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Basal lamina thickness and permeability to horseradish peroxidase of intraneural capillaries in diabetic mice
MICROVASCULAR
RESEARCH
20, 223-232 (1980)
Basal Lamina Thickness and Permeability to Horseradish Peroxidase of lntraneural Capillaries in Diabetic Mice KEITH The Dental Research
A. CARSON' AND JACOB S. HANKERS
Center, School of Dentistry, and Neurobiology Program, Carolina, Chapel Hill, North Carolina 27514 Received
University
of North
September 21, 1979
The fine structural morphology, basal lamina thickness, and permeability to intravenously injected horseradish peroxidase (HRP) of intraneural capillaries have been examined in lo-month-old diabetic mice (C57BL/KsJ, dbldb). The afflicted mice of this strain have previously been shown to develop peripheral neuropathy. In this study we observed that a very small percentage of endothelial cells in sciatic nerves of diabetic mice had abnormal accumulations of membrane whorls in their cytoplasm. Although quantitative measurements of intraneural capillary basal lamina thickness showed that there was no statistically significant increase in basal lamina thickness, occasional sciatic nerve capillaries in diabetic mice did have thickened basal laminae. Cytochemical studies showed that there was no apparent leakage of HRP from capillaries in the sciatic nerve, trigeminal nerve, and cerebral cortex from diabetic mice or their unafthcted littermates. These results suggest that microangiopathy, as assessed by .these measures, is not prominent in diabetic mice and therefore, probably does not play a major role in the development of peripheral neuropathy in these mice.
INTRODUCTION Diabetes mellitus is frequently accompanied by microvascular pathology which may have a primary role in the peripheral neuropathy associated with diabetes. Although the significance of capillary basement membrane thickening and increased capillary permeability observed in diabetes vis a vis ischemia and the supply of metabolites to the nerve have been matters of considerable controversy, the study of these two factors in animal models for diabetic neuropathy continues to be a matter of great interest (Cahill, 1978). Fagerberg (1959) found a close correlation between the presence of microangiopathy in the vasa nervorum and the occurrence of peripheral neuropathy in human diabetics. However, other investigators found that the presence of diabetic microangiopathy correlated poorly with that of the neuropathy (Greenbaum et al., 1964; Thomas and Lascelles, 1966; Chopra et al., 1969). Capillary basal lamina thickening has been used as an anatomical index of microangiopathy and, along with capillary permeability to various tracer molecules, has been frequently used to assess the structural integrity of the microvasculature in diabetes (McMillan, 1975; Williamson and Kilo, 1977). Studies utilizing various animal models for diabetes have demonstrated the I Current Address: Harvard Neurology Unit, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. 2 To whom reprint requests should be addressed. 223 00262862/80/050223-10$02.00/0 Copyright @ 1980 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
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HANKER
existence of peripheral neuropathy which appeared after the onset of chronic hyperglycemia. Alloxan- or streptozotocin-induced diabetes in rats resulted in significantly decreased nerve conduction velocity (Eliasson, 1964; Sharma and Thomas, 1974). Reports of morphological abnormalities in peripheral nerves from these animal models have been contradictory, ranging from nonexistent (Sharma and Thomas, 1974; Sharma et al., 1977)to segmental demyelination (Preston, 1%7; Hildebrand et al., 1968; Seneviratne and Peiris, 1969), pronounced axon and Schwann cell pathology (Powell et al., 1977), axonal dwindling (Jakobsen, 1976a, b), and endoneurial edema (Jakobsen, 1978). The status of the blood nerve barriers in these animal models has also been investigated (Seneviratne, 1972; Jakobsen et al., 1978; Sima and Robertson, 1978b). The diabetic mouse (C57BL/KsJ, dbldb) is afflicted with an hereditary syndrome resembling human maturity-onset diabetes mellitus (Hummel et al., 1966) and its vasculature and peripheral nerves have been previously studied. Peripheral neuropathy has been reported to occur in this diabetic mouse (Sima and Robertson, 1978a; Hanker et al., 1980; Carson et al., 1980). Quantitative investigations of capillary basal lamina thickening in gingiva (Anapolle et al., 1972, 1973), kidney (Like et al., 1972), and muscle (Novis and Korson, 1974) of diabetic mice have produced different results. Sima and Robertson (1978b) reported no differences in intraneural capillary permeability to intravenously injected Evans blue albumin or horseradish peroxidase (HRP) between diabetic mice and unafflicted littermates. These investigators also reported intraneural capillary basal lamina thickening in diabetic mice. Because of this apparent discrepancy, it was decided to undertake further studies of the relationship of capillary morphology, basal lamina thickness and permeability to the peripheral neuropathy in diabetic mice. MATERIALS
AND METHODS
