MICROVASCULAR
RESEARCH
10,76-82
(1975)
Capillary Basement Membrane Thickness: A Comparison of Two Morphometric Methods for Its Estimation’ M. L. BEAUCHEMIN, G. ANTILLE, AND P. M. LEUENBERGER University Eye Clinic and Institut de Statistique mathkmatique universitaire, Geneva, Switzerland
Received November 19, 1974
Retinal capillary basement membrane thickness, BMT, was measured in four different groups of animals, two of which were rendered diabetic by streptozotocin injections and had an additional stress factor on their diet and two nondiabetic control groups, one having the additional stress factor on its diet. Two morphometric methods were used to carry out the measurements of BMT. The first method used was that of Siperstein et al. (1968), in which sites for measuring BMT are chosen at random; in this study 16 sites per capillary were chosen. The second method used was that of Williamson et al. (1969), in which two carefully chosen minimal measurements per capillary are made. A statistical comparison of the two methods was carried out using the nested variance analysis and an extension of this test, the allocation of resources. It was concluded that the more simplified and less time-consuming method of Williamson et al. is the more precise method of the two for the estimation of mean BMT.
Capillary basement membrane thickness (BMT), has been considered a feature generally associated with diabetic microangiopathy ever since Friedenwald’s earlier observations (6,7). However, the resolution of the light microscope ( 2 2000 A), did not allow for an accurate quantitative examination of BMT. Various factors appear to have an influence upon BMT, such as age (24,9-l 1, 13,15), weight (12,14), sex (10,18), anatomical distribution (8, 20, 22), capillary circumference (14), diabetes (for reference see lo), or prediabetic state (5, 16, 18). In addition, numerous pathological conditions other than diabetes seem to be accompanied by BMT (1, 17). Multiple sources of error may influence quantitation of BMT. Some of these are due to artifacts from tissue preparation or section thickness and orientation, others are due to errors in magnification and lastly the statistical method used for evaluating the BMT is of utmost importance. Recently we undertook a study (14), involving a morphometric analysis of retinal capillary BMT of four groups of animals, two of which were rendered diabetic by injections of streptozotocin and two of which were control groups. The morphometric method used for measuring BMT was that of Siperstein et al. (18). Since this is rather a long and tedious method, we repeated the morphometric analysis using the more simplified method of Williamson et al. (21), and compared the two methods determining which of them gives the more precise estimation of the BMT. Williamson et al’s method 1 Supported by SNSF Grant 3.1150.73. Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
76
MORPHOMETRY OF BASEMENT MEMBRANES
77
FIG. 1. Electron micrograph of a retinal capillary from group II, showing a grid similar to that of Siperstein et al. which chooses at random sites for measuring the BMT (arrow); 16 instead of 20 equidistant measurements were used in this study. Calculated mean basement membrane thickness 1154 A. (Magnification: x12,600). E = endothelial cell; L = lumen.
78
BEAUCHEMIN,
ANTILLE
AND
LEUENBERGER
was also chosen because it takes into account the major sources of error mentioned above. As such we thought this method to be potentially interesting for future morphometric studies. MATERIAL
AND
METHODS
The retinas of 20 Wistar rats were used for this study. The animals were divided into four groups, two of which were experimentally diabetic, one having an additional stress factor on its diet (devoid of flavonoids), group I, the second a stress factor control on its diet, i.e., flavonoid-free diet plus flavonoids, group II. The remaining two groups were nondiabetic control groups one having the stress factor on its diet, group III, and the last group having normal rat food, group IV. Details of the method of this study can be found in an earlier publication (14). The first morphometric analysis of BMT was carried out according to Siperstein et al. (18), except that 16 instead of 20 measurements were made per capillary (Fig. 1). Eleven capillaries per animal were used in both methods. Of the four groups examined there were six animals in group I, six in group II, four in group III, and four in group IV. The second morphometric analysis carried out was that of Williamson et al. (21), in which only two minimal measurements per capillary are made (Fig. 2). A form of nested variance analysis (19a), was used to compare these two morphometric methods, classifying them into two statistical groups. Three subsamples were used in this test: animals (a), capillaries in animals (b), and measurements in capillaries (c), each having, respectively, I, m and IZ elements. By this analysis we were able to obtain the variance for the average of the sample, & using the variance of each subsample.
