The aim of this study was to characterize the microvascular flow abnormalities and oxygenation changes that are present following six months of hyperglycemia in the diabetic Ins2(Akita) mouse. Previous studies have shown decreased retinal blood flow in the first several weeks of hyperglycemia in rodents, similar to the decreases seen in the early stages of human diabetes. However, whether this alteration in the mouse retina continues beyond the initial weeks of diabetes has yet to be determined, as are the potential consequences of the decreased flow on retinal oxygenation. In this study, male Ins2(Akita) and age-matched C57BL/6 (non-diabetic) mice were maintained for a period of six months, at which time intravital microscopy was used to measure retinal blood vessel diameters, blood cell velocity, vascular wall shear rates, blood flow rates, and transient capillary occlusions. In addition, the presence of hypoxia was assessed using the oxygen-sensitive probe pimonidazole. The diabetic retinal microvasculature displayed decreases in red blood cell velocity (30%, p < 0.001), shear rate (25%, p < 0.01), and flow rate (40%, p < 0.001). Moreover, transient capillary stoppages in flow were observed in the diabetic mice, but rarely in the non-diabetic mice. However, no alterations were observed in retinal hypoxia as determined by a pimonidazole assay, suggesting the possibility that the decreases seen in retinal blood flow may be dictated by a decrease in retinal oxygen utilization.

Objective In the current study of murine colitis, the potential roles of thromboxane and the thromboxane-prostanoid (TP) receptor were investigated, in as much as thromboxane signaling has been implicated in human inflammatory bowel disease.Methods Colitis was induced in C57BL/6 mice via ingestion of dextran sodium sulfate (DSS), with or without co-administration of the thromboxane synthase inhibitor ozagrel (25 mg/kg/day) or the TP receptor antagonist vapiprost (2.5 mg/kg/day).Results Immunohistochemistry of colonic tissue demonstrated a DSS-induced increase in TP receptor expression, but not of thromboxane synthase. Moreover, tissue levels of the metabolite thromboxane B-2 were unchanged by DSS. Vapiprost, but not ozagrel, partially attenuated histologic signs of inflammation induced by DSS, with vapiprost allowing a smaller increase in colon weight per unit length than ozagrel. Vapiprost also tended to attenuate DSS-induced alterations in intestinal transit.Conclusions In summary, TP receptor antagonism was more effective than thromboxane synthase inhibition in alleviating DSS-induced colitis in mice.

AIMS:The purpose of this study was to investigate the influence of glutathione on the electroretinogram (ERG) in diabetic and non-diabetic rats.MATERIALS AND METHODS:Streptozotocin (STZ: 60 mg/kg) was injected into male RCC Wistar rats to induce hyperglycemia, with buffer instead of STZ injected into age-matched non-diabetic controls. After 8 weeks, ERG measurements were obtained at seven different scotopic flash intensities on the two groups of anesthetized, dark-adapted rats (controls, STZ). Following ERG measurements, eyes were enucleated for measurements of retinal/vitreous GSH and glutathione disulfide (GSSG).RESULTS:Diabetic rats produced delayed b-wave ERG signals (increased implicit times), but had normal a-wave and b-wave amplitudes, a-wave implicit times, and oscillatory potentials. No differences were observed in retinal GSH or GSSG between controls and diabetics; however, correlations between GSH and all ERG parameters (with the exception of b-wave implicit times) were noted, and were not significantly altered by the presence of hyperglycemia.CONCLUSIONS:GSH is likely to play an important role in retinal function as assessed by the ERG, with this role not substantially altered in rats diabetic for 8 weeks.

Hypoxia and the associated hypoxia-inducible factors (HIFs) may be influential in the progression of diabetic retinopathy. However, little is known of the extent of hypoxia and the levels of HIFs early in the progression of the disease. In the current study, we injected the oxygen-dependent probe pimonidazole (Hypoxyprobe™-1) into diabetic rats, and also performed immunohistochemistry to determine the retinal levels of HIF-1α and HIF-2α. The rats were made diabetic using a single injection of streptozotocin (STZ; 60 mg/kg), with vehicle-injected rats used as non-diabetic controls. The measurements of hypoxia and HIF levels were obtained three weeks following STZ injection, at which time we have previously found significant decreases in retinal blood flow in the same model. In the current experiments, no increases in either HIF-1α or hypoxia were observed in the diabetic rats (compared with controls), and there was even a tendency for hypoxia levels to be decreased (tissue more highly oxygenated). However, we did observe an increase in HIF-2α in the retinas of the diabetic rats. Therefore, we conclude that early diabetes-induced increases in HIF-2α occur independently of hypoxia.

Decreases in retinal blood flow in diabetics could render the retina hypoxic. In mouse and rat models of diabetes, a decrease in retinal blood flow occurs early, within 3-4 weeks of the induction of hyperglycemia, although information is scarce on whether this early decrease in flow induces hypoxia. The purpose of the current study was to determine whether hypoxia-inducible factor (HIF) levels increase following 4 and/or 12 weeks of hyperglycemia in streptozotocin (SIZ)-injected mouse (C57BL/6) and rat (Wistar) retinas. Additionally, retinal tissue hypoxia was measured with pimonidazole following 12 weeks of hyperglycemia. These aims were accomplished via immunostaining of cross-sections from enucleated eyes. In mice, staining for HIF-1 alpha and HIF-2 alpha showed a contrasting pattern, with HIF-1 a higher in the inner retina than outer, but HIF-2a higher in the outer retina than inner. However, in rats, staining for both HIF-1 alpha and HIF-2 alpha was more intense in the inner retina. The HIF-1 alpha staining intensities and patterns were similar between diabetic animals and their non-diabetic counterparts following 4 and 12 weeks of hyperglycemia. The same was true for HIF-2 alpha except for a trend toward an increase following 12 weeks of hyperglycemia in mice. Pimonidazole staining showed significant decreases throughout all layers of the central retina and most layers of the peripheral retina of rats (but not mice), following 12 weeks of hyperglycemia. In summary, despite early decreases in flow in rats and mice, retinal HIF-1 alpha and HIF-2 alpha were not found to be increased, and the extent of hypoxia may even decrease after 12 weeks of hyperglycemia in rats. (C) 2009 Elsevier Ltd. All rights reserved.

Experimental models of the diabetic retina have suggested a pathological role for thromboxane. To date however, little information is available as to the cellular locations of retinal thromboxane synthase (TxS), or its receptor, even in non-diabetic controls. In this study, C57BL/6 mice and Wistar rats were injected with streptozotocin to induce diabetes, or with buffer for non-diabetic controls. Four weeks following the injection, eyes were enucleated and labeled for TxS and the thromboxane-prostanoid (TP) receptor. Immunofluorescent intensity was quantified in the ganglion cell plus inner plexiform layers, inner nuclear layer, outer plexiform layer, outer nuclear layer, and photoreceptor inner segment. Even in control mice and rats, all layers of the retina showed immunoreactivity for TxS and the TP receptor: however, the pattern of expression demonstrated an inverse relationship, with the highest TxS staining in the inner retina, and the highest TP receptor staining in the outer retina (more specifically, in the photoreceptor inner segment). Four weeks of hyperglycemia did not increase the retinal levels of TxS or TP receptor; however, TP receptor intensities in the outer retina of diabetic rats were highly variable (mostly high but some low), with no values from the photoreceptor inner segment in the same range as obtained from controls.