Neuroprotective effect of melatonin against hypoxia-induced retinal ganglion cell death in neonatal rats.

The purpose of this study was to determine whether melatonin treatment would mitigate retinal ganglion cell (RGC) death in the developing retina following a hypoxic insult. Lipid peroxidation (LPO), glutathione (GSH), tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) concentrations, expression of vascular endothelial growth factor receptors, Flt-1 and Flk-1, release of cytochrome c from mitochondria, and caspase-3 expression were examined in the retinas of 1-day-old rats at 3 hr to 14 days after a hypoxic exposure. The mRNA and protein expression of Flt-1 and Flk-1 and the tissue concentration of LPO, TNF-α, and IL-1ß were upregulated significantly after the hypoxic exposure, whereas the content of GSH was decreased significantly. RGC cultures also showed increased LPO and decreased GSH levels after hypoxic exposure but these effects were reversed in cells treated with melatonin. TNF-α and IL-1ß expression was specifically located on microglial cells, whereas Flt-1 and Flk-1 was limited to RGCs as confirmed by double immunofluorescence labeling. Cultures of hypoxic microglial cells treated with melatonin showed a significant reduction in the release of these cytokines as compared to untreated hypoxic cells. Hypoxia induced increase in the cytosolic cytochrome c and caspase-3 in RGCs was attenuated with melatonin treatment. The results suggest that, in hypoxic injuries, melatonin is neuroprotective to RGCs in the developing retina through its antioxidative, anti-inflammatory, and anti-apoptotic effects. Melatonin suppressed Flt-1 and Flk-1 expression in retinal blood vessels, which may result in reduced retinal vascular permeability and it also preserved mitochondrial function as shown by a reduction in cytochrome c leakage into the cytosol. The results may have therapeutic implications for the management of retinopathy of prematurity.

Enhanced dopamine D1 and D2 receptor gene expression in the hippocampus of hypoglycaemic and diabetic rats.

Hypoglycaemic coma and brain injury are potential complications of insulin therapy. Hippocampal neurons are particularly vulnerable to hypoglycaemic stress leading to memory impairment. In the present article, we have investigated the dopamine (DA) content, homovanillic acid (HVA)/DA turnover ratio, DA D(1) and DA D(2) receptors in the hippocampus of insulin-induced hypoglycaemic (IIH) and streptozotocin induced diabetic rats where brain functions are impaired. The DA content decreased significantly in hippocampus of diabetic, diabetic +IIH and control +IIH rats compared to control. The HVA/DA turnover ratio also increased significantly in diabetic, diabetic +IIH and control +IIH rats compared to control. Scatchard analysis using [(3)H] DA in the hippocampus showed a significant increase in DA receptors of diabetic, diabetic +IIH and control +IIH rats with decreased affinity. Gene expression studies using Real-time PCR showed an increased expression of DA D(1) and DA D(2) receptors in the hippocampus of hypoglycaemic and diabetic rats. Our results indicate that the dopaminergic system is impaired in the hippocampus of hypoglycaemic and hyperglycaemic rats impairing DA related functions of hippocampus. We observed a prominent dopaminergic functional disturbance in the hypoglycaemic condition than in hyperglycaemia compared to control. This dopaminergic dysfunction in hippocampus during hypoglycaemia and hyperglycaemia is suggested to contribute to cognitive and memory deficits. This will have clinical significance in the treatment of diabetes.

Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the major causes of blindness worldwide. The pathogenesis of DR has been investigated using several animal models of diabetes. These models have been generated by pharmacological induction, feeding a galactose diet, and spontaneously by selective inbreeding or genetic modification. Among the available animal models, rodents have been studied most extensively owing to their short generation time and the inherited hyperglycemia and/or obesity that affect certain strains. In particular, mice have proven useful for studying DR and evaluating novel therapies because of their amenability to genetic manipulation. Mouse models suitable for replicating the early, non-proliferative stages of the retinopathy have been characterized, but no animal model has yet been found to demonstrate all of the vascular and neural complications that are associated with the advanced, proliferative stages of DR that occur in humans. In this review, we summarize commonly used animal models of DR, and briefly outline the in vivo imaging techniques used for characterization of DR in these models. Through highlighting the ocular pathological findings, clinical implications, advantages and disadvantages of these models, we provide essential information for planning experimental studies of DR that will lead to new strategies for its prevention and treatment.

Fluvastatin downregulates VEGF-A expression in TNF-α-induced retinal vessel tortuosity.

PURPOSE: To investigate the effect of tumor necrosis factor alpha (TNF-α) on the mouse retinal vasculature, function, and expression of vascular endothelial growth factor-A (VEGF-A) in the retina and retinal pigment epithelium (RPE) and to evaluate the protective effect of statin therapy (fluvastatin) on retinal vascular and functional changes. METHODS: A single intravenous injection of murine TNF-α (8 µg/kg body weight) was administered to one group of mice (TNF group). In the second group of mice (TNF+Statin group), a single dose of TNF-α was followed by 28 days oral medication of fluvastatin (10 mg/kg/d), and an equivalent volume of saline was administered to the third group (Control group). After 28 days, electroretinography (ERG) and fundus photography were performed. Eyes were collected for cell and molecular studies. Transcript levels of VEGF-A in retina and RPE were quantified using real-time polymerase chain reaction, and protein expression was analyzed by Western blot and immunostaining. RESULTS: TNF-α-injected mice showed retinal vessel tortuosity, structural change, and altered retinal function. Fluvastatin-treated mice exhibited retinal vascular, structural, and functional changes almost similar to those of the control group. VEGF-A expression was significantly upregulated in the retina and RPE of TNF-α-injected mice, and this was significantly downregulated in fluvastatin-treated mice. CONCLUSIONS: This study shows that the TNF-α-induced inflammatory process results in the alteration of retinal microvasculature and function, and fluvastatin could be a potential therapy for treating/preventing retinal microvascular or inflammatory complications.

