This study determined whether retinal degeneration during diabetes includes retinal neural cell apoptosis. Image analysis of retinal sections from streptozotocin (STZ) diabetic rats after 7.5 months of STZ diabetes identified 22% and 14% reductions in the thickness of the inner plexiform and inner nuclear layers, respectively ( P Ͻ 0.001). The number of surviving ganglion cells was also reduced by 10% compared to controls ( P Ͻ 0.001). In situ end labeling of DNA terminal dUTP nick end labeling (TUNEL) identified a 10-fold increase in the frequency of retinal apoptosis in wholemounted rat retinas after 1, 3, 6, and 12 months of diabetes ( P Ͻ 0.001, P Ͻ 0.001, P Ͻ 0.01, and P Ͻ 0.01, respectively).

The early pathophysiology of diabetic retinopathy and the involvement of neural and vascular malfunction are poorly understood. Glial cells provide structural and metabolic support for retinal neurons and blood vessels, and the cells become reactive in certain injury states. We therefore used the streptozotocin rat model of short-term diabetic retinopathy to study glial reactivity and other glial functions in the retina in the first months after onset of diabetes. With a two-site enzyme-linked immunosorbent assay, we measured the expression of the intermediate filament glial fibrillary acidic protein (GFAP). After 1 month, GFAP was largely unchanged, but within 3 months of the beginning of diabetes, it was markedly induced, by fivefold (P < 0.04). Immunohistochemical staining showed that the GFAP induction occurred both in astrocytes and in Müller cells. Consistent with a glial cell malfunction, the ability of retinas to convert glutamate into glutamine, assayed chromatographically with an isotopic method, was reduced in diabetic rats to 65% of controls (P < 0.01). Furthermore, retinal glutamate, as determined by luminometry, increased by 1.6-fold (P < 0.04) after 3 months of diabetes. Taken together, these findings indicate that glial reactivity and altered glial glutamate metabolism are early pathogenic events that may lead to elevated retinal glutamate during diabetes. These data are the first demonstration of a specific defect in glial cell metabolism in the retina during diabetes. These findings suggest a novel understanding of the mechanism of neural degeneration in the retina during diabetes, involving early and possibly persistent glutamate excitotoxicity.

Blood-retinal barrier (BRB) breakdown is a hallmark of diabetic retinopathy, but the molecular changes that cause this pathology are unclear. Occludin is a transmembrane component of interendothelial tight junctions that may regulate permeability at the BRB. In this study, we examined the effects of vascular endothelial growth factor (VEGF) and diabetes on vascular occludin content and barrier function. Sprague-Dawley rats were made diabetic by intravenous streptozotocin injection, and age-matched animals served as controls. After 3 months, BRB permeability was quantified by intravenous injection of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA), Mr 66 kDa, and 10-kDa rhodamine-dextran (R-D), followed by digital image analysis of retinal sections. Retinal fluorescence intensity for FITC-BSA increased 62% (P < or = 0.05), but R-D fluorescence did not change significantly. Occludin localization at interendothelial junctions was confirmed by immunofluorescence, and relative protein content was determined by immunoblotting of retinal homogenates. Retinal occludin content decreased approximately 35% (P < or = 0.03) in the diabetic versus the control animals, whereas the glucose transporter GLUT1 content was unchanged in rat retinas. Additionally, treatment of bovine retinal endothelial cells in culture with 0.12 nmol/l or 12 nmol/l VEGF for 6 h reduced occludin content 46 and 54%, respectively. These data show that diabetes selectively reduces retinal occludin protein expression and increases BRB permeability. Our findings suggest that the elevated VEGF in the vitreous of patients with diabetic retinopathy increases vascular permeability by downregulating occludin content. Decreased tight junction protein expression may be an important means by which diabetes causes increased vascular permeability and contributes to macular edema.

Normal vision depends on the normal function of retinal neurons, so vision loss in diabetes must ultimately be explained in terms of altered neuronal function. However to date relatively little attention has been paid to the impact of diabetes on the neural retina. Instead, the focus of most research has been primarily on retinal vascular changes, with the assumption that they cause altered neuronal function and consequently vision loss. An increasing body of evidence suggests that alterations in neuronal function and viability may contribute to the pathogenic mechanisms of diabetic retinopathy beginning shortly after the onset of diabetes. This view arises from neurophysiological, psychometric, histopathological and biochemical observations in humans and experimental animals. The collective evidence from past and recent studies supports the hypothesis that neurodegeneration, together with functional changes in the vasculature, is an important component of diabetic retinopathy. The authors invite other investigators to include the neural retina as a component of their studies so that the pathogenesis of diabetic retinopathy can be understood more clearly.