where ga, Ok,, and cc represent, respectively, the standard deviation of the subsamples. An extension of this test, “the allocation of resources” (19b), was used to find the ideal number of values in the three subsamples chosen, in order to give the most precise results, taking into account, however, that one knows their CS.The formulas used were the following :
where k, = the time spent to choose the animals, kb = the time spent to choose the capillaries, and k, = the time spent to choose and carry out the measurements. In both methods k, and kb are the same; however, k, is larger in the Siperstein et al. method than in that of Williamson et al. RESULTS A comparison of the methods using the nested variance analysis can be summarized in Table 1 (the results of the total nested variance have not included the constant to convert the numbers in A (105/195).
MORPHOMETRY OF BASEMENT MEMBRANES
79
FIG. 2. Electron micrograph (the same as in Fig. 1) of a retinal capillary from group II showing sites where two minimal measurements of BMT have been chosen (arrows), using Williamson et& method. No grid is used since the minimal sites are carefully selected and not chosen at random as in Fig. 1. Calculated mean basement membrane width: 718 A. (Magnification: x12,600). E = endothelial cell; L = lumen.
80
BEAUCHEMIN,
ANTILLE,
TABLE NESTED VARIANCE
AND
LEUENBERGER
I ANALYSIS
Siperstein et at. Group 2 so 4 2 SC s: Group s,2 4 2 SC s: Group 2 SD s: 2 SC dGroup so2 4 2 SC S:
Williamson
et al.
I: Diabetic rats + flavonoid-free diet 0.052998 0.101050 0.902771 0.011219
0.021558 0.194702 0.053030 0.006945
0.514083 0.173726 2.158458 0.090357
0.102066 0.154546 0.075757 0.019927
0.086578 0.055416 0.661079 0.023843
0.110950 0.075 0.056818 0.030088
0.028262 0.093496 0.336742 0.009669
0.046316 0.073864 0.136363 0.014807
II : Diabetic rats + flavonoid-free diet + flavonoids
III: Nondiabetic control + flavonoid-free diet
IV: Nondiabetic control + normal rat food
a sa = estimated variance within animal subsample; sg = estimated variance within capillary subsample; s: = estimated variance within measurement subsample; s: = estimated variance of the average of BMT. In groups I and II the CT:of Williamson et al’s method is significantly smaller than that of Siperstein et at. ; in group III it is not significantly different, and in group IV the o$ of Siperstein et al’s method is significantly smaller than that of Williamson et al.
DISCUSSION In order to determine the method which yields the more precise estimation of BMT with a minimal variance, we found it preferable to use a nested variance analysis (19). This analysis allowed us to compare the total average variance of the three statistical subsamples, namely, the animals, the capillaries within the animals, and the measurements within the capillaries. This comparative analysis was preferred to a comparison of the coefficient of variation as Williamson et al. (21) used. We found that by employing the latter we were not able to tell at what value the ratio of the coefficient of variation of the two methods would differ significantly from 1. In the nested variance analysis the components of the variability of one group were calculated and it was then possible to determine if the number of blood vessels or measurements should be increased or decreased to give the most precise values of BMT. The number of animals as a factor of variability was not taken into account in this study. The results of this study show that by using the method of Williamson et al. (21) the estimated variance for the average of BMT is significantly smaller (p < 0.05)2 for the two z F-test.