Enhanced [3H] glutamate binding in the cerebellum of insulin-induced hypoglycaemic and streptozotocin-induced diabetic rats.

AIM: Energy deprivation causes neuronal death affecting the cognitive and memory ability of an individual. The kinetic parameters of glutamate dehydrogenase (GDH), the enzyme involved in the production of glutamate, was studied in the cerebellum and liver and the binding parameters of glutamate receptors in the cerebellum of insulin-induced hypoglycaemic and streptozotocin-induced diabetic rats were studied to reveal the role of glutamate excitotoxicity. METHODS: A single intrafemoral dose of streptozotocin was administered to induce diabetes. Hypoglycaemia was induced by appropriate doses of insulin subcutaneously in control and diabetic rats. The kinetic parameters V (max) and K (m) of GDH were studied spectrophotometrically at different substrate concentrations of alpha-ketoglutarate. Glutamate receptor binding assay was done with different concentrations of [3H] Glutamate. RESULTS: The GDH enzyme assay showed a significant increase (P < 0.001) in the V (max) of the enzyme in the cerebellum of hypoglycaemic and diabetic rat groups when compared to control. The V (max) of hypoglycaemic groups was significantly increased (P < 0.001) when compared to diabetic group. In the liver, the V (max) of GDH was significantly increased (P < 0.001) in the diabetic and diabetic hypoglycaemia group when compared to control. The V (max) of GDH increased significantly (P < 0.001) in the diabetic hypoglycaemic rats compared to diabetic group, whereas the control hypoglycaemic rats showed a significant decrease in V (max) (P < 0.001) when compared to diabetic and diabetic hypoglycaemic rats. The K (m) showed no significant change amongst the groups in cerebellum and liver. Scatchard analysis showed a significant increase (P < 0.001) in B (max) in the cerebellum of hypoglycaemic and diabetic rats when compared to control. The B (max) of hypoglycaemic rats significantly increased (P < 0.001) when compared to diabetic group. In hypoglycaemic groups, B (max) of the control hypoglycaemic rats showed a significant increase (P < 0.001) compared to diabetic hypoglycaemic rats. The K (d) of the diabetic group decreased significantly (P < 0.01) when compared to control and control hypoglycaemic rats. There was a significant decrease (P < 0.05) in the K (d) of diabetic hypoglycaemic group when compared to the control hypoglycaemic rats. CONCLUSION: Our studies demonstrated the increased enzyme activity in the hypoglycaemic rats with increased production of extracellular glutamate. The present study also revealed increased binding parameters of glutamate receptors reflecting an increased receptor number with increase in the affinity. This increased number of receptors and the increased glutamate production will lead to glutamate excitotoxicity and neuronal degeneration which has an impact on the cognitive and memory ability. This has immense clinical significance in the management of diabetes and insulin therapy.

Enhanced beta-adrenergic receptors in the brain and pancreas during pancreatic regeneration in weanling rats.

Adrenergic stimulation has an important role in the pancreatic beta-cell proliferation and insulin secretion. In the present study, we have investigated how sympathetic system regulates the pancreatic regeneration by analyzing Epinephrine (EPI), Norepinephrine (NE) and beta-adrenergic receptor changes in the brain as well as in the pancreas. EPI and NE showed a significant decrease in the brain regions, pancreas and plasma at 72 hrs after partial pancreatectomy. We observed an increase in the circulating insulin levels at 72 hrs. Scatchard analysis using [(3)H] propranolol showed a significant increase in the number of both the low affinity and high affinity beta-adrenergic receptors in cerebral cortex and hypothalamus of partially pancreatectomised rats during peak DNA synthesis. The affinity of the receptors decreased significantly in the low and high affinity receptors of cerebral cortex and the high affinity hypothalamic receptors. In the brain stem, low affinity receptors were increased significantly during regeneration whereas there was no change in the high affinity receptors. The pancreatic beta-adrenergic receptors were also up regulated at 72 hrs after partial pancreatectomy. In vitro studies showed that beta-adrenergic receptors are positive regulators of islet cell proliferation and insulin secretion. Thus our results suggest that the beta-adrenergic receptors are functionally enhanced during pancreatic regeneration, which in turn increases pancreatic beta-cell proliferation and insulin secretion in weanling rats.

Increased insulin secretion by muscarinic M1 and M3 receptor function from rat pancreatic islets in vitro.

Parasympathetic system plays an important role in insulin secretion from the pancreas. Cholinergic effect on pancreatic beta cells exerts primarily through muscarinic receptors. In the present study we investigated the specific role of muscarinic M1 and M3 receptors in glucose induced insulin secretion from rat pancreatic islets in vitro. The involvement of muscarinic receptors was studied using the antagonist atropine. The role of muscarinic M1 and M3 receptor subtypes was studied using subtype specific antagonists. Acetylcholine agonist, carbachol, stimulated glucose induced insulin secretion at low concentrations (10(-8)-10(-5) M) with a maximum stimulation at 10(-7) M concentration. Carbachol-stimulated insulin secretion was inhibited by atropine confirming the role of muscarinic receptors in cholinergic induced insulin secretion. Both M1 and M3 receptor antagonists blocked insulin secretion induced by carbachol. The results show that M3 receptors are functionally more prominent at 20 mM glucose concentration when compared to M1 receptors. Our studies suggest that muscarinic M1 and M3 receptors function differentially regulate glucose induced insulin secretion, which has clinical significance in glucose homeostasis.