Retinal Capillary Basement Membrane Research Articles

Prevention of retinal capillary basement membrane thickening in diabetic dogs by a non-steroidal anti-inflammatory drug.

To investigate the effect of treatment with the non-steroidal anti-inflammatory drug Sulindac on the early vascular pathology of diabetic retinopathy in the dog, and it's effect on recognised biochemical indices of hyperglycaemia-related pathophysiology. Experimental diabetes (streptozotocin/alloxan) was induced in 22 male beagle dogs and 12 of the animals were assigned at random to receive oral Sulindac (10 mg/kg daily). Age- and sex-matched control animals were maintained as non-diabetic controls. After 4 years, several morphological parameters were quantified in the retinal microvasculature of each animal group using an established stereological method. Also, the following diabetes-associated biochemical parameters were analysed: accumulation of advanced glycation end products (AGEs), red blood cell polyol levels and antioxidant status. Diabetes increased red blood cell sorbitol levels when compared to non-diabetic controls (p< or =0.05), however, there was no difference in sorbitol levels between the untreated and the treated diabetic animals. No significant differences were found in red blood cell myoinositol levels between the three groups of animals. Pentosidine and other AGEs were increased two- to three-fold in the diabetic animals (p< or =0.001) although treatment with Sulindac did not affect their accumulation in diabetic skin collagen or alter diabetes-induced rises in plasma malondialdehyde. Retinal capillary basement membrane volume was significantly increased in the untreated diabetic dogs compared to non-diabetic controls or Sulindac-treated diabetic animals (p< or =0.0001). This study has confirmed the beneficial effect of a non-steroidal anti-inflammatory drug on the early vascular pathology of diabetic retinopathy. However the treatment benefit was not dependent on inhibition of polyol pathway activity, advanced glycation, or oxidative stress.

Inhibition of advanced glycation end-products protects against retinal capillary basement membrane expansion during long-term diabetes.

The purpose of this study was to investigate the advanced glycation end-product (AGE)-inhibitory properties of aminoguanidine and to determine whether treatment in long-term diabetic rats can prevent basement membrane lesions of diabetic retinopathy. Four groups of male Wistar rats were studied: untreated diabetics injected with 45 mg/kg streptozotocin, aminoguanidine-treated diabetics, untreated controls, and aminoguanidine-treated controls. After 12 months' diabetes, the retinas from six animals were processed for electron microscopy or the retinal microvasculature was isolated using the trypsin digest technique. Stereological analysis was used to estimate quantitative ultrastructural changes in the retinal capillary-associated basement membrane. Serum AGEs were quantified by competitive AGE-ELISA, while microvascular-associated, immunoreactive AGEs were analysed on retinal trypsin digests. Aminoguanidine significantly reduced serum AGEs in the diabetic group (p < 0.001). In the retinal capillaries, there was a marked reduction in AGE immunoreactivity in the aminoguanidine-treated diabetics when compared with untreated diabetics. The surface area and absolute volume of the retinal capillary basement membrane were significantly increased in the diabetic rats when compared with non-diabetic controls (p < 0.001 and p < 0.001, respectively). Aminoguanidine treatment of diabetic rats protected against basement membrane expansion when compared with untreated diabetic counterparts. Aminoguanidine treatment prevents the development of diabetes-induced basement membrane expansion in retinal capillaries. The AGE inhibition properties of aminoguanidine suggest that AGEs play an important role in the complex pathogenesis of basement membrane thickening during diabetic retinopathy.