MORPHOMETRY
OF BASEMENT
MEMBRANES
81
diabetic groups than that obtained with the method of Siperstein et al. (18). In the third group, however, the estimated variance is not significantly different and in the fourth group it was found that with the method of Siperstein et al. (18) it is significantly smaller than with that of Williamson et al. (p < 0.05)2. This must be due to the fact that it is a control group with no basement membrane focal thickenings as are regularly found in the diabetic group (12, 14). The Siperstein et al. (18) method, however, owing to its multiple measuring points, appears to be more sensitive in detecting these focal thickenings which probably also account for the larger estimated variance for the average of BMT in the diabetic groups. In view of the fact that BM focal thickenings have been reported to occur in both human and experimental diabetic microangiopathy even before an increase of the mean BM width is detectable (10, 12, 17), this point certainly deserves particular attention. The two diabetic groups have a BMT which is more heterogeneous than the control groups. Since Williamson et al’s method (21) yielded a better estimation of the variance of the average of BMT of these two diabetic groups, we therefore concluded it to be the more precise method of the two. Its usefulness is further emphasized by the fact that the time spent to choose and carry out the measurements within the capillaries (k,) using Williamson et al’s method is smaller than that of Siperstein et al. By using an extension of the nested variance analysis to compare the two methods it was shown that in order to decrease the variance of the average BMT it is necessary to decrease the number of measurements per capillary and increase the number of small blood vessels to be measured.
REFERENCES J. M., JR., AND SOMMERS, S. C. (1959). “Cirrhotic glomerulosclerosis,” a renal lesion associated with hepatic cirrhosis. Lab. Invest. 8, 962-978.
1. BLOODWORTH, 2. BLOODWORTH,
3. 4.
J. M., JR., ENGERMAN,
R. L., CAMERINI-DAVALOS,
R. A., AND POWERS, K. L. (1970).
Variations in capillary basement membrane width produced by aging and diabetes mellitus. Zn “Early Diabetes” (R. A. Camerini-Davalos and K. L. Powers, eds.), pp. 279-287. Academic Press, New York. BLOOM, P. M., HARTMANN, J. F., AND VERNIER, R. L. (1959). An electron microscopic evaluation of the width of normal glomerular basement membrane in man at various ages. BURDITT, A. F., CAIRO, F. I., AND DRAPER, G. J (1968). The natural history of diabetic retinopathy. Quart. J. Med. 37, 303-317. D. P., AMHERDT, M., LEUENBERGER, P. M., ORCI, L., AND STAUFFACHER, W. (1973). Microvascularalterationsin chronicallystreptozotocin-diabetic rats. In “Vascularand NeurologicalChanges in EarlyDiabetes”(suppl.2to Advances in MetabolicDisorders), (R. A. CameriniDavalosandH. S. Cole,eds.),pp. 257-264.AcademicPress,NewYork. FRIEDENWALD, J. S. (1949).A newapproachto someproblemsof retinalvasculardisease. Amer.
5. CAMERON,
6.
J. Ophthalmol. 32,487-498. 7. FRIEDENWALD, J. S. (1950).Diabeticretinopathy.Amer. J. Ophthalmol. 33, 1187-1199. 8. FRIEDERICI, H. H. R., TUCKER, W. R., AND SCHWARTZ, T. B. (1966).Observarions onsmallblood vessels of skin in the normalandin diabeticpatients.Diabetes 15, 233-250. 9. JORDAN, S. W., AND PERLEY, M. J. (1972).Microangiopathy in diabetesmellitusandaging.Arch.
Pathol. 93,261-265. C., VOGLER, N., AND WILLIAMSON, J. R. (1972).Musclecapillarybasement membrane changes relatedto agingandto diabetesmellitus.Diabetes 21, 881-904. LEUENBERGER, P. M., BABEL, J., AND FULL, C. (1970).Width of retinalcapillarybasement membraneof spinymice(Acomys cahirinus) at variousages.Dot. Ophthalmol.28, 191-200.
10. KILO, 11.
82
BEAUCHEMIN,
ANTILLE
AND
LEUENBERGER
12. LEUENBERGER, P. M., CAMERON, D., STAUFFACHER, W., RENOLD, A. E., AND BABEL, J. (1971). Ocular lesions in rats rendered chronically diabetic with streptozotocin. Ophthalmol. Res. 2, 189-204. 13. LEUENBERGER, P. M. (1972). Ultrastructure of the aging retinal vascular system with special reference to quantitative and qualitative changes of capillary basement membranes. Gerontologiu 19, 1-15. 14. LEUENBERGER, P. M., BEAUCHEMIN, M. L., AND BABEL, J. (1974). Experimental diabeticretinopathy. Arch. Ophthalmol. 34289-302. 15. ORCI, L., STAUFFACHER, W., AMHERDT, M., PICTET, R., RENOLD, A. The kidney of spiny mice (Acomys cahirinus) : Electron microscopy
16. 17. 18. 19. 20. 21. 22.