Male diabetic mice and unafflicted littermates (C57BL/KsJ, dbldb; Jackson Laboratories, Bar Harbor, Maine) 10 months of age were selected for these experiments from a colony housed under standard laboratory animal facility conditions and provided with mouse chow and water ad libitum. The mice were fasted overnight prior to sacrifice and blood glucose determination. Blood glucose levels were determined from samples taken directly from the heart at the time of sacrifice by a modification of the method of Raabo and Terkildsen (1960) using the Sigma Kit No. 510 (Sigma Chemicals, St. Louis, MO.). Diabetic mice used in this study were screened for symptoms of peripheral neuropathy. Only mice with symptoms such as abnormal hindlimb flexion and poor roto-wheel performance were used (see Hanker et al., 1980, for a full description). In addition, during ultrastructural studies of sciatic nerves, the presence of morphological correlates of peripheral neuropathy was verified in all cases (see Carson et al., 1980, for a full description). Studies of Capillary Morphology and Basal Lamina Thickening Five diabetic mice displaying symptoms of peripheral neuropathy and five unafflicted littermates were anesthetized by an intraperitoneal injection of tribromoethanol solution (2.5% aqueous, 0.20 ml/5 g body wt) and sacrificed by
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intracardiac perfusion of 10 ml of room temperature saline followed by 50 ml of a mixture of 2.5% glutaraldehyde and 1.5% freshly depolymerized paraformaldehyde in 0.05 M cacodylate buffer, pH 7.4. The left sciatic nerve was exposed and immersed in fixative for 12-18 hr at 4°C. It was then dissected free, cut into three segments and rinsed in two changes of 0.1 M cacodylate buffer, pH 7.4, for 15 min each change. The nerve tissue was postfixed in 2% osmium tetroxide buffered with 0.1 M sodium cacodylate to pH 7.4 for 3 hr at 4°C. Following osmication the tissue was rinsed twice in 0.1 M cacodylate buffer, pH 7.4, dehydrated through a graded series of ethanols followed by 100% acetone and embedded in a 6 to 4 mixture of Spurr and Epon epoxy resins (Coleman et al., 1976). The blocks were polymerized for 48 hr. Silver thin sections were cut on a Sorvall, Porter-Blum MT-2B ultramicrotome using a diamond knife, picked up on 200-mesh uncoated copper grids, poststained with 5% aqueous uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and viewed with an AEI 801 electron microscope. Ten electron micrographs of whole capillary cross sections from each sciatic nerve were made at a standard magnification of 6300 and enlarged during printing to 16,000. In a blind trial, the prints were numbered and measurements of the basal lamina thicknesses at their two thinnest points around the circumference of the capillary were made. Areas of the endothelium not covered by pericyte processes were selected. This procedure was followed to reduce error due to the potential oblique sectioning of the capillary (Williamson et al., 1969; Kilo et al., 1972), and because this method has been suggested to be rapid, simple, and precise (Beauchemin et al., 1975). The data were statistically evaluated using analysis of variance. Intraneural
Capillary
Permeubility
to Horseradish
Peroxidase
This aspect was studied by a method similar to that of Jakobsen and co-workers (1978) using a tail-vein injection of 5 mg of HRP (Type VI, Sigma Chemicals) dissolved in 0.1 ml saline in three pairs of lo-month-old male diabetic mice exhibiting symptoms of peripheral neuropathy and unafflicted littermates. One hour after injection, the mice were sacrificed under tribromoethanol anesthesia by removal of the heart. Tissues including the left sciatic nerve, trigeminal nerve, and brain were excised and fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 4 hr at room temperature. Following fixation, the tissues were rinsed in several changes of 0.1 M cacodylate buffer, pH 7.4, with 5% sucrose. The tissues were then rapidly frozen in isopentane cooled by a bath of dry ice and 95% ethanol and cut at 20 pm on a cryostat. The sections were picked up on cover slips and allowed to dry for 18 hr in the dark, since long exposure to light inactivates HRP in tissue sections. Horseradish peroxidase activity was localized according to the method of Hanker and co-workers (1977) using paraphenylenediamineipyrocatechol (PPD-PC, Polysciences) as the chromogen. The sections were preincubated in a medium without hydrogen peroxide (15 mg PPD-PC in 10 ml 0.1 M Tris-HCI buffer, pH 7.4) for 20 min at 4°C followed by incubation in the same medium with hydrogen peroxide (0.01%) for 10 min at room temperature. After a brief rinse in buffer, the sections were dehydrated in a graded series of ethanols, cleared in xylene, and mounted in Permount (Fisher).
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1
SCIATIC NERVE CAPILLARY BASAL LAMINA THICKNESS IN DIABETIC MICE AND UNAFFLICTED LITTERMATES
Unafflicted littermates Diabetic mice
Blood glucose” (mg/lOO ml)
Basal lamina thickness* (A)
193 2 15 (n = 8) 620 2 50 (n = 8)
947?51(tl=5) 964 t 37 (n = 5)
a Determined with glucose test kit No. 510, Sigma Chemical Company (St. Louis, MO). These values were included to indicate the degree of hyperglycemia in the diabetic mice. b These values were determined by measuring the basal lamina thickness of 10capillaries from each of five diabetic mice and their unaftlicted littermates. These 10 values were averaged and the averages were then statistically treated with the analysis of variance. The F value was 0.3669 which was not significant even at the 0.25 level.
