase in the basement membrane thickness correlated with age, in basement membranes associated with both mesenchymal (capillaries) and epithelial (testes) tissue. A comparison of capillary basement membrane in the pectoralis muscle and seminiferous tubule basement membrane in the male show a constant increase in thickness until the age of 60 years, following which only slight increases were noted. The basement membrane thickness in capillaries of the pectoralis muscle in females showed linear increases throughout the lifespan, although at a lower rate than in the male; at age 80 years, it had the same thickness as the male. A comparison of basement membrane thickness between the youngest and oldest groups showed an increase of approximately 50% in all tissues studied.
INTRODUCTION Basement membranes, associated with both epithelial and mesenchymal tissue, have been altered in many diseases as well as in the aging process [ 1 - 7 ] . Basement membranes are widely distributed throughout the body as extracellular components of blood vessels and muscle, and are important structures in the genitourinary, respiratory, gastrointestinal and endocrine systems. Although their function is not well understood, they may serve as a vascular mechanical barrier through which all cells and macromolecules entering tissue must pass, and provide a framework for tissue differentiation as well as regeneration following injury. Thickening of basement membrane has been proposed as an inhibitor to neoplastic invasion [8], is seen with arteriosclerosis [9], and is a prominent feature in many of the 0047-6374/82/0000-0000/$02.75
© Elsevier Sequoia/Printed in The Netherlands
316 collagen diseases [9, 10]. Many reports are available indicating that basement membranes thicken with age [1, 2, 4] particularly in association with maturity-onset diabetes [1,2, 11--19]. The latter is often suggested to be an age-related disease. The duration and/or severity of the diabetes has been correlated with the thickness of the muscle basement membrane [1]. This study quantitatively measures the increase in basement membrane thickness correlated with age in seminiferous tubules of the testes. Measurements of basement membranes in capillaries of the pectoralis muscles, in males and females, are also correlated with age.
METHOD AND MATERIALS The thickness of basement membranes was measured in tissues obtained from both autopsies and surgical specimens. The 111 specimens were received from surgical specimens or autopsies performed at the Columbia-Presbyterian Medical Center, St. Luke's Hospital, or from the New York Medical Examiner's Office (Table I). Autopsy specimens were mainly from post mortem examinations performed within 24 hours after death (Table II). Diabetes was ruled out both from the patients' personal histories and laboratory information obtained from patients' charts and autopsy protocols. The specimens were fixed overnight in 2.5% glutaraldehyde in cocadylate buffer at pH 7.4. After rinsing in the same buffer, the tissues were postfixed in 1% osmium tetroxide in cocadylate buffer for 2 hours, processed for embedding through an ascending series of ethanol and embedded in epon. Thin sections were cut with a Sorvall MT-1 ultramicrotome, stained with uranyl acetate and lead citrate [20], and examined and photographed on a Siemens Elmiskop 1 or 1A electron microscope. Ten to twelve basement membranes of capillaries from the pectoral muscle or seminiferous tubules were measured from each specimen and were averaged to obtain the mean basement membrane thickness. The micrographs were enlarged to a final magnification of 30 000 for capillaries and 15 000 for seminiferous tubules. Variations in magnification related to instability of the electron microscope were monitored by calibration plates. Morphometric measurements of basement membranes were employed as described by Underwood [21]. The estimate of basement membrane thickness was calculated as follows: After outlining the basement membrane, a midline between the inner and outer boundary of the basement membrane was drawn. A plastic template (square grid) of known size (D) was placed over the enlarged electron micrograph. To measure the mean thickness (T) two counts were made: (1) the number of intersections between the drawn midline of the basement membrane and the overlaying grid system (N~); (2) the number of nodal points of the grid falling inside the area of the basement membrane (NA). The mean thickness was calculated from the formula NA
4
NL
n
=--X--XDXM
317 TABLE I SOURCE AND NUMBER OF MEASURED SPECIMENS Capillaries in pectoralis
Post mortem Surgery Total
Testes
Male
Female
Total
31 10 41
40 0 40
18 12 30
89* 22 111
*89 Specimens were collected from 59 autopsies. 30 Specimens of testis and pectoralis came from the same case. TABLE II HOURS OF DEATH BEFORE AUTOPSY IN 59 CASES 12 hours
20
1 3 - 2 4 hours
> 24 hours
Unknown *
23
12
4
• Exact time of death unknown, but estimated time definitely more than 24 hours.
where D is the size of the grid ( = 1 / 4 cm) and M is the magnification (/am on the specimen/cm on the image).