E., AND ROUILLER, C. (1970). of glomerular changes associated with aging and tubular glycogen accumulation during hyperglycemia. Diubefologiu 6,343355. ~STERBY, R. (1972). Morphometric studies of the peripheral glomerular basement membrane in early juvenile diabetes. I. Development of initial basement membrane thickening. Diabetologia 8, 84-92. POMETTA, D., AMHERDT, M., AND RUFFENER, C. (1971). Capillary basement membrane and abnormality of carbohydrate metabolism (abstract). Diubetologia 7, 152. SIPERSTEIN, M. D., UNGER, R. H., AND MADISON, L. L. (1968). Studies of muscle capillary basement membranes in normal subjects, diabetic and prediabetic patients. J. Clin. Invest. 47,1937-1999. SNEDECOR, G. W., AND COCHRAN, W. G. (1967). “Statistical Methods”, 6thed.,pp. 285-288,10.16; 531-533, 17.12. Iowa State Univ. Press. Ames, Iowa. VRACKO, R. (1970). Skeletal muscle capillaries in diabetes: A quantitative analysis. Circulation 41, 271-283. WILLIAMSON, I. R., VOGLER, N. J., AND KILO, C. (1969). Estimation of vascular basement membrane thickness. Theoretical and practical considerations. Diabetes 18, 567-578. WILLIAMSON, J. R., VOGLER, N. J., AND KILO, C. (1971). Regional variations in the width of the basement membrane of muscle capillaries in man and giraffe. Amer. .Z. Z’athol. 63, 359-370.
RESEARCH
10,76-82
(1975)
Capillary Basement Membrane Thickness: A Comparison of Two Morphometric Methods for Its Estimation’ M. L. BEAUCHEMIN, G. ANTILLE, AND P. M. LEUENBERGER University Eye Clinic and Institut de Statistique mathkmatique universitaire, Geneva, Switzerland
Received November 19, 1974
Retinal capillary basement membrane thickness, BMT, was measured in four different groups of animals, two of which were rendered diabetic by streptozotocin injections and had an additional stress factor on their diet and two nondiabetic control groups, one having the additional stress factor on its diet. Two morphometric methods were used to carry out the measurements of BMT. The first method used was that of Siperstein et al. (1968), in which sites for measuring BMT are chosen at random; in this study 16 sites per capillary were chosen. The second method used was that of Williamson et al. (1969), in which two carefully chosen minimal measurements per capillary are made. A statistical comparison of the two methods was carried out using the nested variance analysis and an extension of this test, the allocation of resources. It was concluded that the more simplified and less time-consuming method of Williamson et al. is the more precise method of the two for the estimation of mean BMT.
Capillary basement membrane thickness (BMT), has been considered a feature generally associated with diabetic microangiopathy ever since Friedenwald’s earlier observations (6,7). However, the resolution of the light microscope ( 2 2000 A), did not allow for an accurate quantitative examination of BMT. Various factors appear to have an influence upon BMT, such as age (24,9-l 1, 13,15), weight (12,14), sex (10,18), anatomical distribution (8, 20, 22), capillary circumference (14), diabetes (for reference see lo), or prediabetic state (5, 16, 18). In addition, numerous pathological conditions other than diabetes seem to be accompanied by BMT (1, 17). Multiple sources of error may influence quantitation of BMT. Some of these are due to artifacts from tissue preparation or section thickness and orientation, others are due to errors in magnification and lastly the statistical method used for evaluating the BMT is of utmost importance. Recently we undertook a study (14), involving a morphometric analysis of retinal capillary BMT of four groups of animals, two of which were rendered diabetic by injections of streptozotocin and two of which were control groups. The morphometric method used for measuring BMT was that of Siperstein et al. (18). Since this is rather a long and tedious method, we repeated the morphometric analysis using the more simplified method of Williamson et al. (21), and compared the two methods determining which of them gives the more precise estimation of the BMT. Williamson et al’s method 1 Supported by SNSF Grant 3.1150.73. Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britain
76
MORPHOMETRY OF BASEMENT MEMBRANES
77
FIG. 1. Electron micrograph of a retinal capillary from group II, showing a grid similar to that of Siperstein et al. which chooses at random sites for measuring the BMT (arrow); 16 instead of 20 equidistant measurements were used in this study. Calculated mean basement membrane thickness 1154 A. (Magnification: x12,600). E = endothelial cell; L = lumen.