RESULTS Morphology
of Capillaries
and Basal Lamina
Thickness
The means and standard errors of the blood glucose levels for the diabetic mice and their unafflicted littermates used in these experiments are given in Table 1. Examination of the ultrastructure of sciatic nerve capillaries from normal and diabetic mice did not reveal a considerable degree of pathological change in sciatic nerve capillaries from diabetic mice. However, there was an apparent increase in the incidence of abnormal endothelial cell cytoplasmic inclusions which appeared to be made up of whorls of membrane (Fig. 1). These structures were not observed in sciatic nerve capillary endothelial cells from unafflicted mice, although they were observed in those of their diabetic littermates. There was no apparent capillary basal lamina thickening in sciatic nerve capillaries from lo-month-old unafflicted mice (Fig. 2). In the diabetic mice, a very small percentage of the capillaries exhibited basal lamina thickening (Fig. 3). In other respects, the morphology of the intraneural endothelial cells from diabetic and nondiabetic mice was within the limits of variation normally observed. The measurements of sciatic nerve capillary basal lamina thickness for diabetic mice and their unafflicted littermates are shown in Table 1. As the mean and standard error values indicate, there was no statistically significant increase in capillary basal lamina thickness in diabetic mice when compared to their unafflicted littermates. Intraneural
Capillary
Permeability
to Horseradish
Peroxidase
Following intravenous injection of HRP and incubation of the tissue sections for localization of the tracer, intraneural and brain capillaries were filled with dark FIG. 1. Sciatic nerve capillary from a IO-month-old diabetic mouse. Note the normal basal lamina width (arrow) and the abnormal accumulation of whorled membranes in the endothelial cell cytoplasm (*). x22,000. -FIG. 2. Capillary from the sciatic nerve of a IO-month-old normal mouse. There is no abnormal basal lamina thickening (arrow). x 10,800. FIG. 3. A rare instance of a thickened basal lamina surrounding a capillary in the sciatic nerve of a diabetic mouse (arrows). The adjacent Schwann cell cytoplasm contains two Pi granules of Reich (P). x 12,000.
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reaction product (Figs. 4-6). There was no observable reaction product outside the capillaries in the sciatic nerve, trigeminal nerve and cerebral cortex (Figs. 4-6). Incubation of nerve tissue sections from mice not receiving an injection of HRP revealed that, under the conditions of these experiments, no endogenous peroxidase or catalase was demonstrated. DISCUSSION The blood glucose levels of the diabetic mice and their unafflicted littermates used in these experiments were similar to those previously reported for the db mouse (Hummel et al., 1966). These data point out the severe chronic hyperglycemic condition which develops in diabetic mice and indicates the utility of this hereditary model for maturity-onset diabetes in the study of the etiology of the complications of diabetes. The examination of endothelial cell fine structure in the sciatic nerves of diabetic mice revealed only a low incidence of abnormal morphology. The accumulation of whorls of membrane within endothelial cells may be indicative of altered endothelial cell metabolism. osterby and co-workers (1978) reported that the density of micropinocytotic vesicles in capillary endothelium of striated muscle was increased about 30% in streptozotocin-diabetic rats injected with insulin. Insulin apparently may alter the morphology of the endothelial cells. Diabetic mice were shown to have greatly increased plasma insulin levels, at least through the first 6 to 8 weeks after birth (Coleman and Hummel, 1968); plasma immunoreactive insulin levels in diabetic mice may reach six times those of unafflicted heterozygotes. The whorls of membrane observed in the endothelial cells from diabetic mice could reflect altered autolytic metabolism resulting in decreased membrane turnover and subsequent accumulation. Pfeiger and coworkers (1978) have reported that acute, high plasma insulin levels inhibited formation of autophagic vacuoles in rat hepatocytes. Another factor worthy of consideration is the possible effect of the chronic hyperglycemia on intraneural capillaries. Chronic hyperglycemia has been shown to alter the way cells metabolize glucose, resulting in intracellular accumulation of sorbitol (Winegrad et al., 1973), a compound which has been suggested to promote development of microangiopathy, cataracts, and neuropathy in the rat (Van Heyningen, 1958; Drasnin, 1973). However, at this time there is no experimental evidence from work on the diabetic mouse to support either of these suppositions. Thickening and reduplication of the capillary basal lamina has been described in nerves from human diabetics with peripheral neuropathy (Bischoff, 1968; Lapresle, 1968). Most studies with chemical and hereditary animal models for diabetes have examined basal lamina thickness of muscle capillaries, so that there are insufficient data to determine if there is a correlation between the peripheral neuropathy and intraneural microangiopathy in these animal models. Several tissues from this diabetic mouse strain have been examined quantitatively to determine whether or not there is an increase in capillary basal lamina thickness. These include gingiva (Anapolle et al., 1972, 1973), kidney (Like et al., 1972) and muscle (Novis and Korson, 1974). In these studies, gingival capillaries and capillary tufts in glomeruli demonstrated increased basal lamina formation,
INTRANEURAL
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IN
DIABETIC
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229
5b
6b FIGS. 4-6. Following histochemical incubation, the intraneural capillaries of mice injected intravenously with horseradish peroxidase contained dark reaction product. In the cerebral cortex (Fig. 4), trigeminal nerve (Fig. 5), and sciatic nerve (Fig. 6) there was no observable increase in leakage of HRP from capillaries in diabetic mice (b) when compared to their unafflicted littermates (a). Fig. 4, x30. Figs. 5 and 6. x340.