RESULTS The specimens of seminiferous tubules of the testes were divided into four age groups, as were the capillary specimens from the pectoral muscle: group 1 specimens, 1 0 - 2 9 years of age; group 2 specimens, 3 0 - 4 9 years; group 3 specimens, 5 0 - 6 9 years; and group 4 specimens, 7 0 - 8 9 years. Representative examples of basement membranes of capillaries in the pectoralis muscle as well as basement membrane in the testes are shown (Fig. 1). The average basement membrane thickness (BMT) of each specimen (seminiferous tubules) examined and the mean BMT in the four different age groups are shown in Table III. Comparison of the thickness of the basement membrane in the different age groups showed a statistically significant increase in BMT between groups 1 (age 1 0 - 2 9 ) , 2 (age 3 0 - 4 9 ) and 3 (age 5 0 - 6 9 ) , but no statistical difference could be detected between group 3 and group 4 (age 70--89). There was a sharp increase in BMT with increased age (Fig. 2), which reached a plateau at the age of 60 and showed only a slight increase between the ages of 60 and 80. When the average BMT for each specimen was plotted (Fig. 3A), it became apparent that the distribution of values for BMT in the under 40 years of age was small. After the age of 40 the BMT showed a marked increase and with much greater individual variation in distribution.
318
i
Fig. 1. A + B. Basement membrane (arrows) of capillaries of pectoralis muscle with red blood cells. (×30000). (A) Male, 16 years old, thickness of membrane 951 A. (B) Female, 53 years old, thickness of membrane 1752 A. C + D. Basement membrane (arrows) of seminiferous tubules in testes (× 15 000). (C) Male, 23 years old, thickness of membrane 1585 A. (D) Male, 58 years old, thickness of membrane 3775 A. The m e a n m e a s u r e m e n t s o f BMT o f capillaries in s t r i a t e d muscle ( p e c t o r a l i s ) in t h e d i f f e r e n t age g r o u p s are given in T a b l e IV for males a n d in T a b l e V for females. Comparing the BMT in t h e d i f f e r e n t age g r o u p s it was a p p a r e n t t h a t b o t h sexes s h o w e d a statisti-
319
TABLE III BASEMENT MEMBRANE THICKNESS* (A) OF SEMINIFEROUS TUBULES IN D I F F E R E N T AGE (YEARS) GROUPS
Group 1 (16-29)
Group 2 (30-49)
Group 3 (50-69)
Group 4 {70-89)
1329 1721 1738 1524 1675 1513 1621 1883
1168 1728 1819 1580 1693 1680 1628 1946 1937 2063 1864 2400 1834
2625 1956 2581 2116 2413 2979 2481 2271 2222
2140 2360 2897 2596 2206 2136 2244 2190 3281 2859 2382 2060 2446
± ± ± ± ± ± ± ±
123 201 220 176 188 107 142 195
1626 ± 170
± ± ± ± ± ± ± ± ± ± ± ± ±
176 184 161 187 375 196 436 154 287 208 314 354 230
± 273 ± 146 ± 363 ± 247 ± 324 +_456 ± 477 +_439 _+ 119
2405 ± 306
+_ 214 ± 304 _+419 ± 340 ± 251 ± 226 ± 269 +- 184 +- 319 ± 368 ± 195 ± 272 ± 342
*BMT is represented as mean ± S.D. p value between group 1 and group 2 < 0.05.
p value between group 2 and group 3 < 0.001.