Retinal Changes in Otsuka Long-Evans Tokushima Fatty Rats (Spontaneously Diabetic Rat)—Possibility of a New Experimental Model for Diabetic Retinopathy

Purpose: To study retinal changes in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a spontaneously diabetic rat, and evaluate it as a possible new diabetic retinopathy model. Methods: We examined the retinas of OLETF rats and Long-Evans Tokushima Otsuka (LETO) rats as controls under both a transmission electron microscope (TEM) and a scanning electron microscope (SEM). Results: We detected higher blood sugar level in the OLETF rats than in the LETO rats beginning at 5 months of age. The inner nuclear layers of the retina decreased from 3–4 rows to 2 rows, whereas the photoreceptor cell nuclei decreased from 8 rows to 3–6 rows. Retinal pigment epithelial cells decreased in height, and basal infoldings were poorly developed. Retinal capillary basement membranes were significantly thicker in the OLETF rats than in the LETO rats, and endothelial cell damage was observed. The SEM of vascular corrosion casts of OLETF rats showed tortuosity, microaneurysms, and loop formations. Conclusions: The development of spontaneous hyperglycemia in OLETF rats was associated with alterations in retina ultrastructure. Changes were similar to those seen in diabetic retinopathy, but did not include either hemorrhages or exudates. The OLETF rat may be a useful animal model for the study of ocular complications in humans.

Non-uniform distribution of lesions and biochemical abnormalities within the retina of diabetic humans

Objective. Microaneurysms, acellular capillaries, pericyte ghosts, and thickening of retinal capillary basement membrane are characteristic of diabetic retinopathy, and are believed to be sequelae of excessive blood glucose. Previous studies by us in dogs demonstrated that lesions of diabetic retinopathy were not uniformly distributed across the retina, but were significantly more numerous in the superior/temporal areas of the retina. In the present study, the distribution of these lesions and the biochemical abnormalities postulated to play a role in their pathogenesis have been evaluated in retinas collected at autopsy from diabetic patients. Methods. Retinas were divided into quadrants (nasal, temporal, superior, inferior), the vasculature exposed by the trypsin-digest method, and the frequency of the lesions compared among the quadrants. Homogenates taken from the midretina of nasal and temporal quadrants of retina were used to explore regional differences in expression of Glut1, PKCß, and iNOS (Western blots) and caspases (enzymatic activity). Results. Microaneurysms, acellular capillaries and pericyte ghosts were not uniformly distributed across the retina, and were significantly more numerous in the temporal retina than in the nasal retina (P < 0.05). In contrast, the thickness of retinal capillary basement membrane was not found to differ significantly across the retina. In our limited study, activity of the pro-inflammatory protease, caspase 1, was the only biochemical abnormality where there was both a significant diabetes-induced alteration in activity and also a significant difference between retinal quadrants. Expression of the glucose transporter, Glut1, was significanlty decreased in diabetes, but there was no significant difference in expression between the quadrants. Expression of iNOS was increased only in temporal retina in diabetes (but no significant difference between quadrants), and PKCß tended to be greater than normal in both temporal and nasal retina. Conclusions. Retinal microvascular disease does not develop uniformly across the retina of diabetic patients, even though the different regions are exposed to the same level of hyperglycemia.

Increased numbers of caveolae in retinal endothelium and pericytes in hypertensive diabetic rats

Long-term clinical studies have now shown that tight control of blood pressure in type 2 diabetes reduces the risk of diabetes-related death and common diabetic complications, including diabetic retinopathy. However, the mechanisms by which hypertension enhances diabetic microvascular disease, especially diabetic retinopathy, are poorly understood. We developed an experimental model of hypertension in diabetic rats and studied the early ultrastructural changes in retinal capillaries under these conditions. Hypertension was induced in diabetic BioBreeding (BB) rats by unilateral nephrectomy, weekly subcutaneous mineralocorticoid and 0.9% oral saline. Serial blood pressures and ultrastructural features of retinal capillaries were recorded in four groups: normotensive Wistar rats, normotensive diabetic rats, hypertensive Wistar rats and hypertensive diabetic rats. A significant and sustained increase in systolic blood pressure occurred in both groups of nephrectomised rats. There was a significant increase in the number of caveolae (i) in both pericytes and endothelial cells in animals with hypertension and diabetes together compared with all other groups and (ii) in pericytes in animals with diabetes alone. The number of direct contacts between pericytes and endothelial cells was reduced in diabetic and hypertensive diabetic animals. Hypertension and diabetes had an interactive effect in producing retinal capillary basement membrane thickening. In the BB rat hypertension and diabetes have an interactive effect in increasing the number of caveolae in both endothelial cells and pericytes. We speculate that this may be a reflection of changes in calcium and nitric oxide metabolism in these animals.