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CARSON AND HANKER
while the difference in muscle capillary basal lamina thickness was not statistically significant in diabetic mice and their unafllicted littermates. Sima and Robertson (1978b) reported intraneural capillary basal lamina thickening in diabetic mice but did not furnish quantitative data. The present study indicated that at least in the sciatic nerve, where neuropathological changes have been reported (Carson et al., 1980) there was no statistically significant increase in capillary basal lamina thickness in diabetic mice. Recent studies on the effects of chronic hyperglycemia on the permeability of endoneurial vasculature to tracer substances have been conflicting. Seneviratne (1972) reported that in the alloxan-diabetic rat, the permeability of both the perineurium and endoneurial vasculature to tail-vein injected Evans blue-labeled albumin was increased. On the basis of these findings the author suggested that increased extravasation of proteins into the extracellular space of the endoneurium may play a role in the pathogenic mechanism of diabetic neuropathy observed in this animal model. However, Jakobsen and co-workers (1978) showed that the blood nerve barrier in the streptozotocin-diabetic rat was capable of excluding Evans blue-labeled albumin, HRP, and cytochrome c from the nerve parenchyma. The work of Sima and Robertson (1978b) supported Jakobsen’s findings in the streptozotocin-diabetic rat and extended them to the alloxandiabetic rat and the diabetic mouse. The results of the HRP permeability experiments in the present study are in agreement with those of Sima and Robertson (1978b), in that there was no observable leakage of HRP from the intraneural vasculature of both diabetic mice and unafflicted littermates. In conclusion, it is apparent that microangiopathy probably does not play a major role in the etiology of the peripheral neuropathy in the diabetic mouse. The fine structural studies indicated that a certain degree of intraneural capillary pathology may occur. However, at this time, sufficient evidence is not available to determine to what degree the structural abnormalities in the endothelium may reflect functional changes which could be involved in the development of peripheral neuropathy.
ACKNOWLEDGMENTS The authors are grateful to Jodie Kurtz for typing the manuscript. This work was supported by NIH Research Grants DE 04730, DE 02668, and DE 00288 from the National Institute for Dental Research, NIH Grant RR 05333 from the Division of Research Facilities and Resources, and a grant to the Neurobiology Program from the Alfred P. Sloan Foundation.
REFERENCES ANAPOLLE, S., ALBRIGHT, J., AND CRAFT, F. (1972). The ultrastructure of the gingiva in the diabetic mouse. Microvasc. Res. 4, 132. ANAPOLLE, S., ALBRIGHT, J., AND CRAFT, F. (1973). Continued study of the ultrastructure of the gingiva in the diabetic mouse. Microvasc. Res. 6, 44. BEAUCHEMIN, M., ANTILLE, G., AND LEVENBERGER,P. (1975). Capillary, basement membrane thickness: A comparison of two morphometric methods for its estimation. Microvasc. Res. 10,76.
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MICE
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BISCHOFF, A. (1968). Diabetische Neuropathie:
pathologische Anatomie, Pathophysiologie, und Pathogenese auf Grund electronenmikroskopische Untersuchungen. DeuZ. Med. Wochenschr. 93,
237. CAHILL,
G. F., JR. (1978). Diabetes mellitus: A brief overview. The Johns Hopkins Med. J. 143, 155-159. CARSON, K. A., BOSSEN, E. H., AND HANKER, J. S. (1980). Peripheral neuropathy in mouse hereditary diabetes mellitus. II. Ultrastructural correlates of degenerative and regenerative changes. Neuropathol. App. Neurobiol. (in press). CHOPRA, J., HURWITZ, L., AND MONTGOMERY, D. (1%9). The pathogenesis of sural nerve changes in diabetes mellitus. Brain 92, 391. COLEMAN, D. L., AND HUMMEL, K. P. (1967). The mutation diabetes in the mouse. In “Proceedings of the VIth Congress of the International Diabetes Federation,” [pp. 813-8201. Excerpta Medica, Amsterdam. COLEMAN, R. A., RAMP, W., TOVERUD, S., AND HANKER, J. S. (1976). Electron microscopic localization of lactate dehydrogenase in osteoclasts of chick embryo tibia. Histochetn. J. 8, 543. DRASNIN, N. (1973). Sorbitol accumulation: A factor in aging? Letter to the editor. Lancet 1, 1175. ELIASSON, S. (1964). Nerve conduction changes in experimental diabetes. .Z. Clin. Invest. 43, 2353. FAOERBERG, S. (1959). Diabetic neuropathy: A clinical and histological study on the significance of vascular affections. Acta Med. Stand. Suppl. 164, 345. GREENBAUM, D., RICHARDSON, P., SALMON, M., AND ULRICH, H. (1964). Pathological observations on six cases of diabetic neuropathy. Brain 87, 215. HANKER, J. S., YATES, P., METZ, C., AND RUSTIONI, A. (1977). A new, specific, sensitive and noncarcinogenic reagent for the demonstration of horseradish peroxidase. Histochem. J. 9, 789. HANKER, J. S., AMBROSE, W., YATES, P., KOCH, G., AND CARSON, K. A. (1980). Peripheral neuropathy in mouse hereditary diabetes mellitus. I. Comparison of neurologic, histologic and morphometric parameters with dystonic mice. Acta Neuropafhol., (in press). HILDEBRAND, J., JOFFREY, A., GROTT, G., AND COERS, C. (1968). Neuromuscular changes with alloxan hyperglycemia. Electrophysiological, biochemical, and histological study in rats. Arch.
