Survival of allografted pancreatic islets in the subretinal space in rats.

The purpose of this study was to determine whether the subretinal space would provide immune privilege for pancreatic islet transplantation. Islets from outbred Sprague Dawley (SD) rats were isolated by collagenase digestion, and about 200 handpicked islets were transplanted into the subretinal space of SD rats. Similar grafts were transplanted into the subconjunctival space of SD rats as controls. Transplanted eyes were enucleated after 2 to 60 days, fixed and embedded in paraffin for immunoperoxidase staining of insulin, glucagon, and CD8+ lymphocytes. Clinical examination of rat eyes revealed minimal or no inflammation in the anterior chamber or vitreous at any time point. Fifteen of 19 subretinal allografts survived up to 60 days. Few CD8+ lymphocytes were present in the subretinal grafts and the endocrine cells stained intensely for insulin and glucagon at all time points. In contrast, CD8+ lymphocytes were present in subconjunctival grafts in rats by day 14 and all grafts were destroyed by day 21. These results suggest that the subretinal space provides immune privilege for islet allotransplantation by preventing massive lymphocyte infiltration.

Antithrombotic effect of ticlopidine in an experimental model of retinal vein occlusion.

PURPOSE: Ticlopidine inhibits adenosine diphosphate (ADP)-induced platelet aggregation and may be effective in patients with retinal vein occlusions (RVO). This study tests the efficacy of ticlopidine in an animal model of RVO. METHODS: Rose bengal-mediated argon laser photothrombosis of retinal veins was created in rabbits pretreated with oral ticlopidine, aspirin, or saline. The number of laser spots necessary to produce a partial or complete RVO was recorded and tabulated. RESULTS: Pretreatment with ticlopidine significantly increased the number of laser spots needed to produce a partial (P =.02), or a complete (P =.002) RVO as compared to the control group. Pretreatment with ticlopidine significantly increased the number of laser spots needed to produce a partial RVO (P =.02). Pretreatment with aspirin significantly increased the number of laser spots needed to produce a complete RVO (P =.002). CONCLUSION: Ticlopidine may be a useful antiplatelet agent for the treatment of patients with RVO. Patients treated with ticlopidine should be monitored for the possible development of hematologic disorders.

Photocrosslinkable polysaccharides for in situ hydrogel formation.

In situ photopolymerization is an exciting new technique for tissue engineering. Two photocrosslinkable polysaccharides composed of alginate and hyaluronan are described that upon photolysis form soft, flexible, and viscoelastic hydrogels. The degree of methacrylate modification and thus covalent affects mechanical properties such as swelling, compression, and creep compliance. Significant swelling is observed in aqueous solution; these hydrogels can swell up to 14 times their dry weight. Both hydrogels exhibit low phase angles and (G*) values indicative of viscoelastic materials. The hyaluronan based hydrogel is stronger and more resilient than the corresponding alginate gel. SEM and AFM studies on both hydrogels show smooth and uniform surfaces at the macroscopic level with salient features observed only on the nanometer scale. Rapid polymerization by an optical trigger allows for controlled in situ photopolymerization in a minimally invasive manner, indicating that these hydrogels are relevant for biomedical applications such as sealing wounds and reconstructing soft tissues.

Electron immunocytochemical analysis of posterior hyaloid associated with diabetic macular edema.

BACKGROUND: Tangential traction in the macula from a thickened posterior hyaloid of the vitreous has been implicated as a cause of diffuse diabetic macular edema. Vitrectomy with peeling of the posterior hyaloid has been shown to reduce retinovascular leakage and improve vision in select patients. We report a clinicopathologic correlation using electron microscopy and electron immunocytochemistry to characterize the membrane infiltrating the posterior hyaloid in two such patients. METHODS: Two patients presented with vision loss associated with diffuse diabetic macular edema and an attached, thickened, and taut posterior hyaloid. The patients underwent vitrectomy with peeling of the posterior hyaloid. The premacular posterior hyaloid specimens then were analyzed by electron microscopy with immunocytochemical staining for cytokeratin and glial fibrillary acidic protein. RESULTS: Both posterior hyaloid specimens contained collagen and a large cellular component. Immunogold labeling showed cells positive for glial fibrillary acidic protein or cytokeratin. With double labeling, no cells expressed both proteins simultaneously. Clinically, both patients had vision improvement and macular edema resolution after surgery. CONCLUSIONS: The thickened, taut posterior hyaloid observed in our patients with diabetic macular edema contained cells of glial and epithelial origin. This cellular infiltration may contribute to abnormal vitreomacular adherence and could play a role in the pathogenesis of macular edema in some patients with diabetes.

