Evaluation of the impact of diabetes on retinal metabolites by NMR spectroscopy.

PURPOSE/AIM OF THE STUDY: Diabetic retinopathy (DR) is a leading cause of blindness in working age adults in developed countries. Changes in metabolites and in metabolic pathways of the retina caused by hyperglycemia may compromise the physiology of the retina. Using nuclear magnetic resonance (NMR) spectroscopy, we aimed to investigate the effect of diabetes on the levels of intermediate metabolites in rat retinas and the metabolic pathways that could be affected. MATERIALS AND METHODS: Diabetes was induced in male Wistar rats with a single injection of streptozotocin (65 mg/Kg, i.p.). Metabolic alterations were analyzed in streptozotocin-induced diabetic rat retinas by (1)H NMR spectroscopy. Glucose uptake was measured with 2-deoxy-D-[1-(3)H]glucose. Lactate production was evaluated by (1)H NMR spectroscopy using [U-(13)C]glucose. RESULTS: Tissue levels of several metabolic intermediates were quantified, but no significant changes in the levels of most metabolites were detected, with the exceptions of glucose, significantly increased, and lactate, significantly reduced in diabetic rat retinas, as compared to age-matched controls. The cytosolic redox ratio, indirectly evaluated by lactate-to-pyruvate ratio, was significantly reduced in diabetic rat retinas, as well as glucose uptake. Parallel studies demonstrated that lactate production rates were significantly diminished, suggesting a reduction in the glycolytic flux. CONCLUSIONS: These results suggest that diabetes may significantly decrease glycolysis in the retina since higher intracellular glucose levels do not translate into higher intracellular lactate levels or into higher rates of lactate production. These changes may alter the normal functioning of the retina during diabetes and may contribute for vision loss in DR.

High glucose changes extracellular adenosine triphosphate levels in rat retinal cultures.

Diabetic retinopathy (DR) is the leading cause of blindness in adults. In diabetes, there is activation of microglial cells and a concomitant release of inflammatory mediators. However, it remains unclear how diabetes triggers an inflammatory response in the retina. Activation of P2 purinergic receptors by adenosine triphosphate (ATP) may contribute to the inflammatory response in the retina, insofar as it has been shown to be associated with microglial activation and cytokine release. In this work, we evaluated how high glucose, used as a model of hyperglycemia, considered the main factor in the development of DR, affects the extracellular levels of ATP in retinal cell cultures. We found that basal extracellular ATP levels were not affected by high glucose or mannitol, but the extracellular elevation of ATP, after a depolarizing stimulus, was significantly higher in retinal cells cultured in high glucose compared with control or mannitol-treated cells. The increase in the extracellular ATP was prevented by application of botulinum neurotoxin A or by removal of extracellular calcium. In addition, degradation of exogenously added ATP was significantly lower in high-glucose-treated cells. It was also observed that, in retinal cells cultured under high-glucose conditions, the changes in the intracellular calcium concentrations were greater than those in control or mannitol-treated cells. In conclusion, in this work we have shown that high glucose alters the purinergic signaling system in the retina, by increasing the exocytotic release of ATP and decreasing its extracellular degradation. The resulting high levels of extracellular ATP may lead to inflammation involved in the pathogenesis of DR.

High glucose induces caspase-independent cell death in retinal neural cells.

Diabetic retinopathy is a leading cause of blindness among adults in the western countries. It has been reported that neurodegeneration may occur in diabetic retinas, but the mechanisms underlying retinal cell death are poorly understood. We found that high glucose increased the number of cells with condensed nuclei and the number of TUNEL-positive cells, and caused an increase in the translocation of phosphatidylserine to the outer leaflet of the plasma membrane, indicating that high glucose induces apoptosis in cultured retinal neural cells. The activity of caspases did not increase in high glucose-treated cells, but apoptosis-inducing factor (AIF) levels decreased in the mitochondria and increased in the nucleus, indicating a translocation to the nucleus where it may cause DNA fragmentation. These results demonstrate that elevated glucose induces apoptosis in cultured retinal neural cells. The increase in apoptosis is not dependent on caspase activation, but is mediated through AIF release from the mitochondria.

Inflammation and neurogenesis in temporal lobe epilepsy.

The aim of the present review is to discuss the evidence supporting the hypothesis that inflammation and neurogenesis play an important role in temporal lobe epilepsy (TLE) and to examine whether possible strategies that involve the pharmacological manipulation of inflammation/neurogenesis can lead to the development of novel approaches for the treatment of epilepsy. Since it is not yet clear whether the neuron-glia response obtained in this pathology is a secondary effect of an aggressive inflammation or if it is somehow related to the cause of the epileptic condition, with the present review we guide the readers through the complex and ambiguous crosstalk between neuroimmunology and epilepsy.