Neural. 18, 638. HUMMEL, K., DICKIE, M., AND COLEMAN, D. L. (1966). Diabetes, a new mutation in the mouse. Science 153, 1127. JAKOBSEN, J. (1976a). Axonal dwindling in early experimental diabetes. I. A study of cross sectioned 12, 539. nerves. Diabetologia JAKOBSEN, J. (1976b). Axonal dwindling in early experimental diabetes. II. A study of isolated nerve fibers. Diaberologia 12, 547. JAKOBSEN, J., MALMGREN, L., AND OLSSON, Y. (1978). Permeability of the blood-nerve barrier in the streptozotocin-diabetic rat. Exp. Neural. 60, 277. KILO, C., VOGLER, N., AND WILLIAMSON, J. R. (1972). Muscle capillary basement membrane changes related to aging and to diabetes mellitus. Diabetes 21, 881. LAPRESLE, J. (1968). Etude anatomique des neuropathies peripheriques du diabete sucre. J. Ann. Diabetol. Hotel Dieu 9, 101. LIKE, A., LAVINE, R., POTFENBARGER, L., AND CHICK, W. (1972). Studies in the diabetic mutant mouse. VI. Evolution of glomerular lesions and associated proteinuria. Amer. J. Pathol. 66, 193. MCMILLAN, D. E. (1975). Deterioration of the microcirculation in diabetes. Diabetes 24, 944. NOVIS, D., AND KORSON, R. (1974). Capillary basement membranes of muscle in diabetic mice (genetic and virally induced). Fed. Proc. 33, 607. ~STERBY, R., GUNDERSEN, H. J. G., AND CHRISTENSEN, N. J. (1978). The acute effect of insulin on capillary endothelial cells. Diabetes 27, 745. POWELL, H., KNOX, D., LEE, S., CHARTERS, A., ORLOTT, M., GARRET, R., AND LAMPERT, P. (1977). Alloxan diabetic neuropathy: electron microscopic studies. Neurology 27, 60. PRESTON, G. (1967). Peripheral neuropathy in the alloxan-diabetic rat. J. Physiol. 189, 49. RAABO, E., AND TERKILDSEN, T. (1960). On the enzymatic determination of blood glucose. Stand J. C/in. Lab. Invest. 12, 402. REYNOLDS, E. (1963). The use of lead at high pH as an electron-opaque stain in electron microscopy. J. Cell. Biol. 17, 208.
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SENEVIRATNE, K. (1972). Permeability of blood nerve barriers in the diabetic rats. J. Neural. Neurosurg. Psychiat. 35, 156. SENEVIRATNE, K., AND PEIRIS, 0. (1968). The effect of ischaemia on the excitability of sensory nerves in diabetes mellitus. J. Neurol. Neurosurg. Psychiat. 31, 348. SHARMA, A., AND THOMAS, P. (1974). Peripheral nerve structure and function in experimental diabetes. J. Neurol. Sci. 23, 1. SHARMA, A., THOMAS, P., AND DE MOLINA, A. (1977). Peripheral nerve fiber size in experimental diabetes. Diabetes 26, 689. SIMA, A., AND ROBERTSON, D. (1978a). Peripheral neuropathy in mutant diabetic mice (C57BL/KsJ, db-db). Acta Neuropathol. 41, 85. SIMA, A. AND ROBERTSON, D. (1978b). The perineurial and blood-nerve barriers in experimental diabetes. Acta Neuropathol. 44, 189. THOMAS, P., AND LASCELLES, R. (1966). The pathology of diabetic neuropathy. Quart. J. Med. 35, 489. VAN HEYNINGEN, R. (1959). Formation of polyol in the lens of the rat with “sugar” cataract. Nature (London) 184, 194. WATSON, M. (1958). Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4, 475. WILLIAMSON, J., VOGLER, N., AND KILO, C. (1969). Estimation of vascular basement membrane thickness. Diabetes 18, 567. WILLIAMSON, J., AND KILO, C. (1977). Current status of capillary basement membrane disease in diabetes mellitus. Diabetes 26, 65. WINEGRAD, A. I., MORRISON, A., AND CLEMENTS, R. (1973). Polyol pathway activity in aorta. Adv. Met. Disorders (Suppl. 2), 117.
RESEARCH
20, 223-232 (1980)
Basal Lamina Thickness and Permeability to Horseradish Peroxidase of lntraneural Capillaries in Diabetic Mice KEITH The Dental Research
A. CARSON' AND JACOB S. HANKERS
Center, School of Dentistry, and Neurobiology Program, Carolina, Chapel Hill, North Carolina 27514 Received
University
of North
September 21, 1979
The fine structural morphology, basal lamina thickness, and permeability to intravenously injected horseradish peroxidase (HRP) of intraneural capillaries have been examined in lo-month-old diabetic mice (C57BL/KsJ, dbldb). The afflicted mice of this strain have previously been shown to develop peripheral neuropathy. In this study we observed that a very small percentage of endothelial cells in sciatic nerves of diabetic mice had abnormal accumulations of membrane whorls in their cytoplasm. Although quantitative measurements of intraneural capillary basal lamina thickness showed that there was no statistically significant increase in basal lamina thickness, occasional sciatic nerve capillaries in diabetic mice did have thickened basal laminae. Cytochemical studies showed that there was no apparent leakage of HRP from capillaries in the sciatic nerve, trigeminal nerve, and cerebral cortex from diabetic mice or their unafthcted littermates. These results suggest that microangiopathy, as assessed by .these measures, is not prominent in diabetic mice and therefore, probably does not play a major role in the development of peripheral neuropathy in these mice.