Increased nonenzymatically glycosylated proteins in the vitreous humor of diabetic animals.

One of the earliest pathologic changes of diabetes mellitus is increased nonenzymatic glycosylation (i.e., glycation) of proteins, which results in abnormal aggregation of collagen fibrils and production of superoxide radicals. These abnormalities may be responsible for the precocious senescence of connective tissue associated with the disease. We sought to determine whether glycation is increased in the vitreous humor of short-term diabetic cats (6 months' duration) and rabbits (2 months' duration), using a nitroblue tetrazolium colorimetric assay for fructosamine. Vitreous protein fructosamine concentration was significantly higher in diabetic cats and rabbits, compared with that in control (nondiabetic) animals. These results indicate that glycation is increased in the vitreous humor of short-term diabetic animals, and therefore may be one of the initial triggers for clinically apparent diabetic retinopathy.

Retinal hypoxia in long-term diabetic cats.

PURPOSE: To determine whether the retina is hypoxic in early stages of diabetic retinopathy in cats and to correlate intraretinal PO2 with fluorescein angiographic and histologic alterations. METHODS: Intraretinal PO2 was measured with microelectrodes in three cats with long-standing diabetes (>6 years) that had been followed with fluorescein angiographs every 6 months. Average PO2 in the inner vascularized half of the retina was compared with similar measurements in 21 control animals. Photoreceptor oxygen consumption was also compared. The retinal vascular endothelium of the diabetic animals was stained for ADPase activity in flatmounts, and transverse sections were used to visualize microscopic alterations in vascular structure. RESULTS: PO2 in the inner half of the retina was abnormally low in the diabetic cats, 7.7+/-5.2 mm Hg (35 penetrations in 3 cats) versus 16.4+/-9.3 mm Hg in normal cats (85 penetrations in 21 cats) (P << 0.001). Oxygenation was almost normal in some regions of the diabetic retinas, but little evidence of oxygen supply from the retinal circulation was observed in other regions. Inner retinal hypoxia was present in areas with no detectable capillary dropout in fluorescein angiograms or flatmounts. The worst changes histologically were microaneurysms, leukocyte and platelet plugging of aneurysms and venules, and degenerating endothelial cells in capillary walls. These histologic abnormalities were confined to small regions, some of which could be positively correlated with markedly abnormal PO2 profiles. Photoreceptor oxygen utilization was not affected in two diabetic cats, but was below normal in one animal in which choroidal PO2 was low. CONCLUSIONS: This is the first direct demonstration of retinal hypoxia in early diabetic retinopathy, before capillary dropout was evident clinically. Hypoxia was correlated with endothelial cell death, leukocyte plugging of vessels, and microaneurysms.

Aminoguanidine inhibits reactive oxygen species formation, lipid peroxidation, and oxidant-induced apoptosis.