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TABLE IV BASEMENT MEMBRANE THICKNESS* (A) OF CAPILLARIES IN PECTORALIS IN D I F F E R E N ] AGE (YEARS) GROUPS IN MALES
Group 1 (16-29)
Group 2 (30-49)
Group 3 (50-69)
Group 4 (70-89)
920 944 967 994 938 1219 1048 1028 1081 1208 1017
1213 1128 1206 1117 1232 1181 1087 1186 1203 1356 1367 1512 1232
1641 1788 1601 1778 1433 1639 1424 1363 1560
1400 1453 1956 1447 1745 1470 1671 1615
-+ 87 +_ 147 +_ 81 -+ 148 _+ 101 _+ 112 _+ 138 _+ 108 + 99 -+ 116 _+ 116
1022 _+ 102
± 117 -+ 69 _+ 201 -+ 57 _+ 119 +_ 97 _+ 166 + 139 _+ 115 +_ 263 ~ 97 +_ 169 _+ 122
*BMT is represented as mean +_ S.D. p value between group 1 and group 2 < 0.001. p value between group 2 and group 3 < 0.001.
± 361 _+ 147 ~ 247 _+ 230 ~ 156 _+ 303 +_ 119 _+ 194 _+ 284
1582 +_ 152
+- 127 _+ 121 +- 205 ± 318 -+ 205 ± 146 ± 214 -+ 128
1595 -+ 191
321 TABLE V BASEMENT MEMBRANE THICKNESS* (A) OF CAPILLARIES IN PECTORALIS IN DIFFERENT AGE (YEARS) GROUPS IN FEMALES Group 1 (16-29)
Group 2 (30-49)
Group 3 (50- 69)
Group 4 (70-89)
1046 _+136 1029 _+86 1043 ± 75 998 ± 101
1144 ± 153 1145 ± 116 1122 ± 159 1086 ± 233 1053 +_160 1360 ± 224 1138 ± 95 1509 ± 172 1355 ± 119 1174 ± 142
1631 ± 201 1684 _+267 1620 _+175 1318 ± 150
1029 _+22
1209 +_148
1623 ± 161 1315 ± 137 1503 +_168 1405 _+93 1493 +_167 1312 ± 89 1253 ± 205 1410 ± 185 1230 ± 123 1555 ± 236 1267 ± 185 1268 ± 100 1386 ± 132
1563 ± 166
*BMT is represented as mean ± S,D. p value between group 1 and group 2 < 0.05. p value between group 2 and group 3 < 0.01. p value between group 3 and group 4 < 0.05. cally significant increase between age groups 1, 2, and 3. Only the females showed a significant increase between groups 3 and 4. A comparison of the mean BMT in the same age groups in males and females (Tables IV and V) showed that the BMT was similar in both sexes in group 1 (1022 vs. 1029), group 2 (1232 vs. 1209), and group 4 (1595 vs. 1563); only group 3 (1582 vs. 1386) showed a statistical difference in the BMT. The capillary BMT in males showed little increase between group 3 (age 5 0 - 6 9 ) and group 4 (age 7 0 - 8 9 ) and the results were similar to that of the seminiferous tubules. As seen in Fig. 4, the basement membrane in males steadily increased before the age of 60 and reached a plateau with only a slight increase after the age of 60. In contrast, in females a linear increase in capillary basement membranes was observed throughout the different age groups, reaching, by the age of 80, the same thickness as seen in males (Fig. 4). There appears to be a close clustering of values within a fairly narrow range before the age of 40. After 40, individual variation becomes a much more prominent feature (Fig. 3B and C). This also correlates with findings in the testes (Fig. 3A) previously described.
DISCUSSION This study included some autopsy specimens from post mortems performed more than 24 hours after death. Although these specimens were not suitable for electronmicroscopic studies of fine intracellular details, the morphology of the basement membranes remained intact [22].