Ca2+ stores in the chick embryo retina cells.

The Ca2+ stores of digitonin permeabilized chick embryo retina cells in culture were characterized, by using the fluorescence of Fluo-3 potassium salt to follow continuously the free [Ca2+] in the medium. After ATP dependent Ca2+ accumulation, the Ca2+ release was induced by several agents; 10 microM cyclic-ADP-ribose (cADPR), 40 microM Ins (1,4,5)P3 10 microM thapsigargin (Th), 25 microM ionomycin (Ion), 15 microM CCCP together with 4.5 micrograms/ml oligomycin (CCCP/Olig), 50 microM arachidonic acid (AA). Neither Ins(1,4,5)P3 nor cADPR were able to mobilize Ca2+ from internal stores in these cells, but Th and AA were effective in releasing Ca2+. Four major Ca2+ stores in chick embryo retina cells were distinguished: i) the thapsigargin sensitive Ca2+ store, most likely the ER; ii) the Ca2+ store sensitive to oligomycin and CCCP, most likely the mitochondrial Ca2+ store, iii) an AA sensitive Ca2+ store, which is distinct from the previous two; and, iv) the Ca2+ store only sensitive to ionomycin. The capacities of these different Ca2+ stores of the chick embryo retina cells, relative to the total intracellular stores, are: 63.3%, 14.1%, 8.2%, for the ER, the mitochondrial and for the AA sensitive Ca2+ stores, respectively.

Voltage-sensitive Ca2+ channels in rat striatal synaptosomes: role on the [Ca2+]i responses to membrane depolarization.

The fluorescent Ca2+ indicator Indo-1 was used to study the effect of depolarization evoked by KCl or 4-aminopyridine (4-AP) on the intracellular free calcium concentration responses (delta[Ca2+]i) in rat striatal synaptosomes. Depolarization of the synaptosomes with [KCl] > 7.5 mM induced a rapid increase of the [Ca2+]i followed by a decay towards a plateau. The size of the [Ca2+]i response varied sigmoidally with the synaptosomal membrane potential, with a transition potential of -27.3 mV. Depolarization with 4-AP evoked a dose-dependent sustained increase of the [Ca2+]i. Nitrendipine, omega-Conotoxin GVIA (omega-CgTx) and omega-Agatoxin IVA (omega-Aga IVA) were used to evaluate the relative role of L-, N-, P- and possibly Q-type voltage-sensitive Ca2+ channels (VSCCs) on the [Ca2+]i changes evoked by each of the two depolarizing agents. Nitrendipine caused only about 10% inhibition of the effect of either agent on the [Ca2+]i, suggesting that the L-type VSCCs have a modest contribution. The omega-CgTx decreased the response to KCl and 4-AP by 15 and 30%, respectively, but the latter effect may be partially due to a non-specific effect on Na+ channels. The omega-Aga IVA reduced the response to 4-AP by 26.5%, and this effect was additive to that of omega-CgTx, further suggesting that the striatal nerve terminals possess P- and/or Q-type, in addition to N-type Ca2+ channels. Neomycin (0.35 mM), tentatively used as an antagonist of the P-type channels, had a potent effect, decreasing the response to K(+)-depolarization and to 4-AP by, respectively, 32.5 and 48.5%. It is suggested that at the concentration used the antibiotic also partially blocks VSCCs which do not belong to the L-, N-, P- or Q-type VSCCs. We conclude that striatal nerve endings are equipped with at least four to five pharmacologically distinct classes of VSCCs, which are sensitive to well known antagonists of the L-, N-, P-, and Q-type VSCCs.

Ins(1,4,5)P3 induces Ca2+ release from brain microsomes loaded either by the Ca2+ ATPase or by the Na+/Ca2+ exchanger.

In this study we investigated the release of Ca2+ in brain microsomes after Ca2+ loading by the Ca(2+)-ATPase or by the Na+/Ca2+ exchanger. The results show that in microsomes loaded with Ca2+ by the Ca(2+)-ATPase, Ins(1,4,5)P3 (5 microM) released 21 +/- 2% of the total Ca2+ accumulated, and that in the microsomes loaded with Ca2+ by the Na+/Ca2+ exchanger, Ins(1,4,5)P3 released 28 +/- 3% of the total Ca2+ accumulated. These results suggest that receptors of Ins(1,4,5)P3 may be co-localized with the Na+/Ca2+ exchanger in the endoplasmic reticulum membrane or that there are Ins(1,4,5)P3 receptors in the plasma membrane where the Na+/Ca2+ exchanger is normally present, or both. We also found that Ins(1,4,5)P3 inhibited the Ca(2+)-ATPase by 33.7%, but that it had no significant effect on the Na+/Ca2+ exchanger.