Absence of degenerative changes in retinal and uveal capillary pericytes in diabetic rats

Ultrastructural morphometric techniques were used to quantify pericyte degeneration in retinal and uveal capillaries of streptozotocin-diabetic rats in order to assess the suitability of this small rodent model of diabetes for studies of the pathogenesis of microvascular eye disease in diabetic humans. Male, Sprague-Dawley rats were killed by intraaortic perfusion of fixative 6 and 9 mos after induction of diabetes with 50 mg/kg streptozotocin. No differences were evident between diabetics and age-matched controls in capillary circumference, numbers of endothelial cells per capillary, and capillary cytoplasmic area of retinal, choroidal, and iridial vessels. Capillary basement membrane width and the percentage of the capillary circumference covered by pericytes were increased in retinas of diabetic vs age-matched control rats after 9 mos of diabetes (P less than 0.05), but no differences were evident in the number of pericyte processes per capillary and the percentage of vessels with pericyte nuclei. No differences in pericyte distributions were observed between control and diabetic rats in the choriocapillaris and iris after 9 mos of diabetes. These findings indicate that retinal capillary basement membrane thickening precedes any evidence of pericyte degenerative changes and suggest that pericyte degeneration analogous to that associated with human diabetic microangiopathy does not occur in this experimental animal model.     

Pericyte coverage is greater in the retinal than in the cerebral capillaries of the rat

Using electron microscopic morphometry, we demonstrate here that, in the albino rat, the pericytes of the retinal microcirculation over the capillary circumference much more extensively than in five widely separate regions of cerebral cortex. The ratio of pericyte plasma membrane length in contact with the vascular circumference, to the outer circumference of the endothelial cell tube, was 0.41 +/- 0.02 (SEM) for the retinal microvessels, and ranged from 0.22 +/- 0.02 to 0.30 +/- 0.03 for the five regions of cerebral cortex. The values of this ratio for the retina differed significantly from those for the various cortical regions. Differences of comparable magnitude were obtained between ratios of pericyte to endothelial cell areas in retina and cerebral cortex. The substantial quantitative difference in pericyte coverage of the microvessel circumference between the retinal and cerebral microcirculations may have important implications for normal and pathological microcirculatory dynamics in these two organs.     

Pericyte form and distribution in rat retinal and uveal capillaries

Ultrastructural morphometric techniques were used to assess differences in endothelial cells and in pericyte structure and distribution in rat retinal and uveal capillaries. Retinal capillaries were significantly smaller than those in the three different uveal vascular beds, all of which were similar in size. Approximately 10% of the capillaries in the retina and choroid were formed by three endothelial cells, compared with 30% and 46% of capillaries sampled from ciliary processes and iris, respectively. The percentage of the capillary circumference covered by pericytes (46-58%) and the percentage of capillary sections with pericyte nuclei (12-16%) were similar in retina, iris, and ciliary processes. Corresponding data for the choriocapillaris indicated that pericyte coverage of these capillaries was approximately 50% of that observed in the other eye microcirculations. The number of pericyte processes per capillary varied markedly in the different vasculatures, with an average of three for capillaries in the retina and choriocapillaris and nine to eleven for capillaries in the iris and ciliary processes. These marked differences in capillary dimensions are consistent with the well-known capillary hemodynamic and functional differences of these tissues; however, the significance of the differences in pericyte shape, frequency and distribution in the different vasculatures of the eye is less clear.     

Pericyte changes in branch retinal vein occlusion

The model of experimental branch vein occlusion (BVO) in the monkey offers the opportunity to examine retinal capillaries under stress. Electron microscopic morphometry was done on 812 capillaries of 13 eyes of cynomolgus monkeys, comparing 579 capillary collaterals of 9 BVO eyes with 233 normal capillaries of 4 control eyes. The tissue underwent the myosin subfragment-1 technique to decorate and quantify bundles of actin filaments in capillary pericytes. The duration of BVO was 2-48 months. Capillary collaterals of BVO eyes had an enlarged caliber, endothelial hyperplasia, and pericyte hypertrophy, but no proportional increase in basement membrane area. Collaterals near the inner plexiform layer (IPL) had a greater wall thickness, pericyte coverage, and actin coverage than collaterals near the outer plexiform layer (OPL). Pericyte hypertrophy was proportionate to caliber increase in OPL vessels and exceeded caliber increase only in IPL vessels. Actin coverage was proportional with the vessel dilation and size of pericyte cytoplasm in all vessels. These findings indicate that capillary collaterals in BVO are not equipped morphologically for an increased regulatory role in microvascular flow beyond their normal function.     