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In this study we have quantitated BMT in seminiferous tubules and skeletal muscle (pectoralis) capillaries in different age groups by morphometry. A comparison of basement membranes derived from specimens obtained more than 24 hours after death with those obtained less than 24 hours was impossible, because the 16 cases were scattered in the different age and sex groups making a statistical analysis impossible. Our aim was twofold: (1) to correlate the change in BMT with age in epithelial and mesenchymal associated basement membranes, and (2) to compare the change with age in males and females. We chose the seminiferous tubules of the testis for the epithelial associated basement membrane. The testes are under the influence of hormonal control which decreases with age and are well known to show an increase in BMT with increased age [8]. The attempt to correlate the testis with a female reproductive organ failed, because the basement membranes of the endometrium, fallopian tubes and ovarian follicles were found to show with age either a multilaminar, a diffusely fragmented, or too thin a structure to be measured by the method used. The mesenchymal tissue selected for quantitative analysis for both sexes was the capillary of skeletal muscle. Unlike lung, kidney, liver and other organs, the pectoralis muscle is relatively immune from primary neoplasia and inflammation and in contrast to the reproductive organs is not under as prominent an endocrine influence. Any changes noted in the basement membranes of the capillaries selected could be most likely ascribed to the aging process per se, rather than to a specific disease process. An increase in BMT in diabetes has been reported by many investigators [1, 2, 1 1 - 1 9 ] . In this study, diabetes was ruled out by either the patients' histories or from laboratory results from patients' charts and autopsy protocols. Cases from the New York Medical Examiner's Office, in which insufficient information was available, involved mostly young people dying accidental deaths. Since the average BMT in these cases was found to be in the normal range for that particular age group, it was assumed that longstanding or severe diabetes was not present.
323 The morphometric method described [21] made it possible to measure the BMT of capillaries and the seminiferous tubules by the same method, since it does not require the total outline of the basement membrane for the measurement as do methods described by Siperstein et al. [13] and Williamson e t al. [11]. The error due to tangential sectioning is also minimized since the average BMT is derived by considering both the area and the length of the basement membrane. Although most of the basement membranes in capillaries and seminiferous tubules are homogeneous in structure, we occasionally observed, as described by Kilo et al. [ 1 ], diffuse, segmental or focal asymmetrical basement membrane thickening in different age groups. These findings are rather critical in morphometric studies. Thickening of the basement membrane may not involve the whole length or circumference of the vessels or seminiferous tubules. By measuring the most representative portion of the capillaries and seminiferous tubules, we obtained the best average BMT for all age groups. Our large number of random measurements in each specimen provided increased validity for our findings. The basement membrane width increased with age both in seminiferous tubules and capillaries. The data confirm the results obtained by other investigators who also observed an increase, with age, in BMT in animal and human tissue [ 1 - 7 ] . However, Deckert e t al. [ 19], while measuring basement membrane thickening in muscle capillaries in long-standing, insulin-dependent diabetics, found no correlation between BMT and age in the non-diabetic control group (ages 30-59). Our data clearly show an increase in capillary BMT in the 3 0 - 5 9 age range in males and females (Fig. 4). Comparison of the BMT from the pectoralis muscle in males and females shows that the total increase in BMT from the youngest to the oldest age group is very similar. Examination of the thickened basement membrane in males and females (Fig. 4) showed that, in males, the increase is faster than in females until it reaches a plateau at age 60. In females the increase seems to be a nonstop, continuous process throughout the female's life span as measured in these studies. There is a remarkable similarity in the increase in BMT whether the basement membrane is associated with mesenchymal or epithelial tissue or whether the surrounding cells are under direct hormonal influence (testis vs. muscle). A direct comparison of basement membrane from the two tissues in males (Fig. 2) shows a parallel increase until the age of 60 and only a slight increase in both organs after age 60. The increase in BMT from the youngest samples (age 10-29) to the oldest age groups (age 7 0 - 8 9 ) was 50.4% in seminiferous tubules, 54.4% in capillaries of the pectoralis muscle in males, and 51.9% in females--a remarkable similarity. The distribution of individual values of BMT seen in Fig. 3 for seminiferous tubules in males and capillaries in males and females showed clustering in the under 40 years of age but a wide variation over the age of 40. These findings cannot be explained in terms of strict chronological age [23]. Aging appears as a biologically independent process, rather than a strictly chronologically dependent process, with considerable variation in both males and females, especially in the middle and older age groups.