Acarbose partially inhibits microvascular retinopathy in the Zucker Diabetic Fatty rat (ZDF/Gmi-fa).

We compared quantitative capillary retinopathic changes between non-insulin-dependent diabetic Zucker Diabetic Fatty (ZDF) rats and heterozygous nondiabetic Zucker controls and evaluated the effect of an orally administered glucosidase inhibitor, acarbose, on retinopathy in these animals. Four groups of eight rats were analyzed: treated and untreated ZDF and treated and untreated Zuckers. Retinal capillary basement membrane thickening and retinal capillary cell density were determined from transmission electron microscopy and trypsin digestion preparations. ZDF rats had thicker basement membranes (p<0.0001) and more cells per unit capillary length (p=0.0003) compared to Zuckers. Acarbose treatment significantly reduced basement membrane thickening in the treated ZDF rats (p=0.001), but the effects on cell density showed only a favorable trend. Acarbose treatment has an ameliorative effect on the development of microvascular retinopathy in the ZDF rat, probably due to lessening of hyperglycemia.

Retinal vascular changes induced by the oxidative stress of alpha-tocopherol deficiency contrasted with diabetic microangiopathy.

It has been proposed that oxidative tissue damage is involved in the development of diabetic angiopathies. To evaluate this hypothesis, experiments were conducted to identify the retinal vessel changes induced by the oxidative stress related to alpha-tocopherol deficiency and examine possible similarities with the lesions characteristic of diabetic retinopathy. Twenty-one-day-old male Fisher 344 albino rats were divided randomly to receive a basal, chemically defined diet either with (adequate group) or without (deficient group) alpha-tocopherol. After 6 and 8 months, some rats (n = 3 per group) were killed and the eyes removed. In order to evaluate cell integrity and localization of lipofuscin-specific autofluorescence by light and fluorescence microscopy, some of the retinas were prepared for cryostat-sections while others were digested by elastase to isolate intact retinal vasculatures. After 8 and 14 months, the central retina of one eye per rat (n = 6 to 8 per group) was examined by electron microscopy for retinal capillary basement membrane (RCBM) thickening and other ultrastructural changes. At 6 and 8 months, the deficient rats exhibited extensive shortening and disarray of rod outer segments (ROS), marked loss of photoreceptor cells, and pronounced increases in the numbers of granules with lipofuscin-specific autofluorescence in the retinal pigment epithelium (RPE) and retinal vessels. At 14 months, the ultrastructure revealed that the damage to ROS involved disruption of membranes and that the capillary lipofuscin was contained mainly within the endothelial cells. Membrane remnants were found in the lipofuscin granules of both the RPE and retinal vessels. In addition, there was an increase in RCBM thickness (98.7 +/- 2.6 nm vs. 86.9 +/- 2.9 nm). RCBM thickening was the only finding common with diabetic retinopathy, and the thickening was 13.6%, significantly less than that reported in diabetic rat models with 8 and 14 months durations (34% and 53.1%, respectively). Capillary lipofuscin accumulation, which was prominent in the deficient rats, is not notable in diabetes. Both the moderate RCBM thickening and marked lipofuscin accumulations seen in alpha-tocopherol-deficient rats were similar to changes occurring in the aging process, though more pronounced. The spectrum of microangiopathies characteristic of diabetic retinopathy did not develop in alpha-tocopherol-deficient rats. These findings suggest that oxidative damage, though probably involved, is unlikely to play a predominant role in the development of diabetic retinal microangiopathies.

Retinal capillary dilation: early diabetic-like retinopathy in the galactose-fed rat model.