78
BEAUCHEMIN,
ANTILLE
AND
LEUENBERGER
was also chosen because it takes into account the major sources of error mentioned above. As such we thought this method to be potentially interesting for future morphometric studies. MATERIAL
AND
METHODS
The retinas of 20 Wistar rats were used for this study. The animals were divided into four groups, two of which were experimentally diabetic, one having an additional stress factor on its diet (devoid of flavonoids), group I, the second a stress factor control on its diet, i.e., flavonoid-free diet plus flavonoids, group II. The remaining two groups were nondiabetic control groups one having the stress factor on its diet, group III, and the last group having normal rat food, group IV. Details of the method of this study can be found in an earlier publication (14). The first morphometric analysis of BMT was carried out according to Siperstein et al. (18), except that 16 instead of 20 measurements were made per capillary (Fig. 1). Eleven capillaries per animal were used in both methods. Of the four groups examined there were six animals in group I, six in group II, four in group III, and four in group IV. The second morphometric analysis carried out was that of Williamson et al. (21), in which only two minimal measurements per capillary are made (Fig. 2). A form of nested variance analysis (19a), was used to compare these two morphometric methods, classifying them into two statistical groups. Three subsamples were used in this test: animals (a), capillaries in animals (b), and measurements in capillaries (c), each having, respectively, I, m and IZ elements. By this analysis we were able to obtain the variance for the average of the sample, & using the variance of each subsample.
where ga, Ok,, and cc represent, respectively, the standard deviation of the subsamples. An extension of this test, “the allocation of resources” (19b), was used to find the ideal number of values in the three subsamples chosen, in order to give the most precise results, taking into account, however, that one knows their CS.The formulas used were the following :
where k, = the time spent to choose the animals, kb = the time spent to choose the capillaries, and k, = the time spent to choose and carry out the measurements. In both methods k, and kb are the same; however, k, is larger in the Siperstein et al. method than in that of Williamson et al. RESULTS A comparison of the methods using the nested variance analysis can be summarized in Table 1 (the results of the total nested variance have not included the constant to convert the numbers in A (105/195).
MORPHOMETRY OF BASEMENT MEMBRANES
79
FIG. 2. Electron micrograph (the same as in Fig. 1) of a retinal capillary from group II showing sites where two minimal measurements of BMT have been chosen (arrows), using Williamson et& method. No grid is used since the minimal sites are carefully selected and not chosen at random as in Fig. 1. Calculated mean basement membrane width: 718 A. (Magnification: x12,600). E = endothelial cell; L = lumen.
80
BEAUCHEMIN,
ANTILLE,
TABLE NESTED VARIANCE
AND
LEUENBERGER
I ANALYSIS
Siperstein et at. Group 2 so 4 2 SC s: Group s,2 4 2 SC s: Group 2 SD s: 2 SC dGroup so2 4 2 SC S:
Williamson
et al.