Aminoguanidine (AG) treatment, like nerve growth factor (NGF) treatment, prevents diabetes-induced apoptosis of retinal Müller cells in the rat eye, but the mechanism involved is unknown. In this study, the effects of preincubation with AG on oxidant-induced apoptosis, oxidant-induced intracellular reactive oxygen species (ROS) production, and lipid peroxidation were determined in rat retinal Müller cells and compared with the effects of NGF, a protein that protects neuronal cells from oxidative stress. The effect of AG on rabbit vitreous lipid peroxide levels was also determined. After exposure to increasing concentrations of H2O2, there was a corresponding increase in the percentage of apoptotic Müller cells. Preincubation with AG for 48 h completely inhibited oxidant-induced apoptosis in response to 10 micromol/l H2O2 (+AG 0 vs. 10 micromol/l, NS), and reduced the percentage of apoptotic cells in response to 50 micromol/l H2O2 by 50% (+AG vs. -AG, P < 0.01). Longer preincubation did not increase the antiapoptotic effect of AG. The effect of AG was dose-dependent. Similar results were obtained after preincubation with NGF. Both AG and NGF preincubation prevented the twofold increase in oxidant-induced lipid peroxides. The fivefold increase in oxidant-induced ROS production was decreased 100% by NGF, but only 61% by AG preincubation. The twofold increase in vitreous lipid peroxide level in diabetic rabbits was completely prevented by AG treatment. AG reduced H2O2-induced benzoate hydroxylation in a dose-dependent manner. Intracellular glutathione content was unchanged. These data demonstrate that AG can act as an antioxidant in vivo, quenching hydroxyl radicals and lipid peroxidation in cells and tissues and preventing oxidant-induced apoptosis.

In vivo photoactivation of caged-thrombin.

Aberrant ocular neovascularization is a major cause of blindness in the world. Abnormal blood vessels in the eye may produce corneal opacification, corneal transplant rejection, neovascular glaucoma, vitreous hemorrhage, traction retinal detachment, and subretinal scars from choroidal neovascular membranes (1-5). Light-induced clotting of blood within these abnormal vessels could provide a novel method for the ablation of deleterious neovascularization. Thrombin is a serine proteinase that participates in the final stages of the coagulation cascade. An inhibitor of thrombin, p-Amidinophenyl-(E)-4-diethylamino-2-hydroxy-alpha-methylcinnamate hydrochloride, MeCINN (1), covalently attaches to the active site serine hydroxyl, inhibiting or caging, the enzyme. Photolysis of the caged-thrombin in vitro causes a trans-cis isomerization of MeCINN which leads to regeneration of active enzyme and cleaving of fibrinogen into fibrin (6). Using a rabbit model of corneal neovascularization, we found that light at 366 nm safely and effectively photoactivates intravenous caged-thrombin and produces localized thrombosis in vivo. These results suggest that intravascular photoactivation of caged-thrombin could be used to occlude abnormal blood vessels in the human eye.

Quantification of erythrocyte flow in the choroid of the albino rat.

Choroidal blood flow is one of the highest in the body on a global volume basis. Little is known, however, about flow through individual vessels, which has important consequences for ocular blood delivery and oxygen transport. The purpose of this study was to use a new epifluorescent technique to view, record, and quantify erythrocyte (RBC) flow in individual rat choroidal vessels through the intact sclera. With the Sprague-Dawley rats under urethan anesthesia, rhodamine-labeled liposomes were injected intravenously and served as a plasma marker. Rat RBC were labeled ex vivo with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and then infused intravenously. The flow of the fluorescent RBC through 69 choroidal vessels with diameters between 12 and 52 microm in six rats was recorded on videotape, and the images were used to determine vascular diameter, RBC flux and velocity, and microvessel hematocrit (Hct). RBC flux and RBC velocity were positively correlated with vessel diameter (r = 0.67, P < 0.001 and r = 0.29, P = 0.016, respectively). Microvascular Hct ranged between 4 and 32% (8 and 67% of systemic Hct) and was negatively correlated with diameter (r = -0.28, P = 0.018). The relationships of RBC flux and RBC velocity with vessel diameter were the same as found in other tissues. However, in other vascular beds, microvascular Hct and diameter are positively correlated. Because microvascular Hct is a determinant of relative viscosity and oxygen delivery, this relatively high Hct in small choroidal vessels could have significant implications for local blood flow and oxygen transport.

Alterations in stimulus-induced integrin expression in peripheral blood neutrophils of patients with diabetic retinopathy.