Vascular architecture of degenerated retina in WBN/Kob rats: corrosion cast and electron microscopic study.

The purpose of the present study is to determine the changes of vascular architecture in the degenerated retina. We used mainly corrosion casts of the retinal vasculature and scanning electron microscopy to obtain a wide three-dimensional view. WBN/Kob rats (a spontaneously diabetic strain) were used because their outer retinas degenerate and become very thin with age. In 15-month-old rats, localized constriction and irregular caliber of the capillaries were evident in the vascular casts. Two layers of capillary network in the retina were maintained, but the capillaries were decreased in number. Numerous loop formations were present in the superficial capillary networks. Neither microaneurysms nor arteriovenous shunts were seen in young and old rats. Transmission electron microscopy revealed that capillary pericytes in the inner and outer plexiform layers had thickened basement membranes and that endothelial cells had many vesicles in their cytoplasm. Thus the retinal capillary changes in WBN/Kob rats are nondiabetic but due to hereditary retinal degeneration, although the systemic and pancreatic abnormalities in this rat strain are diabetic. Even when the retina becomes very thin, two layer capillary networks remain.     

In vitro proliferation of endothelial cells from kitten retinal capillaries

When microvessel (predominantly capillary) fragments freshly isolated from the retinas of young kittens are incubated in tissue culture medium, we observe the slow outgrowth of cells that appear to be derived from the capillary endothelium. The cells grow as a monolayer with a tightly packed, mosaic-like array. Electron microscopy of these cells reveals "tight junctions" (zonulae occludentes) resembling those found in the endothelium of intact capillaries. The availability of in vitro preparations of retinal capillary endothelial cells should facilitate investigations of the causes of abnormal endothelial cell proliferation in various diseases and of factors that modify cell junctions and capillary permeability in the retina. Since we have previously demonstrated proliferation in culture of intramural pericytes ("mural cells") from retinal capillaries, it should now be possible to carry out compratative studies of biochemical and functional properties of retinal capillary pericytes and endothelial cells.     

Diabetic-like retinopathy in rats prevented with an aldose reductase inhibitor

The earliest histopathologic signs of diabetic retinopathy include selective loss of intramural pericytes and thickening of capillary basement membranes. Previous evidence from animal models indicated that aldose reductase inhibitors could prevent these capillary wall lesions, but only recently have aldose reductase inhibitors been tested for prevention of the subsequent retinal complications of diabetes, such as microaneurysms. In the present study, Sprague-Dawley rats were fed diets containing 50% galactose with or without an aldose reductase inhibitor (tolrestat). After 28 months of galactose feeding, the retinal capillaries in whole mounts exhibited a marked increase in periodic acid-Schiff (PAS) staining, extensive pericyte loss, endothelial cell proliferation, acellularity, diffuse dilation, occluded lumens, microaneurysms, and complex microvascular abnormalities including gross dilation and formation of multiple shunt networks. The PAS hyperchromaticity of basement membrane material and pericyte loss occurred throughout the retinal vasculature, while while the microaneurysms and complex lesions were limited to the capillaries of the central and paracentral retina. The changes were associated with both the arterial and venous portions of the capillary plexus. Treatment with orally administered tolrestat prevented essentially all of the vessel abnormalities. Thus, long-term galactose feeding of rats induced microvascular lesions simulating those occurring in background diabetic retinopathy in humans, and these lesions were prevented by treatment with an aldose reductase inhibitor.     

The process of occlusion and re-construction of the choriocapillaries following dye laser photocoagulation

In order to elucidate the histopathological changes in the choriocapillaries following laser photocoagulation, moderate laser burns were performed on monkey retinas by an orange dye laser beam (595nm). Immediately after photocoagulation, the choriocapillaries were occluded by the intraluminal thrombus, however, the basement membrane of the endothelial cells remained. The newly-formed immature endothelial cells began to migrate along the pre-existing basement membrane toward the center from the edge of the lesion after 3 days. They formed new vascular lumens by coupling of intercellular junctions at 5 days. At 7 days, newly-formed capillaries were formed beneath the Bruch's membrane. After more than 7 days, the choriocapillaries in the lesion were re-constructed and required an almost normal structure with wide patent lumens, basement membrane, pericytes and fenestrations. It was showed that choriocapillaries occluded by moderate laser photocoagulation were re-constructed by the process of re-endothelialization along the preexisting basement membrane.     