I: Diabetic rats + flavonoid-free diet 0.052998 0.101050 0.902771 0.011219
0.021558 0.194702 0.053030 0.006945
0.514083 0.173726 2.158458 0.090357
0.102066 0.154546 0.075757 0.019927
0.086578 0.055416 0.661079 0.023843
0.110950 0.075 0.056818 0.030088
0.028262 0.093496 0.336742 0.009669
0.046316 0.073864 0.136363 0.014807
II : Diabetic rats + flavonoid-free diet + flavonoids
III: Nondiabetic control + flavonoid-free diet
IV: Nondiabetic control + normal rat food
a sa = estimated variance within animal subsample; sg = estimated variance within capillary subsample; s: = estimated variance within measurement subsample; s: = estimated variance of the average of BMT. In groups I and II the CT:of Williamson et al’s method is significantly smaller than that of Siperstein et at. ; in group III it is not significantly different, and in group IV the o$ of Siperstein et al’s method is significantly smaller than that of Williamson et al.
DISCUSSION In order to determine the method which yields the more precise estimation of BMT with a minimal variance, we found it preferable to use a nested variance analysis (19). This analysis allowed us to compare the total average variance of the three statistical subsamples, namely, the animals, the capillaries within the animals, and the measurements within the capillaries. This comparative analysis was preferred to a comparison of the coefficient of variation as Williamson et al. (21) used. We found that by employing the latter we were not able to tell at what value the ratio of the coefficient of variation of the two methods would differ significantly from 1. In the nested variance analysis the components of the variability of one group were calculated and it was then possible to determine if the number of blood vessels or measurements should be increased or decreased to give the most precise values of BMT. The number of animals as a factor of variability was not taken into account in this study. The results of this study show that by using the method of Williamson et al. (21) the estimated variance for the average of BMT is significantly smaller (p < 0.05)2 for the two z F-test.
MORPHOMETRY
OF BASEMENT
MEMBRANES
81
diabetic groups than that obtained with the method of Siperstein et al. (18). In the third group, however, the estimated variance is not significantly different and in the fourth group it was found that with the method of Siperstein et al. (18) it is significantly smaller than with that of Williamson et al. (p < 0.05)2. This must be due to the fact that it is a control group with no basement membrane focal thickenings as are regularly found in the diabetic group (12, 14). The Siperstein et al. (18) method, however, owing to its multiple measuring points, appears to be more sensitive in detecting these focal thickenings which probably also account for the larger estimated variance for the average of BMT in the diabetic groups. In view of the fact that BM focal thickenings have been reported to occur in both human and experimental diabetic microangiopathy even before an increase of the mean BM width is detectable (10, 12, 17), this point certainly deserves particular attention. The two diabetic groups have a BMT which is more heterogeneous than the control groups. Since Williamson et al’s method (21) yielded a better estimation of the variance of the average of BMT of these two diabetic groups, we therefore concluded it to be the more precise method of the two. Its usefulness is further emphasized by the fact that the time spent to choose and carry out the measurements within the capillaries (k,) using Williamson et al’s method is smaller than that of Siperstein et al. By using an extension of the nested variance analysis to compare the two methods it was shown that in order to decrease the variance of the average BMT it is necessary to decrease the number of measurements per capillary and increase the number of small blood vessels to be measured.
REFERENCES J. M., JR., AND SOMMERS, S. C. (1959). “Cirrhotic glomerulosclerosis,” a renal lesion associated with hepatic cirrhosis. Lab. Invest. 8, 962-978.
1. BLOODWORTH, 2. BLOODWORTH,
3. 4.
J. M., JR., ENGERMAN,
R. L., CAMERINI-DAVALOS,
R. A., AND POWERS, K. L. (1970).
Variations in capillary basement membrane width produced by aging and diabetes mellitus. Zn “Early Diabetes” (R. A. Camerini-Davalos and K. L. Powers, eds.), pp. 279-287. Academic Press, New York. BLOOM, P. M., HARTMANN, J. F., AND VERNIER, R. L. (1959). An electron microscopic evaluation of the width of normal glomerular basement membrane in man at various ages. BURDITT, A. F., CAIRO, F. I., AND DRAPER, G. J (1968). The natural history of diabetic retinopathy. Quart. J. Med. 37, 303-317. D. P., AMHERDT, M., LEUENBERGER, P. M., ORCI, L., AND STAUFFACHER, W. (1973). Microvascularalterationsin chronicallystreptozotocin-diabetic rats. In “Vascularand NeurologicalChanges in EarlyDiabetes”(suppl.2to Advances in MetabolicDisorders), (R. A. CameriniDavalosandH. S. Cole,eds.),pp. 257-264.AcademicPress,NewYork. FRIEDENWALD, J. S. (1949).A newapproachto someproblemsof retinalvasculardisease. Amer.