Adhesion molecule expression on peripheral blood leukocytes from diabetic patients with severe retinopathy and age-matched control subjects was assessed. Expression of CD11b, CD18, and L-selectin was measured on granulocytes and lymphocytes in whole blood within 1 hour of blood collection. Adhesion molecule expression was determined at 4 degrees C, 37 degrees C, and after stimulation with one of the chemotactic peptides, N-formyl-methionyl-leucyl-phenyl-alanine or beta-phorbol 12-myristate 13-acetate. There were no differences between diabetics and controls in CD11b expression in neutrophils at 4 degrees C, 37 degrees C, or after N-formyl-methionyl-leucyl-phenylalanine stimulation. However, during stimulation with beta-phorbol 12-myristate 13-acetate, the increase in CD11b expression in neutrophils from patients with diabetes was significantly less than in controls. In neutrophils, there was no difference between the control and diabetic participants in CD18 expression at 4 degrees C, but after warming the cells to 37 degrees C, the expression was significantly higher in patients with diabetes. The difference became even more apparent after N-formyl-methionyl-leucyl-phenyl-alanine stimulation. The increase in CD18 expression after beta-phorbol 12-myristate 13-acetate stimulation of neutrophils was similar in control and diabetic participants. There was no difference in L-selectin expression in neutrophils under any conditions. There was no difference in adhesion molecule expression on lymphocytes under similar conditions. In summary, these observations indicate that integrin expression of neutrophils from patients with diabetes and retinopathy is altered after stimulation with neutrophil-activating agents. The changes were integrin-, stimulus-, and cell-specific, which suggests that the signal transduction mechanisms may be altered in diabetic neutrophils. These alterations may be responsible for abnormal leukocyte/endothelial interactions and microvascular complications in diabetic retinopathy.

Decreased deformability of polymorphonuclear leukocytes in diabetic cats.

OBJECTIVE: Polymorphonuclear leukocytes (PMN) have been found to be less deformable in humans with non-insulin-dependent diabetes. It has therefore been hypothesized that white blood cells (WBC) may affect the development of diabetic microangiopathy. This study was designed to determine whether PMN or small and large lymphocytes were less deformable in a large animal model of diabetes-chronically hyperglycemic pancreatectomized cats. METHODS: Venous blood was withdrawn from 13 normal and 14 diabetic cats. The diabetic cats had been kept in poor metabolic control since their pancreatectomy (7-113 months before this study). Blood cell counts, hemoglobin concentration, hematocrit, erythrocyte volume, and WBC differential counts were obtained from the blood samples. Purified mononuclear WBC and PMN fractions were obtained by separating the blood on a discontinuous Percoll gradient. The deformability of each cell fraction was determined using a Cell Transit Analyzer (ABX, Montpellier, France) that measures the transit time of cells through 7.5-microns pores. By varying the sampling rate of the CTA and the pressure difference across the filter, the subpopulations of the mononuclear fractions (small and large lymphocytes) could be identified and each subpopulation analyzed separately. RESULTS: Median transit times for the PMN and small lymphocytes were significantly greater for the diabetic cats, but no difference was found in the large lymphocyte fractions. CONCLUSIONS: These results are in accordance with the finding that PMN are less deformable in humans with diabetes. We also showed that the small lymphocytes from diabetic cats have prolonged transit times. The results suggest that PMN may contribute to the development of microangiopathies like diabetic retinopathy. Diabetic cats may prove useful for testing potential therapies to improve WBC deformability.

Role of neutrophils in breakdown of the blood-retinal barrier following intravitreal injection of platelet-activating factor.

Breakdown of the blood-retinal barrier occurs in inflammatory conditions and in ischemic retinal diseases such as diabetic retinopathy. Platelet-activating factor (PAF) is a potent inflammatory mediator which increases vascular permeability. The purpose of this study was to determine if intravitreally-injected PAF would cause breakdown of the blood-retinal barrier and, if so, by what mechanism. Fluorescein angiography was performed before and at 0.5, 1, 2, 3 and 4 hr after PAF injection into the vitreous cavity of rabbit eyes and the eyes were enucleated immediately for light and electron microscopy. Slow flowing thrombi were observed in all PAF-injected eyes. Complete vascular occlusion was observed in 10 of 16 eyes after 3 and 4 hr. There was no fluorescein leakage in any of eyes before or at 0.5 or 1 hr after PAF injection. Fourteen of 20 eyes had fluorescein leakage at 2, 3 and 4 hr after PAF injection. The extent of fluorescein leakage correlated with the degree of polymorphonuclear leukocyte (PMN) margination, disruption of the endothelial cell layer, infiltration into vascular walls and migration into the vitreous cavity. PMNs appeared to migrate by both intercellular and transcellular routes across the endothelium. Pretreatment of rabbits with a PAF inhibitor, BN52021, prevented most of the abnormal findings.