INTRODUCTION Diabetes mellitus is frequently accompanied by microvascular pathology which may have a primary role in the peripheral neuropathy associated with diabetes. Although the significance of capillary basement membrane thickening and increased capillary permeability observed in diabetes vis a vis ischemia and the supply of metabolites to the nerve have been matters of considerable controversy, the study of these two factors in animal models for diabetic neuropathy continues to be a matter of great interest (Cahill, 1978). Fagerberg (1959) found a close correlation between the presence of microangiopathy in the vasa nervorum and the occurrence of peripheral neuropathy in human diabetics. However, other investigators found that the presence of diabetic microangiopathy correlated poorly with that of the neuropathy (Greenbaum et al., 1964; Thomas and Lascelles, 1966; Chopra et al., 1969). Capillary basal lamina thickening has been used as an anatomical index of microangiopathy and, along with capillary permeability to various tracer molecules, has been frequently used to assess the structural integrity of the microvasculature in diabetes (McMillan, 1975; Williamson and Kilo, 1977). Studies utilizing various animal models for diabetes have demonstrated the I Current Address: Harvard Neurology Unit, Beth Israel Hospital, 330 Brookline Ave., Boston, MA 02215. 2 To whom reprint requests should be addressed. 223 00262862/80/050223-10$02.00/0 Copyright @ 1980 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in U.S.A.
224
CARSON
AND
HANKER
existence of peripheral neuropathy which appeared after the onset of chronic hyperglycemia. Alloxan- or streptozotocin-induced diabetes in rats resulted in significantly decreased nerve conduction velocity (Eliasson, 1964; Sharma and Thomas, 1974). Reports of morphological abnormalities in peripheral nerves from these animal models have been contradictory, ranging from nonexistent (Sharma and Thomas, 1974; Sharma et al., 1977)to segmental demyelination (Preston, 1%7; Hildebrand et al., 1968; Seneviratne and Peiris, 1969), pronounced axon and Schwann cell pathology (Powell et al., 1977), axonal dwindling (Jakobsen, 1976a, b), and endoneurial edema (Jakobsen, 1978). The status of the blood nerve barriers in these animal models has also been investigated (Seneviratne, 1972; Jakobsen et al., 1978; Sima and Robertson, 1978b). The diabetic mouse (C57BL/KsJ, dbldb) is afflicted with an hereditary syndrome resembling human maturity-onset diabetes mellitus (Hummel et al., 1966) and its vasculature and peripheral nerves have been previously studied. Peripheral neuropathy has been reported to occur in this diabetic mouse (Sima and Robertson, 1978a; Hanker et al., 1980; Carson et al., 1980). Quantitative investigations of capillary basal lamina thickening in gingiva (Anapolle et al., 1972, 1973), kidney (Like et al., 1972), and muscle (Novis and Korson, 1974) of diabetic mice have produced different results. Sima and Robertson (1978b) reported no differences in intraneural capillary permeability to intravenously injected Evans blue albumin or horseradish peroxidase (HRP) between diabetic mice and unafflicted littermates. These investigators also reported intraneural capillary basal lamina thickening in diabetic mice. Because of this apparent discrepancy, it was decided to undertake further studies of the relationship of capillary morphology, basal lamina thickness and permeability to the peripheral neuropathy in diabetic mice. MATERIALS
AND METHODS
Male diabetic mice and unafflicted littermates (C57BL/KsJ, dbldb; Jackson Laboratories, Bar Harbor, Maine) 10 months of age were selected for these experiments from a colony housed under standard laboratory animal facility conditions and provided with mouse chow and water ad libitum. The mice were fasted overnight prior to sacrifice and blood glucose determination. Blood glucose levels were determined from samples taken directly from the heart at the time of sacrifice by a modification of the method of Raabo and Terkildsen (1960) using the Sigma Kit No. 510 (Sigma Chemicals, St. Louis, MO.). Diabetic mice used in this study were screened for symptoms of peripheral neuropathy. Only mice with symptoms such as abnormal hindlimb flexion and poor roto-wheel performance were used (see Hanker et al., 1980, for a full description). In addition, during ultrastructural studies of sciatic nerves, the presence of morphological correlates of peripheral neuropathy was verified in all cases (see Carson et al., 1980, for a full description). Studies of Capillary Morphology and Basal Lamina Thickening Five diabetic mice displaying symptoms of peripheral neuropathy and five unafflicted littermates were anesthetized by an intraperitoneal injection of tribromoethanol solution (2.5% aqueous, 0.20 ml/5 g body wt) and sacrificed by
INTRANEURAL
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IN DIABETIC
MICE
225