5. CAMERON,
6.
J. Ophthalmol. 32,487-498. 7. FRIEDENWALD, J. S. (1950).Diabeticretinopathy.Amer. J. Ophthalmol. 33, 1187-1199. 8. FRIEDERICI, H. H. R., TUCKER, W. R., AND SCHWARTZ, T. B. (1966).Observarions onsmallblood vessels of skin in the normalandin diabeticpatients.Diabetes 15, 233-250. 9. JORDAN, S. W., AND PERLEY, M. J. (1972).Microangiopathy in diabetesmellitusandaging.Arch.
Pathol. 93,261-265. C., VOGLER, N., AND WILLIAMSON, J. R. (1972).Musclecapillarybasement membrane changes relatedto agingandto diabetesmellitus.Diabetes 21, 881-904. LEUENBERGER, P. M., BABEL, J., AND FULL, C. (1970).Width of retinalcapillarybasement membraneof spinymice(Acomys cahirinus) at variousages.Dot. Ophthalmol.28, 191-200.
10. KILO, 11.
82
BEAUCHEMIN,
ANTILLE
AND
LEUENBERGER
12. LEUENBERGER, P. M., CAMERON, D., STAUFFACHER, W., RENOLD, A. E., AND BABEL, J. (1971). Ocular lesions in rats rendered chronically diabetic with streptozotocin. Ophthalmol. Res. 2, 189-204. 13. LEUENBERGER, P. M. (1972). Ultrastructure of the aging retinal vascular system with special reference to quantitative and qualitative changes of capillary basement membranes. Gerontologiu 19, 1-15. 14. LEUENBERGER, P. M., BEAUCHEMIN, M. L., AND BABEL, J. (1974). Experimental diabeticretinopathy. Arch. Ophthalmol. 34289-302. 15. ORCI, L., STAUFFACHER, W., AMHERDT, M., PICTET, R., RENOLD, A. The kidney of spiny mice (Acomys cahirinus) : Electron microscopy
16. 17. 18. 19. 20. 21. 22.
E., AND ROUILLER, C. (1970). of glomerular changes associated with aging and tubular glycogen accumulation during hyperglycemia. Diubefologiu 6,343355. ~STERBY, R. (1972). Morphometric studies of the peripheral glomerular basement membrane in early juvenile diabetes. I. Development of initial basement membrane thickening. Diabetologia 8, 84-92. POMETTA, D., AMHERDT, M., AND RUFFENER, C. (1971). Capillary basement membrane and abnormality of carbohydrate metabolism (abstract). Diubetologia 7, 152. SIPERSTEIN, M. D., UNGER, R. H., AND MADISON, L. L. (1968). Studies of muscle capillary basement membranes in normal subjects, diabetic and prediabetic patients. J. Clin. Invest. 47,1937-1999. SNEDECOR, G. W., AND COCHRAN, W. G. (1967). “Statistical Methods”, 6thed.,pp. 285-288,10.16; 531-533, 17.12. Iowa State Univ. Press. Ames, Iowa. VRACKO, R. (1970). Skeletal muscle capillaries in diabetes: A quantitative analysis. Circulation 41, 271-283. WILLIAMSON, I. R., VOGLER, N. J., AND KILO, C. (1969). Estimation of vascular basement membrane thickness. Theoretical and practical considerations. Diabetes 18, 567-578. WILLIAMSON, J. R., VOGLER, N. J., AND KILO, C. (1971). Regional variations in the width of the basement membrane of muscle capillaries in man and giraffe. Amer. .Z. Z’athol. 63, 359-370.