Retinal vascular changes in rats with inherited hypercholesterolemia--corrosion cast demonstration.

PURPOSE. To demonstrate specific hypercholesterolemic changes in the retinal vascular architecture. METHODS. Corrosion casts of 12- to 18-month-old rats with inherited hypercholesterolemia (RICO) and of control Wistar Kyoto (WKy) rats were examined with a scanning electron microscope (SEM). The diameters of the retinal arteries, veins and capillaries were measured in photographs with a caliber micrometer. The capillary branches were counted in the micrographs with the use of Adobe Photoshop. The retinal capillaries were examined by transmission electron microscopy (TEM). RESULTS. SEM examination of the vascular casts of 15-month-old RICO rats showed slight tortuosity of large vessels at the posterior pole of the retina. The precapillary arterioles branching from the major artery were longer and straighter than normal. Retinal capillary changes such as caliber irregularity and narrowing in the capillary network were more severe in 18-month-old RICO rats. The most prominent finding was marked straightening of the capillaries in the inner and outer layers of the capillary network, which looked like fine strings. Intercapillary spaces became wider, and finally capillaries looked scattered. The diameter of the retinal capillaries lumen in RICO rats was significantly narrower than that in WKy rats (p < 0.0001). The capillary branches were fewer in 18-month-old RICO rats than that in 18-month-old WKy rats (p < 0.0001). Neither local stenosis or obstruction in the arterioles and venules nor any arteriovenous crossing defect was seen in young and old RICO rats. Transmission electron microscopy of 16-month-old RICO rat retinas revealed that the capillaries in the inner and outer plexiform layers contained scarce cytoplasmic components, vacuoles in endothelial cells and basement membranes of irregular thickness. Capillary pericytes were swollen and irregular in shape, contained vacuolated mitochondria and scarce cytoplasmic components. CONCLUSIONS. These findings indicate that the retinal capillary changes are probably related to hypercholesterolemia.     

Increased capillary vulnerability in diabetic retinopathy

The retinal capillary network is isolated by Cogan and Kuwabara's method. During prolonged trypsinization the capillaries lose their endothelial cells and pericytes and are transformed into cell-free tubes consisting of basement membranes. This occurs earlier in patients with stage I or II retinopathy than in controls with normal carbohydrate metabolism and patients with stage I hypertension. Therefore, the intercellular junctions and the adhesion between endothelial cells and subendothelial basement lamella are more loosely structured in diabetics than normal subjects, and the cells do not resist the tryptic attack as well. The higher capillary vulnerability demonstrated in these experiments contributes to bleeding and exudation. Whether the local loss of cells in the microcirculation of diabetics occurs primarily in vivo or is only unmasked in vitro manifesting pre-existing cellular damage is discussed.     

Retinal vessels of canine eyes at different ages--a qualitative and quantitative study.

Diameter and number of cells per unit length of vessel wall and endothelial cell frequency were determined in capillaries of trypsin-digested Foxhound retinas from different age groups. Capillary diameters increased and number of cells per unit length of capillary wall decreased with age and distance from the optic disc. Endothelial cell frequency was constant at approximately 79% of the total cells in capillary walls in all areas measured. Peripheral cystoid degeneration and peripheral annular and focal degeneration were found in aged dog retinas. Sclerosis of retinal arterioles was observed ophthalmoscopically, histologically, and in trypsin-digested retinas from aged dogs. The significance of this change in relation to the peripheral retinal degeneration is undetermined. It is proposed that thickening of basement membranes observed in peripheral capillaries of retinas causes chronic, low-level hypoxia leading to peripheral retinal degeneration in aged retinas.     

Freeze-fracture and lanthanum studies of the retinal microvasculature in diabetic rats.