Growth inhibition of human Tenon's capsule fibroblasts and rabbit dermal fibroblasts with non-carcinogenic N-alkylated anthracyclines.

Bleb fibrosis after glaucoma filtering surgery and proliferative vitreoretinopathy after retinal detachment surgery are complications caused by proliferation of fibroblasts or fibroblastlike cells. The anthracycline daunomycin (DNM) has been used for treatment of those proliferative processes in humans. However, complications such as conjunctival necrosis and corneal or scleral ulcerations have been reported after administration of DNM to glaucoma patients. Intravitreal administration of DNM in rabbit eyes resulted in morphological and functional retinal damage. DNM also has the undesired general effect of carcinogenicity. N-Alkylation of the aminosugar moiety of DNM results in reduction or loss of carcinogenicity. We evaluated the inhibitory effect of the new non-carcinogenic N-alkylated analogues aclacinomycin A (ACA), N,N-dimethyladriamycin (AD280), and N-trifluoroacetyladriamycin-14-O-hemiadipate (AD143) on the growth of cultured human Tenon's capsule fibroblasts and rabbit dermal fibroblasts. Using DNM as a positive control, we conducted proliferation assays that demonstrated that ACA and AD280 inhibited fibroblast growth as effectively as DNM. AD143 was less efficacious. The magnitude of cellular growth inhibition was concentration dependent for all drugs tested. Extension of exposure times resulted in increased rates of cell death. Our in vitro studies suggest that further evaluation of ACA and AD280 should be carried out in animal models of ocular proliferative disorders.

Increased adhesion of neutrophils to retinal vascular endothelial cells exposed to hyperosmolarity.

Recent studies suggest that leukocytes may contribute to capillary occlusion and endothelial cell injury in diabetic retinopathy. The present study is an attempt to determine whether high glucose concentration/hyperosmolar conditions would increase neutrophil adhesion to retinal endothelial cell monolayers. Confluent monolayers of bovine retinal endothelial cells were incubated for 24 hr in 96-well microtiter plates in 5.5, 20, 50 or 100 mM glucose using 20, 50 or 100 mM mannitol as osmotic controls. After 24 hr the cells were observed by phase microscopy, washed, and then incubated with isolated human neutrophils for 20 min. Non-adherent neutrophils were washed from the monolayers, and the number of adherent neutrophils was determined using an Elisa assay for neutrophil elastase. Adhesion of neutrophils to confluent monolayers of human umbilical vein and bovine aortic endothelial cells at all levels of glucose were assayed in a similar fashion for comparison. Neutrophil adherence to bovine retinal endothelial cells was significantly increased (P < 0.05, n = 8; paired t-test) at all elevated glucose concentrations compared with adherence at control (5.5 mM) glucose concentration. The effect was also concentration dependent with 20, 50 and 100 mM glucose resulting in 35, 57 and 70% increases respectively over controls. However, mannitol at equal concentrations produced similar increases in neutrophil adherence, indicating that the increases in adhesion caused by elevated glucose concentration were due to hyperosmolarity and not some special effect of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukocyte-capillary plugging and network resistance are increased in skeletal muscle of rats with streptozotocin-induced hyperglycemia.