The relationship between neuronal glutamate turnover, the glutamate/glutamine cycle and de novo glutamate synthesis was examined using two different model systems, freshly dissected rat retinas ex vivo and in vivo perfused rat brains. In the ex vivo rat retina, dual kinetic control of de novo glutamate synthesis by pyruvate carboxylation and transamination of a-ketoglutarate to glutamate was demonstrated. Rate limitation at the transaminase step is likely imposed by the limited supply of amino acids which provide the a-amino group to glutamate. Measurements of synthesis of 14 C-glutamate and of 14 C-glutamine from H 14 CO 3 have shown that 14 C-amino acid synthesis increased 70% by raising medium pyruvate from 0.2 to 5 mM. The speci®c radioactivity of 14 C-glutamine indicated that ,30% of glutamine was derived from 14 CO 2 ®xation. Using gabapentin, an inhibitor of the cytosolic branched-chain aminotransferase, synthesis of 14 C-glutamate and 14 C-glutamine from H 14 CO 3 2 was inhibited by 31%. These results suggest that transamination of a-ketoglutarate to glutamate in Mu È ller cells is slow, the supply of branchedchain amino acids may limit¯ux, and that branched-chain amino acids are an obligatory source of the nitrogen required for optimal rates of de novo glutamate synthesis. Kinetic analysis suggests that the glutamate/glutamine cycle accounts for 15% of total neuronal glutamate turnover in the ex vivo retina. To examine the contribution of the glutamate/ glutamine cycle to glutamate turnover in the whole brain in vivo, rats were infused intravenously with H 14 CO 3 2 . 14 C-metabolites in brain extracts were measured to determine net incorporation of 14 CO 2 and speci®c radioactivity of glutamate and glutamine. The results indicate that 23% of glutamine in the brain in vivo is derived from 14 CO 2 ®xation. Using published values for whole brain neuronal glutamate turnover, we calculated that the glutamate/glutamine cycle accounts for ,60% of total neuronal turnover. Finally, differences between glutamine/glutamate cycle rates in these two model systems suggest that the cycle is closely linked to neuronal activity.

Diabetes leads to a wide array of complications in humans, including kidney failure, vascular disease, peripheral nerve degeneration, and vision loss. Diabetic retinopathy causes blindness in more working-age people in the United States than any other disease and contributes greatly to blindness in the young and old as well. The increasing rate of diabetes occurring in our society can only bring about a further decrease in the visual health of this country unless new modalities are discovered to prevent and cure diabetic retinopathy. Breakdown of the blood-retinal barrier and the resultant vascular permeability remains one of the first observable alterations in diabetic retinopathy and strongly correlates with vision loss. In this article, we examine the molecular components that form this blood-retinal barrier and explore how changes in the production of growth factors in the neural parenchyma cause an increase in vascular permeability and contribute to retinal degeneration.

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A novel hypothesis for the role of branched-chain amino acids (BCAA) in regulating levels of the major excitatory neurotransmitter glutamate in the central nervous system is described. It is postulated that the branched-chain aminotransferase (BCAT) isoenzymes (mitochondrial BCATm and cytosolic BCATc) are localized in different cell types and operate in series to provide nitrogen for optimal rates of de novo glutamate synthesis. BCAA enter the astrocyte where transamination is catalyzed by BCATm, producing glutamate and branched-chain alpha-keto acids (BCKA). BCKA, which are poorly oxidized in astrocytes, exit and are taken up by neurons. Neuronal BCATc catalyzes transamination of the BCKA with glutamate. The products, BCAA, exit the neuron and return to the astrocyte. The alpha-ketoglutarate product in the neurons may undergo reductive amination to glutamate via neuronal glutamate dehydrogenase. Operation of the shuttle in the proposed direction provides a mechanism for efficient nitrogen transfer between astrocytes and neurons and synthesis of glutamate from astrocyte alpha-ketoglutarate. Evidence in favor of the hypothesis is: 1) The two BCAT isoenzymes appear to be localized separately in the neurons (BCATc) or in the astroglia (BCATm). 2) Inhibition of the shuttle in the direction of glutamate synthesis can be achieved by inhibiting BCATc using the neuroactive drug gabapentin. Although gabapentin does not inhibit BCATm, it does block de novo glutamate synthesis from alpha-ketoglutarate. 3) Conversely, gabapentin stimulates oxidation of glutamate. Inhibition of BCATc may allow BCKA to accumulate in the astroglia, thus facilitating conversion of glutamate to alpha-ketoglutarate.