The purpose of this study was to determine whether capillary dilation is one of the earliest structural changes in the diabetic-like retinopathy of the galactose-fed rat model and thus may represent a stage where intervention treatment might still be effective. Weanling female Sprague-Dawley rats were randomized into 3 groups and fed Purina laboratory chow plus one of the following for 4 months: 50% starch (CONTROL); 50% D-galactose (Galactose); or 50% D-galactose with ARI-509 (25 mg/kg body wt/day) (Inhibitor). One eye from each of 5 rats per treatment group was processed for retinal vasculature wholemounts using elastase digestion, stained with a standard periodic-acid-Schiff reaction and counterstained with hematoxylin. Average capillary width, overall capillary density and total capillary length were measured, using computerized image analysis, within an arc-shaped area (4.4 mm2) of each vasculature surrounding, but separated from, the optic disc margin by approximately 0.7 mm. Galactose rats exhibited significant (p < 0.001) increases in capillary width (Mean +/- SEM: 7.56 +/- 0.07 microm) and density (42.78 +/- 0.37%) compared with CONTROL rats (6.68 +/- 0.11 microm and 37.18 +/- 0.30%, respectively). These increases were prevented with inhibitor treatment (6.58 +/- 0.16 microm and 35.88 +/- 0.97%, respectively). Capillary length remained unchanged at 4 months ( 246.66 +/- 2.46 mm; Galactose: 250.75 +/- 1.26 mm; Inhibitor: 242.25 +/- 8.43 mm). Retinal capillary dilation, expressed as increased width and density, is one of the earliest detectable lesions in galactose-fed rats. In these rats, the lesion occurs as early as retinal capillary basement membrane thickening (RCBMT), one of the earliest reported changes in human diabetic retinopathy. Like RCBMT, capillary dilation can be prevented in rats with aldose reductase inhibitor treatment. Unlike RCBMT, capillary dilation could be clinically detectable and may be useful for the diagnosis of early retinopathy and for determining the timing of therapeutic intervention.

Lens and Retinal Changes in the WBN/Kob Rat (Spontaneously Diabetic Strain)

Male WBN/Kob rats represent a spontaneously diabetic strain with hyperglycemia, cataracts, nephropathy, neurophathy, pancreatic fibrosis and hyperlipemia. Cataracts and retinal changes in WBN/Kob rats were examined by light and electron microscopy to evaluate the ocular complications. Lens opacity was present in the posterior subcapsular and center of the anterior cortex of male 14-month-old WBN/Kob rats. Light and transmission electron microscopy showed swelling and irregularity of lens fibers. Scanning electron microscopy revealed that lens fibers were irregular and had many granules and bulging processes of various sizes on the cortical side of the opacified region. The nuclear side of the opacified region showed spongy changes and complete absence of lens fibers. Electron microscopy showed retinal degeneration in the photoreceptor outer segments of 1-month-old male WBN/Kob rats. Light microscopy showed thin outer segments and outer nuclear layers in 5-month-old rats, and electron microscopy revealed severe degeneration in the outer segments. The retinas of 11-month-old rats were thinner; the outer plexiform layer was very thin; the photoreceptor cell nuclei in the outer nuclear layer had decreased to one layer and were almost in contact with the inner nuclear layer nuclei, while the visual cells had disappeared. Retinal degeneration had progressed even further in 14-month-old rats, and very few photoreceptor cell nuclei remained. The retinal capillary lumens were small, and their pericytes had thickened basement membranes. The basement membranes of retinal capillaries from WBN/Kob rats were significantly thicker than those from control Wistar rats (p < 0.0001). Although this rat has spontaneous diabetic features, such as cataracts, its retinal changes look more degenerative.