intracardiac perfusion of 10 ml of room temperature saline followed by 50 ml of a mixture of 2.5% glutaraldehyde and 1.5% freshly depolymerized paraformaldehyde in 0.05 M cacodylate buffer, pH 7.4. The left sciatic nerve was exposed and immersed in fixative for 12-18 hr at 4°C. It was then dissected free, cut into three segments and rinsed in two changes of 0.1 M cacodylate buffer, pH 7.4, for 15 min each change. The nerve tissue was postfixed in 2% osmium tetroxide buffered with 0.1 M sodium cacodylate to pH 7.4 for 3 hr at 4°C. Following osmication the tissue was rinsed twice in 0.1 M cacodylate buffer, pH 7.4, dehydrated through a graded series of ethanols followed by 100% acetone and embedded in a 6 to 4 mixture of Spurr and Epon epoxy resins (Coleman et al., 1976). The blocks were polymerized for 48 hr. Silver thin sections were cut on a Sorvall, Porter-Blum MT-2B ultramicrotome using a diamond knife, picked up on 200-mesh uncoated copper grids, poststained with 5% aqueous uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and viewed with an AEI 801 electron microscope. Ten electron micrographs of whole capillary cross sections from each sciatic nerve were made at a standard magnification of 6300 and enlarged during printing to 16,000. In a blind trial, the prints were numbered and measurements of the basal lamina thicknesses at their two thinnest points around the circumference of the capillary were made. Areas of the endothelium not covered by pericyte processes were selected. This procedure was followed to reduce error due to the potential oblique sectioning of the capillary (Williamson et al., 1969; Kilo et al., 1972), and because this method has been suggested to be rapid, simple, and precise (Beauchemin et al., 1975). The data were statistically evaluated using analysis of variance. Intraneural
Capillary
Permeubility
to Horseradish
Peroxidase
This aspect was studied by a method similar to that of Jakobsen and co-workers (1978) using a tail-vein injection of 5 mg of HRP (Type VI, Sigma Chemicals) dissolved in 0.1 ml saline in three pairs of lo-month-old male diabetic mice exhibiting symptoms of peripheral neuropathy and unafflicted littermates. One hour after injection, the mice were sacrificed under tribromoethanol anesthesia by removal of the heart. Tissues including the left sciatic nerve, trigeminal nerve, and brain were excised and fixed by immersion in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for 4 hr at room temperature. Following fixation, the tissues were rinsed in several changes of 0.1 M cacodylate buffer, pH 7.4, with 5% sucrose. The tissues were then rapidly frozen in isopentane cooled by a bath of dry ice and 95% ethanol and cut at 20 pm on a cryostat. The sections were picked up on cover slips and allowed to dry for 18 hr in the dark, since long exposure to light inactivates HRP in tissue sections. Horseradish peroxidase activity was localized according to the method of Hanker and co-workers (1977) using paraphenylenediamineipyrocatechol (PPD-PC, Polysciences) as the chromogen. The sections were preincubated in a medium without hydrogen peroxide (15 mg PPD-PC in 10 ml 0.1 M Tris-HCI buffer, pH 7.4) for 20 min at 4°C followed by incubation in the same medium with hydrogen peroxide (0.01%) for 10 min at room temperature. After a brief rinse in buffer, the sections were dehydrated in a graded series of ethanols, cleared in xylene, and mounted in Permount (Fisher).
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1
SCIATIC NERVE CAPILLARY BASAL LAMINA THICKNESS IN DIABETIC MICE AND UNAFFLICTED LITTERMATES
Unafflicted littermates Diabetic mice
Blood glucose” (mg/lOO ml)
Basal lamina thickness* (A)
193 2 15 (n = 8) 620 2 50 (n = 8)
947?51(tl=5) 964 t 37 (n = 5)
a Determined with glucose test kit No. 510, Sigma Chemical Company (St. Louis, MO). These values were included to indicate the degree of hyperglycemia in the diabetic mice. b These values were determined by measuring the basal lamina thickness of 10capillaries from each of five diabetic mice and their unaftlicted littermates. These 10 values were averaged and the averages were then statistically treated with the analysis of variance. The F value was 0.3669 which was not significant even at the 0.25 level.
RESULTS Morphology
of Capillaries
and Basal Lamina
Thickness
The means and standard errors of the blood glucose levels for the diabetic mice and their unafflicted littermates used in these experiments are given in Table 1. Examination of the ultrastructure of sciatic nerve capillaries from normal and diabetic mice did not reveal a considerable degree of pathological change in sciatic nerve capillaries from diabetic mice. However, there was an apparent increase in the incidence of abnormal endothelial cell cytoplasmic inclusions which appeared to be made up of whorls of membrane (Fig. 1). These structures were not observed in sciatic nerve capillary endothelial cells from unafflicted mice, although they were observed in those of their diabetic littermates. There was no apparent capillary basal lamina thickening in sciatic nerve capillaries from lo-month-old unafflicted mice (Fig. 2). In the diabetic mice, a very small percentage of the capillaries exhibited basal lamina thickening (Fig. 3). In other respects, the morphology of the intraneural endothelial cells from diabetic and nondiabetic mice was within the limits of variation normally observed. The measurements of sciatic nerve capillary basal lamina thickness for diabetic mice and their unafflicted littermates are shown in Table 1. As the mean and standard error values indicate, there was no statistically significant increase in capillary basal lamina thickness in diabetic mice when compared to their unafflicted littermates. Intraneural
Capillary
Permeability
to Horseradish
Peroxidase
Following intravenous injection of HRP and incubation of the tissue sections for localization of the tracer, intraneural and brain capillaries were filled with dark FIG. 1. Sciatic nerve capillary from a IO-month-old diabetic mouse. Note the normal basal lamina width (arrow) and the abnormal accumulation of whorled membranes in the endothelial cell cytoplasm (*). x22,000. -FIG. 2. Capillary from the sciatic nerve of a IO-month-old normal mouse. There is no abnormal basal lamina thickening (arrow). x 10,800. FIG. 3. A rare instance of a thickened basal lamina surrounding a capillary in the sciatic nerve of a diabetic mouse (arrows). The adjacent Schwann cell cytoplasm contains two Pi granules of Reich (P). x 12,000.
