ABSTRACT
Recent studies support a role for excitotoxicity in the development of retinal ganglion cell (RGC) damage in subjects suffering from glaucoma. Coenzyme Q10 (CoQ10), an essential cofactor of the electron transport chain, has been reported to afford neuroprotection, preventing the formation of the mitochondrial permeability transition pore. Using an established animal model of retinal ischemia/reperfusion here, we show that synaptic glutamate increases at 130min from beginning of reperfusion and delayed apoptosis in the RGC layer is seen at 24h. Intraocular administration of CoQ10 minimizes glutamate increase and affords neuroprotection, suggesting that oxidative stress and energy failure might be implicated in the mechanisms of RGC death.
Subject(s)
Intraocular Pressure/physiology , Ischemia/pathology , Retinal Diseases/prevention & control , Ubiquinone/analogs & derivatives , Administration, Topical , Animals , Coenzymes/administration & dosage , Coenzymes/therapeutic use , Glutamic Acid/toxicity , In Situ Nick-End Labeling , Male , Microdialysis , Rats , Rats, Wistar , Retinal Diseases/pathology , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/pathology , Ubiquinone/administration & dosage , Ubiquinone/therapeutic useABSTRACT
PURPOSE: To evaluate whether high intraocular pressure (IOP)-induced ischemia is associated with modifications in the retinal endocannabinoid metabolism and to ascertain whether drugs that interfere with the endocannabinoid system may prevent retinal damage due to ischemic insult. METHODS: Anandamide (AEA) synthesis, transport, hydrolysis, and AEA endogenous levels were assessed by means of high-performance liquid chromatography in the retinas of rats undergoing 45 minutes of ischemia followed by 12 hours of reperfusion. Under these experimental conditions, binding to cannabinoid (CB1R) and vanilloid (TRPV1) receptor was assessed with rapid-filtration assays. AEA-hydrolase (FAAH, fatty acid amide hydrolase), CB1R and TRPV1 protein content was determined by enzyme-linked immunosorbent assay. Finally, to characterize the neuroprotective profile of drugs that interfere with the endocannabinoid system, cell counting in the retinal ganglion cell (RGC) layer and real-time polymerase chain reactions for Thy-1 mRNA expression were used. RESULTS: In rat retina, ischemic insult followed by reperfusion resulted in enhanced FAAH activity and protein expression paralleled by a significant decrease in the endogenous AEA tone, whereas the AEA-membrane transporter or the AEA-synthase NAPE-PLD (N-acyl-phosphatidylethanolamine-hydrolyzing-phospholipase-d) were not affected. Retinal ischemia-reperfusion decreased the expression of cannabinoid (CB1) and vanilloid (TRPV1) receptors. Systemic administration of a specific FAAH inhibitor (e.g., URB597) reduced enzyme activity and minimized the retinal damage observed in ischemic-reperfused samples. Similarly, intravitreal injection of the AEA stable analogue, R(+)-methanandamide, reduced cell loss in the RGC layer, and this was prevented by systemic administration of a CB1 or TRPV1 selective antagonist (e.g., SR141716 and capsazepine, respectively). CONCLUSIONS: The original observation that retinal ischemia-reperfusion reduces endogenous AEA via enhanced expression of FAAH supports the deduction that this is implicated in retinal cell loss caused by high IOP in the RGC layer.
Subject(s)
Cannabinoid Receptor Modulators/physiology , Endocannabinoids , Intraocular Pressure , Reperfusion Injury/metabolism , Retinal Diseases/metabolism , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/metabolism , Animals , Arachidonic Acids/metabolism , Benzamides/pharmacology , Capsaicin/analogs & derivatives , Capsaicin/pharmacology , Carbamates/pharmacology , Cell Count , Chromatography, High Pressure Liquid , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Hydrolysis , Male , Ocular Hypertension/complications , Piperidines/pharmacology , Polyunsaturated Alkamides/metabolism , Pyrazoles/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/metabolism , Reperfusion Injury/etiology , Reperfusion Injury/pathology , Reperfusion Injury/prevention & control , Retinal Diseases/etiology , Retinal Diseases/pathology , Retinal Diseases/prevention & control , Retinal Ganglion Cells/pathology , Retinal Vessels/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Rimonabant , TRPV Cation Channels/antagonists & inhibitors , TRPV Cation Channels/metabolism , Thy-1 Antigens/geneticsABSTRACT
High intraocular pressure (IOP)-induced ischemia is a model for retinal neurodegeneration that recapitulates pathological features almost identical to those seen in patients after central retinal or ophthalmic artery occlusion and may also represent a model of acute angle closure glaucoma. Using this experimental model, we present data indicating that acute IOP elevation for 45 min is followed by a progressive decline in the number of retinal ganglion cells (RGC) which appear to die via an apoptotic mechanism. The observation that systemic treatment with MK801, a N-methyl-d-aspartate (NMDA) receptor antagonist, with GYKI52466, a non-NMDA receptor antagonist, or with l-NAME, an inhibitor of nitric oxide synthase (NOS), prevents the RGC loss observed 24 after IOP elevation strongly suggests an excitotoxic, glutamate-mediated, mechanism of RGC death. The latter deduction is strengthened by the evidence that a microdialysis probe placed into the retinal tissue of rats bearing IOP elevation revealed an increase (90% as compared to baseline value) in glutamate levels that peaked 130 min after the beginning of reperfusion and was reversed by a pre-treatment with MK801. Collectively, our data suggest that acute elevation of IOP increases intraretinal levels of glutamate with consequent abnormal activation of NMDA and non-NMDA subtypes of glutamate receptors and increased NOS activity leading to excitotoxic, glutamate-mediated, RGC death.