Heparan sulfate proteoglycan synthesis and its expression are decreased in the retina of diabetic rats

PURPOSE. Heparan sulfate proteoglycan (HSPG) is an integral component of all basement membranes and is implicated in the charge-selective properties of these basement membranes. Studies have shown a decrease in the number of HSPG anionic sites in the retinal capillary and glomerular basement membrane of diabetic animals. This study examined whether a decrease in both HSPG synthesis and its expression as perlecan are reduced in the retina of streptozotocin-diabetic rats. METHODS. Diabetes was induced in male Wistar rats by intra-peritoneal injection of streptozotocin and the rats were sacrificed 20 weeks later. Retinas were isolated and HSPG synthesis was assessed by incorporation of 35 S-sulfate into heparan sulfate. The expression of mRNA for perlecan was quantified by RNase protection assay. RESULTS. Both the synthesis of HSPG and mRNA expression for perlecan were decreased in the retina of diabetic compared to normal rats. CONCLUSIONS. The decrease in HSPG synthesis may account for the reported decrease in retinal basement membrane anionic sites and increased capillary permeability that occurs in diabetes.

Diabetic retinopathy: quantitative variation in capillary basement membrane thickening in arterial or venous environments.

Diabetes mellitus was induced in male beagles by a single injection of an alloxan and streptozotocin cocktail and fasting blood sugar levels maintained between 15 and 20 mmol/l. Five years after induction of diabetes, three diabetic animals were sacrificed, together with sex and age-matched controls, and the retinas fixed for either transmission electron microscopy (TEM) or trypsin digestion. In TEM specimens, capillaries in close proximity to the major vessels were designated as either AE (arterial environment) or VE (venous environment) and the thickness of their basement membranes (BMs) measured using an image analyser based two dimensional morphometric analysis system. Results show that the BMs of retinal capillaries from the diabetic dogs were significantly thicker than those from control dogs. Furthermore, within the diabetic group the AE capillaries had thicker BMs than VE capillaries (p < or = 0.05). The controls, however, showed no significant difference in BM thickness between AE and VE capillaries. Although many of the capillaries designated as AE or VE would actually have been derived from the opposite side of the circulation, with respect to BM thickness, they conformed to values of their specific group. The conclusion is that diabetic capillaries are more vulnerable to BM thickening in an arterial environment than in a venous environment.

Aldose Reductase Inhibition Fails to Prevent Retinopathy in Diabetic and Galactosemic Dogs

To investigate a possible role of excessive polyol production in the pathogenesis of diabetic retinopathy, 16 ALX-induced diabetic dogs and 20 experimentally galactosemic dogs were randomly assigned to 5 yr of treatment with either sorbinil, an aldose reductase inhibitor, or a placebo. The severity of hyperglycemia in sorbinil-treated and placebo groups was monitored throughout the 5-yr study by assay of glycosuria and nonenzymatically glycated plasma protein and HbA 1 needed in an effort to avoid confounding possible group differences in hyperglycemia severity with possible drug effects. Inhibition of polyol production by sorbinil was monitored in erythrocytes throughout the study and also in retina and other tissue obtained at autopsy. Trypsin digests of retinal vessels were compared after 60 mo of diabetes and after 42 and 60 mo of galactosemia. In diabetic dogs, development of retinopathy was not significantly influenced by a sorbinil dose (20 mg · kg −1 · day −1 ) sufficient to prevent elevation of sorbitol levels in retina and other tissue. Likewise, in dogs made experimentally galactosemic for 42–60 mo, administration of sorbinil (60–80 mg · kg −1 day −1 ) had no significant effect on the development of retinopathy notwithstanding prevention of 93–96% of the polyol elevation in retina and other tissue. Retinal capillary basement membrane was significantly thicker than normal in diabetic and in galactosemic dogs and was not significantly influenced by administration of sorbinil in either dog model. Thus, no evidence was found that the development of retinopathy is critically dependent on excessive polyol production or accumulation.