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MICE
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reaction product (Figs. 4-6). There was no observable reaction product outside the capillaries in the sciatic nerve, trigeminal nerve and cerebral cortex (Figs. 4-6). Incubation of nerve tissue sections from mice not receiving an injection of HRP revealed that, under the conditions of these experiments, no endogenous peroxidase or catalase was demonstrated. DISCUSSION The blood glucose levels of the diabetic mice and their unafflicted littermates used in these experiments were similar to those previously reported for the db mouse (Hummel et al., 1966). These data point out the severe chronic hyperglycemic condition which develops in diabetic mice and indicates the utility of this hereditary model for maturity-onset diabetes in the study of the etiology of the complications of diabetes. The examination of endothelial cell fine structure in the sciatic nerves of diabetic mice revealed only a low incidence of abnormal morphology. The accumulation of whorls of membrane within endothelial cells may be indicative of altered endothelial cell metabolism. osterby and co-workers (1978) reported that the density of micropinocytotic vesicles in capillary endothelium of striated muscle was increased about 30% in streptozotocin-diabetic rats injected with insulin. Insulin apparently may alter the morphology of the endothelial cells. Diabetic mice were shown to have greatly increased plasma insulin levels, at least through the first 6 to 8 weeks after birth (Coleman and Hummel, 1968); plasma immunoreactive insulin levels in diabetic mice may reach six times those of unafflicted heterozygotes. The whorls of membrane observed in the endothelial cells from diabetic mice could reflect altered autolytic metabolism resulting in decreased membrane turnover and subsequent accumulation. Pfeiger and coworkers (1978) have reported that acute, high plasma insulin levels inhibited formation of autophagic vacuoles in rat hepatocytes. Another factor worthy of consideration is the possible effect of the chronic hyperglycemia on intraneural capillaries. Chronic hyperglycemia has been shown to alter the way cells metabolize glucose, resulting in intracellular accumulation of sorbitol (Winegrad et al., 1973), a compound which has been suggested to promote development of microangiopathy, cataracts, and neuropathy in the rat (Van Heyningen, 1958; Drasnin, 1973). However, at this time there is no experimental evidence from work on the diabetic mouse to support either of these suppositions. Thickening and reduplication of the capillary basal lamina has been described in nerves from human diabetics with peripheral neuropathy (Bischoff, 1968; Lapresle, 1968). Most studies with chemical and hereditary animal models for diabetes have examined basal lamina thickness of muscle capillaries, so that there are insufficient data to determine if there is a correlation between the peripheral neuropathy and intraneural microangiopathy in these animal models. Several tissues from this diabetic mouse strain have been examined quantitatively to determine whether or not there is an increase in capillary basal lamina thickness. These include gingiva (Anapolle et al., 1972, 1973), kidney (Like et al., 1972) and muscle (Novis and Korson, 1974). In these studies, gingival capillaries and capillary tufts in glomeruli demonstrated increased basal lamina formation,
INTRANEURAL
CAPILLARIES
IN
DIABETIC
MICE
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5b
6b FIGS. 4-6. Following histochemical incubation, the intraneural capillaries of mice injected intravenously with horseradish peroxidase contained dark reaction product. In the cerebral cortex (Fig. 4), trigeminal nerve (Fig. 5), and sciatic nerve (Fig. 6) there was no observable increase in leakage of HRP from capillaries in diabetic mice (b) when compared to their unafflicted littermates (a). Fig. 4, x30. Figs. 5 and 6. x340.
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while the difference in muscle capillary basal lamina thickness was not statistically significant in diabetic mice and their unafllicted littermates. Sima and Robertson (1978b) reported intraneural capillary basal lamina thickening in diabetic mice but did not furnish quantitative data. The present study indicated that at least in the sciatic nerve, where neuropathological changes have been reported (Carson et al., 1980) there was no statistically significant increase in capillary basal lamina thickness in diabetic mice. Recent studies on the effects of chronic hyperglycemia on the permeability of endoneurial vasculature to tracer substances have been conflicting. Seneviratne (1972) reported that in the alloxan-diabetic rat, the permeability of both the perineurium and endoneurial vasculature to tail-vein injected Evans blue-labeled albumin was increased. On the basis of these findings the author suggested that increased extravasation of proteins into the extracellular space of the endoneurium may play a role in the pathogenic mechanism of diabetic neuropathy observed in this animal model. However, Jakobsen and co-workers (1978) showed that the blood nerve barrier in the streptozotocin-diabetic rat was capable of excluding Evans blue-labeled albumin, HRP, and cytochrome c from the nerve parenchyma. The work of Sima and Robertson (1978b) supported Jakobsen’s findings in the streptozotocin-diabetic rat and extended them to the alloxandiabetic rat and the diabetic mouse. The results of the HRP permeability experiments in the present study are in agreement with those of Sima and Robertson (1978b), in that there was no observable leakage of HRP from the intraneural vasculature of both diabetic mice and unafflicted littermates. In conclusion, it is apparent that microangiopathy probably does not play a major role in the etiology of the peripheral neuropathy in the diabetic mouse. The fine structural studies indicated that a certain degree of intraneural capillary pathology may occur. However, at this time, sufficient evidence is not available to determine to what degree the structural abnormalities in the endothelium may reflect functional changes which could be involved in the development of peripheral neuropathy.
ACKNOWLEDGMENTS The authors are grateful to Jodie Kurtz for typing the manuscript. This work was supported by NIH Research Grants DE 04730, DE 02668, and DE 00288 from the National Institute for Dental Research, NIH Grant RR 05333 from the Division of Research Facilities and Resources, and a grant to the Neurobiology Program from the Alfred P. Sloan Foundation.
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