To see whether or not blood-retinal barrier breakdown during diabetes was associated with breakdown of the endothelial cell tight junctions or with other membrane alterations in the cells comprising the wall of the retinal microvasculature, streptozotocin-induced diabetic rat retinas were studied using lanthanum tracer and freeze-fracture electron microscopic morphometry. This study showed that endothelial cell tight junction permeability to lanthanum and luminal surface area were normal in these diabetic rats. However, freeze-fracture morphometry showed several alterations in the diabetic retinal microvessels. First, the endothelial cell membranes had abnormally large (80-120 nm) plasmalemmal vesicles not evident in the control retinas, suggesting that membrane turnover was abnormal. Second, endothelial cell P-face membranes at the blood front contained more larger particles than those in the control rats (P less than 0.05), implying an alteration in endothelial cell luminal membrane composition. Third, endothelial cell P-face membranes in areas of close apposition with pericyte membranes showed abnormal areas of particle clearing not seen in the control animals, suggesting a change in pericyte-endothelial cell interactions. Finally, pericyte membranes facing the neural retina contained increased numbers of plasmalemmal vesicles compared with control membranes (P less than 0.05). Moreover, the association of these vesicles with collagen fibrils in the extracellular space suggested an alteration in extracellular matrix turnover.     

Age-dependent retinal capillary pericyte degeneration in galactose-fed dogs.

The galactose-fed beagle develops diabetes-like microvascular changes that are histologically and clinically similar in appearance to all stages of human diabetic retinopathy. This animal model is extremely useful for evaluating drugs for the treatment of diabetic retinopathy; however, the time required to develop the various retinal lesions (24-72 months for background to the proliferative stage) may be considered prohibitive. Retinal vascular changes begin with an initial degeneration of capillary pericytes, which has been linked to the aldose reductase catalyzed formation of galactitol. Because aldose reductase-linked sugar cataract formation is known to be age dependent, with the onset and severity of cataract higher in younger diabetic and galactose-fed animals, retinal capillary changes in the eyes of initially 2- versus 9-month-old beagles fed a diet containing 30% galactose were compared. Eyes were enucleated after 36 months of galactose feeding, the intact retinal capillaries were isolated by trypsin digestion, and defined retinal regions were evaluated by computer image analysis. Nicotinamide adenine dinucleotide phosphate-dependent reductase activity, using DL-glyceraldehyde and D-xylose as substrates, was also compared in the lenses and whole retinas of eyes from the 2- and 9-month-old beagles. Significantly (P相似文献   

Galactose-induced retinal capillary basement membrane thickening: prevention by Sorbinil

Normotensive and spontaneously hypertensive rats fed a 30% galactose diet until 15-21 months of age developed significant (P less than 0.05) retinal capillary basement membrane thickening, compared with animals fed a standard test diet. Animals on the high galactose diet containing 250 mg/kg of the aldose reductase inhibitor, Sorbinil, did not develop basement membrane thickening. No cytologic abnormalities of pericytes or endothelial cells were noted, and pericyte:endothelial cell nuclear ratios did not differ in the various experimental groups. This model reproduces a characteristic lesion of diabetes mellitus in non-diabetic animals, and should facilitate study of the biochemical mechanisms of basement membrane thickening.     

HRP/trypsin technique for studies of the retinal vasculature

The HRP/trypsin technique is a new histologic method for the light microscopic study of the retinal blood vessels. A two-stage procedure, the first step results in a retinal whole amount preparation which permits visualization of the three-dimensional architecture of perfused vessels and their relationship within the retina. This allows analysis of gross vessel morphology and differentiation of deep and superficial vascular beds. The second step involves digesting the whole mount with trypsin and staining with hematoxylin. This permits detailed evaluation of the density of retinal capillary endothelial cells and pericytes, recognition of basement membrane ghosts, microaneurysms, and other intraretinal microvascular abnormalities, with intravascular horseradish peroxidase as a perfusion marker. This technique has been employed with success in studies of the retinal vasculature in both normal and RCS rat retinas, and in a monkey model of branch retinal vein occlusion.     