Regulation of Fibronectin and Laminin Synthesis by Retinal Capillary Endothelial Cells and Pericytes In Vitro

Retinal capillaries are composed of endothelial cells resting on a basement membrane, in which are embedded pericytes. In diabetes mellitus, the basement membrane becomes thickened, and there is a loss of pericytes. The relative contributions of endothelial cells and pericytes to the synthesis of extracellular matrix proteins which are components of the basement membrane are not well-characterized. To determine how a selective loss of pericytes might affect the composition of retinal capillary basement membranes, we used primary cultures of bovine retinal capillary endothelial cells and pericytes to determine the forms and quantify the amounts of laminin and fibronectin synthesized and secreted by these cell types as well as to determine how high glucose concentrations alter these parameters. Results of ELISAs showed that pericyte cell/matrix layers contained nearly ten times more fibronectin than endothelial cells (288 ± 24 vs. 34 ± 5 ng μg -1 DNA, P < 0·001), but the amounts of laminin were similar. d -glucose (40 mM) tripled the amount of fibronectin incorporated into the endothelial cell/matrix layer (102 ± 4 vs. 34 ± 5 ng μg -1 DNA, P < 0·05), but had a lesser effect on pericytes. The non-metabolizable analogue l-glucose, also increased the amount of fibronectin incorporated in both pericyte and endothelial cell/matrix layers. The effects of d- and l-glucose on fibronectin secreted into the medium by both cell types were similar to the effects on incorporation of fibronectin into cell/matrix layers. Glucose had no effect on laminin synthesis. [ 35S]methionine radiolabeling and immunoprecipitation showed that pericytes and endothelial cells synthesize different forms of fibronectin. Both pericytes and endothelial cells synthesized an A and two B chains of laminin which were of similar apparent size, but the two cell types post-translationally modified the subunits differently. We conclude that pericytes and endothelial cells may contribute different forms and amounts of fibronectin and laminin to the retinal capillary basement membrane, so the preferential loss of pericytes in diabetes could result in basement membrane abnormalities which might lead to endothelial cell dysfunction.

Dietary myo-inositol supplementation does not prevent retinal and glomerular vascular structural changes in chronically diabetic rats

To assess effects of dietary myo-inositol supplementation on diabetes-induced vascular structural lesions, diabetes was induced in Sprague-Dawley rats with streptozotocin; one-third of these rats was fed a 2% myo-inositol diet for 9 months, one-third was left untreated for 5 months then treated with myo-inositol for the last 4 months, and one-third was untreated for the entire 9 months. Controls included untreated and myo-inositol-treated groups. Weight gain was impaired and plasma glucose, glycosylated hemoglobin, food consumption, urine volume, and albuminuria were increased significantly in diabetic versus age-matched control rats. Plasma myo-inositol levels were increased approximately fivefold in controls and approximately six- to eightfold in diabetic rats treated with myo-inositol. In general, myo-inositol did not affect any of the above parameters in control or diabetic rats. Retinal capillary basement membrane width (CBMW) was increased significantly (∼50% versus controls) after 9 months of diabetes. In the control group myo-inositol increased CBMW to the level of untreated diabetic rats; myo-inositol had no effect on CBMW in each diabetic group. The number of retinal capillaries containing pericyte nuclei and pericyte capillary coverage were increased in untreated as well as myo-inositol-treated diabetic rats and in the myo-inositol-treated control group. Glomerular CBMW was increased after 5 and 9 months of diabetes versus age-matched controls, and was increased even more by myo-inositol. Mesangial fractional volume of the glomerulus was increased 36% by diabetes and was decreased slightly but significantly by myo-inositol. These results indicate that diets supplemented with 2% myo-inositol (1) cause capillary basement membrane (CBM) thickening and pericyte changes in retinal capillaries of normal rats, (2) are ineffective in preventing or reversing diabetes-induced retinal CBM thickening, and (3) cause further thickening of glomerular CBM in diabetic rats.