Although diabetic retinopathy is a leading cause of blindness, the mechanisms underlying the disorder remain unresolved. Recent studies have reported both an increase in viscosity and a decrease in filterability of blood in diabetes, as well as increased activation of monocytes and granulocytes. These rheological effects have been implicated in capillary closure which is an early pathological change in diabetic retinopathy. The objective of this study was to quantitatively measure in vivo for the first time the resistance increase in capillary networks due to leukocyte-capillary plugging during experimental diabetes. Intravital measurements of plugging durations and frequencies were made throughout capillary networks in the spinotrapezius muscle of anesthetized rats subject to streptozotocin (STZ) induced hyperglycemia. These data were used to estimate the increase in microvascular flow resistance due to leukocyte plugging. The increase averaged 13.0% which is significantly different (p < 0.05) from the 1.1% observed in previous experiments with normal rats. Although the total white cell count was normal, the diabetic animals exhibited a significantly increased percentage of monocytes. A small but significant decrease in capillary diameter in the diabetic animals was also observed. Thus, leukocytes have a significant impact on microvascular hemodynamics in diabetic animals, and leukocyte-capillary plugging may be an important mechanism of capillary closure and subsequent microvascular dysfunction in diabetic retinopathy.

Neutrophils plug capillaries in acute experimental retinal ischemia.

Capillary plugging by neutrophils appears to be the mechanism responsible for the no reflow phenomenon following experimental ischemia in many tissues. The purpose of this study was to determine if neutrophils plug capillaries in experimental retinal ischemia. Unilateral retinal ischemia was produced in albino rats by focally exposing three adjacent retinal arterioles to argon blue-green laser light at 100 mW. Total occlusion was achieved in at least two vessels in each eye. The animals were euthanized at 3, 6, 8, or 24 hr following laser treatment. Nonlasered eyes of 17 animals served as controls. Trypsin digests were prepared of the retinal vasculature following formalin fixation. The total number of neutrophils present in the capillaries of each retina was counted using a light microscope and expressed as the number of cells per retina. Other eyes were fixed in glutaraldehyde:paraformaldehyde and the retinal tissue prepared for light and electron microscopy. The mean number of neutrophils in capillaries of retinas subjected to laser treatment was significantly higher than that in untreated control retinas at all time periods studied and increased with time from 3 to 6 hr. Intact as well as degranulated PMNs were present in the capillaries. The presence of large numbers of neutrophils plugging capillaries and their increased number with duration of ischemia supports the hypothesis that they contribute to capillary nonperfusion following acute retinal ischemia.

Granulocyte deformability and F-actin content: relationship to diabetes mellitus and retinopathy.

Capillary closure in diabetic retinopathy may be initiated by lumenal occlusion by granulocytes. To determine whether subjects with diabetes mellitus have less deformable granulocytes than healthy subjects, granulocyte deformability was measured by mean entry time into a model capillary system in 16 diabetic-nondiabetic pairs. Granulocyte F-(filamentous) actin content between groups was compared, under basal conditions and after cellular stimulation. The relationship of granulocyte deformability to several diabetes-related variables was examined. Diabetic granulocytes were only 9% +/- 22% less deformable than normal granulocytes (p = 0.16). Deformability was increased in subjects with retinopathy and those with the worst glycemic control (r = 0.61, p = 0.026); both findings were in the opposite direction from that predicted. Basal and stimulated granulocyte F-actin content did not differ between the two groups (p > 0.2 for all assays). Although granulocytes may be important in the pathogenesis of diabetic retinopathy, granulocyte deformability (measured by mean entry time) and F-actin content are not significantly different between healthy patients and those with diabetes.

Pentoxifylline increases retinal capillary blood flow velocity in patients with diabetes.

OBJECTIVE: The purpose of this study was to determine whether oral pentoxifylline would improve retinal microvascular hemodynamics and blood rheology in patients with diabetes. DESIGN: Patients were enrolled in a double-masked, placebo-controlled trial of pentoxifylline at 2000 mg/d. Retinal capillary blood velocity and leukocyte density, filterability, viscosity, and fibrinogen content were measured by the blue-field entoptic phenomenon simulation, filtration, rotational viscosimetry, and heat precipitation techniques, respectively, before, during, and after drug therapy. RESULTS: Treatment with pentoxifylline resulted in a 23.2%, 26.8%, and 37.8% increase in capillary blood flow velocity at 1, 2, and 3 months of therapy, respectively, with a return to pretreatment baseline levels 1 month after cessation of therapy. There were no apparent effects on the remaining variables during treatment. CONCLUSION: Pentoxifylline increases retinal capillary blood flow velocity in patients with diabetes.