Fenestrated subendothelial basement membranes in human retinal capillaries

A correlated TEM and SEM study of human retinal capillaries and their associated basement membranes (BMs) was carried out. Control tissues show that these vessels are comprised of a continuous layer of endothelial cells separated from overlying intramural pericytes by a discontinuous subendothelial BM (EBM) which accommodates endothelial cell-pericyte (periendothelial) junctions. Three types of junctions exist, including: (1) "peg-and-socket" arrangements where cytoplasmic processes of the two cell layers interdigitate; (2) adhering plaques similar to desmosomes; and (3) cell/cell contacts where adjacent cell membranes appear to fuse or remain separated by a approximately 2 nm space. Following detergent solubilization, acellular retinal capillaries maintain their cylindrical histoarchitectures and all BM components are imaged by TEM and SEM. Topographical (SEM) studies of cryofractured samples show EBM surfaces with numerous (approximately 1.5/microns 2) oval fenestrations (100-450 nm diameter) that correlate well with EBM discontinuities occupied by periendothelial junctions in control tissues. It seems possible that these structures may play an important role in diabetic retinal neovascularization where pericytes are known to degenerate selectively. In this condition, preformed EBM deficiencies could facilitate endothelial cell migration and sprout formation, leading ultimately to the sequelae of proliferative diabetic retinopathy.     

Effects of aldose reductase inhibitor on retinal microangiopathy in streptozotocin-diabetic rats

Wistar strain rats were made diabetic by injection of streptozotocin and divided into three groups fed on different diets: conventional solid foods, a fructose-rich diet and a fructose-rich diet mixed with ONO 2235, an aldose reductase inhibitor. The retinas of these rats were examined in flat mount preparations after trypsinization. Microvascular changes such as capillary tortuosity, microaneurysms, pericyte loss, that are typical of diabetic retinal microangiopathy, were seen most frequently in the rats fed on the fructose-rich diet. The rats fed on the fructose-rich diet with ONO 2235 showed much less vascular change than the diabetic rats fed on the conventional food. Electron microscopy of the retina revealed localized thickening of the basement membrane of the retinal capillaries, and this was most frequent in the fructose-fed rats. However, in rats fed on fructose with ONO 2235 the changes of the basement membrane were slight. It was concluded that the aldose reductase inhibitor, ONO 2235, prevented development of diabetic microangiopathy, probably by suppressing the enzymatic activation of aldose reductase in the retina.     

Aldose reductase activity in retinal and cerebral microvessels and cultured vascular cells

Isolated microvessels (primarily capillaries) from bovine retina and cerebral cortex, as well as cultured bovine retinal capillary pericytes and porcine and canine retinal capillary endothelial cells contain apparent aldose reductase activity. This conclusion is based on the ability of these cultured cells and vessel fragments to reduce DL-glyceraldehyde in preference to D-glucuronate at low (0.1 mM) substrate concentrations, in the presence of NADPH, and in the accumulation of high levels of sorbitol or galactitol when retinal pericytes and endothelial cells are cultured in media enriched in glucose or galactose. The quantitative similarities of these activities in bovine retinal and cerebral microvessels, as well as the quantitatively similar ability of these two sets of microvessels to oxidize 14C-labeled glucose with the label either in the C-1 or the C-6 position, suggests that aldose reductase may not be a major causal factor in diabetic retinopathy. This conclusion is suggested because, while these metabolic activities are similar in bovine retinal and cerebral microvessels, only the retinal microvasculature suffers major anatomic and functional damage in diabetes. This conclusion must be viewed with caution, however, because other metabolic pathways that we have not investigated may be altered by an excess of sugar alcohols, and be present in differing activities in retinal and cerebral microvessels; species differences may exist; and similar experiments have not been conducted using human microvessels.     

糖尿病早期大鼠视网膜毛细血管细胞凋亡研究

目的：研究早期糖尿病视网膜病变大鼠视网膜毛细血管细胞凋亡及其特征。方法：腹腔内注射链脲佐菌素诱导糖尿病大鼠模型。应用PAS染色、末端脱氧核苷酸转移酶介导的dUTP缺口标记（TUNEL）法标记和透镜观察进行研究。结果：在糖尿病3月和6月,可见周细胞和内皮细胞鬼影;周细胞和内皮细胞核异染色质聚集靠边并随病程进展而加重;被TUNEL法标记的细胞核染色质分布不均,表现为环形核、新月形核等。结论：早期糖尿病视网膜病变大鼠视网膜毛细血管周细胞丧失的性质为细胞凋亡,内皮细胞亦存在凋亡。     

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 mid-retina of nasal and temporal quadrants of retina were used to explore regional differences in expression of Glut1, PKCbeta, 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 PKCbeta 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.