Aldose Reductase Inhibition Fails to Prevent Retinopathy in Diabetic and Galactosemic Dogs

To investigate a possible role of excessive polyol production in the pathogenesis of diabetic retinopathy, 16 ALX-induced diabetic dogs and 20 experimentally galactosemic dogs were randomly assigned to 5 yr of treatment with either sorbinil, an aldose reductase inhibitor, or a placebo. The severity of hyperglycemia in sorbinil-treated and placebo groups was monitored throughout the 5-yr study by assay of glycosuria and nonenzymatically glycated plasma protein and HbA1 needed in an effort to avoid confounding possible group differences in hyperglycemia severity with possible drug effects. Inhibition of polyol production by sorbinil was monitored in erythrocytes throughout the study and also in retina and other tissue obtained at autopsy. Trypsin digests of retinal vessels were compared after 60 mo of diabetes and after 42 and 60 mo of galactosemia. In diabetic dogs, development of retinopathy was not significantly influenced by a sorbinil dose (20 mg.kg-1 x day-1) sufficient to prevent elevation of sorbitol levels in retina and other tissue. Likewise, in dogs made experimentally galactosemic for 42-60 mo, administration of sorbinil (60-80 mg.kg-1 x day-1) had no significant effect on the development of retinopathy notwithstanding prevention of 93-96% of the polyol elevation in retina and other tissue. Retinal capillary basement membrane was significantly thicker than normal in diabetic and in galactosemic dogs and was not significantly influenced by administration of sorbinil in either dog model. Thus, no evidence was found that the development of retinopathy is critically dependent on excessive polyol production or accumulation.

The effect of acarbose on diabetes- and age-related basement membrane thickening in retinal capillaries of the [formula omitted

The effect of the α-glucosidase inhibitor acarbose on retinal capillary basement membrane thickening was examined in the spontaneously diabetic BB W -rat . Four months of diabetes resulted in significant thickening of the basement membranes of both the superficial and deep capillary nets of the retina. This characteristic change of the retinal microvasculature in diabetes was completely prevented by acarbose treatment that substantially reduced postprandial hyperglycemia. A similar but less pronounced effect was seen on the age-related increase in basement membrane thickening in acarbose-treated non-diabetic control rats who demonstrated decreased glycated hemoglobin levels compared to non-treated control rats. Significant positive correlations between basement membrane thickness and glycated hemoglobin area suggest that diabetic retinal microangiopathy may be prevented by lowering the cumulative glucose exposure to the microvasculature, and that age-related basement membrane thickening is mediated by long-term exposure to normal glucose levels.

Alterations in Viscosity and Filterability of Whole Blood and Blood Cell Subpopulations in Diabetic Cats

Capillary closure and venous dilatation occur early in diabetic retinopathy, and altered blood rheology may play a role. For example previous studies have shown leukocytes are less deformable, are more activated, and occlude retinal capillaries more often in diabetic subjects. The purpose of this study was to determine if rheologic changes developed in diabetic eats, a model in which we have documented retinal capillary basement membrane thickening, microaneurysms, and intraretinal hemorrhage characteristic of early diabetic retinopathy. Viscosity of blood, plasma and purified erythrocyte suspensions was measured by rotational viscometry and plasma fibrinogen content was deterarmed by heat precipitation. Filterability of blood and purified erythrocyte, mononuclear leukocyte, and granulocyte suspensions was determined at constant flow, measuring the increase in pressure over 2 min relative to the pressure generated by buffer alone. Viscosity of whole blood and plasma, but not erythrocytes, was significantly elevated ( P < 0·005) in the diabetic cats over normals at all shear rates studied (450, 225 and 90 sec -1). Furthermore, fibrinogen levels were significantly elevated in diabetic cats compared to normals ( P < 0·004), and were positively correlated with the viscosity of whole blood and plasma. The filterability of mononuclear leukocytes and whole blood in diabetic cats was decreased 56% and 74% when compared to normals, P > 0·0006 and P < 0·025, respectively. In contrast, the filterability of granulocytes and erythrocytes was not significantly different between the two groups. These results suggest that the rheologic alterations in diabetes are numerous, and involve both cellular and plasma protein changes. The changes in the fibrinogen levels, plasma and whole blood viscosity, and mononuclear cell filterability may play important roles in the genesis of diabetic microangiopathy.

