We investigated a method for isolating sufficient feline islets of Langerhans to restore normoglycaemia following transplantation into the subretinal space of pancreatectomized cats. Collagenase digestate of feline pancreas was maintained in serum-free tissue culture medium for 1–9 days. Viability of islet-like cell clusters (ICC) was assessed with ethidium bromide and fluorescein diacetate staining; cell types were identified immunohistochemically. After nine days, the ICC were transplanted. We estimated viable ICC in tissue culture at nine days as 3800 ± 2000 (mean ± SD) per pancreas. While numbers of β cells decreased over time in culture, ductal cells increased. Bromodeoxyuridine labelling showed no proliferation of β cells but extensive proliferation of ductal cells. Subretinal transplants of cultured ICC in the diabetic cats maintained normoglycaemia for up to 12 days, while they provoked massive lymphocyte infiltration indicating rejection. Islet transplantation into the feline subretinal space temporarily restored normoglycaemia. Our current method of culture achieved sufficient reduction of acinar cells but an insufficient yield of insulin-producing cells.

Survival of pancreatic islet cells transplanted into the subretinal space in rat eyes was studied in relation with apoptosis of infiltrating lymphocytes. Islets isolated from inbred DA rats were transplanted into the subretinal space of fully MHC-mismatched Lewis rats. Syngeneic subretinal transplants and allogeneic subretinal transplants from Sprague-Dawley (SD) rats were performed as controls. From 7 to 60 days after transplantation, immunoperoxidase staining for insulin, glucagon, CD4, CD8, and interleukin (IL)-2, and evaluation of apoptosis using nick-end labeling (TUNEL) were performed. Fully MHC-mismatched grafts were rejected by day 14, showing massive infiltration by CD4+, CD8+, and IL-2+ lymphocytes. Syngeneic transplants and allografts in SD rats survived up to 60 days without appreciable lymphocytic infiltration. Endocrine cells were the major component of surviving transplanted tissue. No difference in distribution of TUNEL+ cells was seen between syngeneic transplants and subretinal allografts in SD rats on day 60. A few TUNEL+ CD8+ lymphocytes were found in rejected MHC-mismatched transplants, but apoptotic lymphocytes were not seen in surviving or syngeneic grafts. Pancreatic islet survival in the subretinal space is MHC dependent. Accumulation of infiltrating lymphocytes may be suppressed by a modified immune response.

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.

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.

Diabetes mellitus is a common endocrinopathy in cats that is associated with pancreatic islets lesions. Research on isolated islets contributed to the understanding of the pathophysiology of human diabetes. Therefore, by improving the existing methods of isolation in cats, we aimed at increasing islet yield, purity and viability of feline isolated islets.Islet isolation was accomplished by pancreas perfusion with 80 ml of Collagenase type IV through the pancreatic duct at the site of the major papilla. The enzymatic digestion was combined with mechanical disruption and controlled by dithizone staining. Purification was performed by filtration and handpicking. Purified islets were plated on extracellular matrix pre-coated plates and cultured for 48 h.Feline islets with a high degree of viability and purity were isolated and cultured for the first time. Although the percentage of islet free from the acinar tissue relative to the total number of isolated islets was low compared to other species, the suggested protocol represents a promising progress in the procedure of islet isolation in cats.

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.

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.

ABSTRACTObjective: 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‐μm 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.

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.

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.

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.

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)