324 REFERENCES 1 C. Kilo, N. Vogler and J, R, Williamson, Muscle capillary basement membrane Changes related to aging and to diabetes mellitus. Diabetes, 21 (1972) 881-905. 2 J. M. B. Bloodworth, Jr., R. L. Engerman, R. A. Camerini-Davalos and K. L. Powers, Variations in capillary basement membrane width produced by aging and diabetes mellitus. Adv. Metab. Disord. Suppl., 1 (1970) 279-295. 3 W. Andrew and D. Pruett, Senile changes in the kidneys of Wistar Institute rats. Am. J. Anat., 100 (1957) 5 1 - 7 9 . 4 C. T. Ashworth, R. R. Erdmann and N. J. Arnold, Age changes in the renal basement membrane in rats. Am. J. Pathol., 36 (1960) 165-179. 5 S. Yagihashi and N. Kaseda, Age-related changes of glomerular basement membrane in normal rats. Tohoku J. Exp. Med., 126 (1978) 2 7 - 4 9 . 6 S. Yagihashi, Quantitative estimation of age-related thickening of glomerular basement membrane in normal rats. Tohoku J. Exp. Med., 126 (1978) 8 5 - 9 3 . 7 R. Vracko, Effects of aging and diabetes on basal lamina thickness of six cell types. In N. A. Kefalides (ed.), Biology and Chemistry of Basement Membranes, Academic Press, New York, 1978, pp. 4 8 3 - 4 9 3 . 8 D. W. King, C. M. Fenoglio and J. H. Lefkowitch, General Pathology. Principles and Dynamics, Lea & Fegiber, 1982, in the press. 9 S. L. Robbins and R. S. Cotran, Pathologic Basis of Diseases, W. B. Saunders, Philadelphia, London, Toronto, 1979, pp. 302 and 1169. 10 M. G. Farquhar, R. L. Vernier and R. A. Good, An electron microscope study of the glomerulus in nephrosis, glomerulonephritis and lupus erythematosus. J. Exp. Med., 106 (1957) 649-660. 11 J. R. Williamson, N. J. Vogler and C. Kilo, Estimation of vascular basement membrane thickness. Diabetes, 18 (1969) 5 6 7 - 5 7 8 . 12 S. A. Bencosme, R. D. West, J. W. Kerr and D. L. Wilson, Diabetic capillary angiopathy in human skeletal muscles. Am. J. Med., 40 (1966) 6 7 - 7 7 . 13 M. D. Siperstein, R. H. Unger and L. L. Madison, Studies of muscle capillary basement membranes in normal subjects, diabetic, and prediabetic patients. J. Clin lnvest., 47 (1968) 1973-1999. 14 R. Vracko, Skeletal muscle capillaries in diabetics: A quantitative analysis. Circulation, 41 (1970) 271-283. 15 J. R. Williamson and C. Kilo, Current status of capillary basement membrane disease in diabetes mellitus. Diabetes, 26 (1977) 6 5 - 7 5 . 16 T. S. Danowski, R. C. Khuranae, A. R. Gonzalez and E. R. Fisher, Capillary basement membrane thickness and the pseudodiabetes of myopathy. Am. J. Med., 51 (1971) 757-766. 17 V. W. Fischer, H. B. Barner and M. L. Leskiw, Capillary basal laminar thickness in diabetic human my0cardium. Diabetes, 28 (1979) 7 1 3 - 7 1 9 . 18 G. E. Peterson and P. H. Fursham, Variation in thickness of the capillary basement membrane in single muscles of diabetic subjects. Diabetes, 28 (1979) 548-551. 19 T. Deckert, J. Egerberg, C. Frimodf-M¢ller, E. Sander and A. Svejgaard, Basement membrane thickness, insulin antibodies and HLA-antigens in long standing insulin dependent diabetics with and without severe retinopathy. Diabetologia, 17 (1979) 9 1 - 9 6 . 20 E. S. Reynolds, The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol., 1 7 (1963) 208-213. 21 E. E. Underwood, Quantitative Stereology, Addison-Wesley, Massachusetts, 1970, pp. 23-45. 22 J. M. B. Bloodworth, Jr., Diabetic microangiography. Diabetes, 12 (1963) 99-114. 23 D. A. Hall, Theories o f Ageing in the Ageing of Connective Tissue, Academic Press, New York, 1976, pp. 1-12.