Intervention with the aldose reductase inhibitor, tolrestat, in renal and retinal lesions of streptozotocin-diabetic rats

The progressive increase in urinary albumin excretion, which precedes the development of diabetic nephropathy, can be prevented in diabetic rats if the aldose reductase inhibitor, tolrestat, is administered at the initiation and throughout the duration of hyperglycaemia. We therefore determined the ability of tolrestat to intervene in the further progression of already established urinary albumin excretion of streptozotocin-diabetic female Wistar rats. Two months after streptozotocin injection, diabetic rats were grouped as low-urinary albumin excretion (0.2-1.0 mg albumin/day) or high-urinary albumin excretion (1.9-5.9 mg albumin/day), at which time tolrestat intervention (25 mg/kg per day) was begun for half of the diabetic rats in each urinary albumin excretion group. After six months of treatment tolrestat caused a significant reduction in the urinary albumin excretion rate of the low-urinary albumin excretion group only. The diabetes-induced rise of total urinary protein in both groups was significantly reduced by tolrestat. Furthermore, the diabetes-induced increase (49%) in the thickness of the basement membranes of retinal capillaries from the outer plexiform layer was significantly diminished by tolrestat administration. In conclusion, intervention therapy with the aldose reductase inhibitor, tolrestat, can reduce the progression of urinary albumin excretion and retinal basement membrane thickening in long-term diabetic rats.

Anionic sites in diabetic basement membranes and their possible role in diffusion barrier abnormalities in the BB-rat

Basement membrane anionic sites, thought to be responsible for charge selective permeability barriers, were investigated in retinal, endoneurial, and muscle capillary basement membranes and in Bruch's membrane of diabetic, and age- and sex-matched non-diabetic BB-rats using an ultrastructural quantitative histochemical technique. Six months of diabetes was associated with significant basement membrane thickening which was linearly related to a decrease in anionic site density suggesting a relative loss of proteoglycans. Calculation of anionic sites per unit length of basement membrane, reflecting their absolute number, revealed a significant loss in basement membrane, constituting part of normal blood-tissue barrier systems such as retinal and endoneurial capillary basement membranes, and the basement membrane of the retinal pigment epithelium. The absolute number of anionic sites in normally permeable microvessels, such as those of muscle and choriocapillaries, was unaltered by diabetes. We conclude that this specific loss of anionic sites in basement membranes of tissues affected by chronic diabetic complications may in part be responsible for permeability abnormalities seen in these tissues.

Increases in collagen type IV and laminin in galactose-induced retinal capillary basement membrane thickening—Prevention by an aldose reductase inhibitor

Biochemical alterations in the composition of retinal capillary basement membrane components were investigated in galactosemic rats, an animal model that develops basement membrane lesions comparable to those of diabetic retinopathy. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for 9 months developed significant thickening of retinal capillary basement membranes by comparison with animals fed a control test diet ( P < 0·001), or animals on a diet containing 30% galactose and 250 mg kg −1 of the aldose reductase inhibitor sorbinil ( P < 0·001). A quantitative electron microscopic immunogold technique applied on ultrathin sections of the retinas of these animals showed that the labeling densities of collagen type IV and laminin per unit cross-sectional area (which is presumably proportional to the concentrations of these molecules) were significantly increased in the retinal capillary basement membranes of galactose-fed rats, compared with animals on the control test diet. Increases in these two components of basement membranes were prevented by addition of sorbinil to the diet. However, there was no significant change in the labeling density of heparan sulfate proteoglycan (HSPG) core protein in the basement membranes of galactose-fed rats in comparison to animals on either the control diet or galactose-sorbinil diet. Two types of striated fibrillar materials were frequently found in areas of focal thickening of basement membranes of galactose-fed rats only. Thinner fibrils reacted strongly with collagen type III antibody, whereas thicker fibrils reacted weakly with collagen type I antibody. Our results indicate that there is an increase in labeling densities of collagen type IV and laminin in thickened basement membranes of retinal capillaries of galactosemic rats along with the expression of interstitial collagens like collagen type III and an abnormal collagen that weakly cross-reacts with antibody to collagen type I, and these effects of galactosemia on the basement membranes are preventable by an aldose reductase inhibitor.