The study of the intracellular mechanism and its modulatory factor in the ischemic retina

本实验在体外培养的视网膜神经元中证实，一氧化氮（NO）在NMDA受体介导的神经毒性中具有双重作用。即低浓度的NO通过抑制NMDA通道的活性来保护视网膜神经元，但过量的NO产生，与氧自由基相互作用，导致NMDA神经毒性下视网膜神经元的死亡。我们进一步评估了这些发现如何参与体内视网膜缺血。基于我们对猫视网膜的体内微透析研究，在缺血期间发生大量谷氨酸的释放，在再灌注期间更显着。MK-801或NOS抑制剂L-NAME可明显抑制缺血再灌注引起的神经节细胞层细胞数减少和内丛状层厚度减少。玻璃体内注射L-NAME可抑制NMDA诱导的视网膜神经毒性。非选择性NOS抑制剂L-NAME不仅抑制神经元型NOS（nNOS）的神经毒性作用，而且抑制内皮型NOS（eNOS）的血管舒张作用。因此，我们的研究结果表明，L-NAME也抑制NMDA诱导的神经毒性，表明NO诱导的神经毒性在视网膜缺血性损伤的发病机制中的主要作用，尽管NO的潜在改善作用作为血管扩张剂。因此，通过NMDA受体产生的NO在缺血引起的视网膜内层损伤中起关键作用。我们的分子遗传学研究表明，短暂性视网膜缺血后，在视网膜内层可见TUNEL阳性细胞。此外，在DNA琼脂糖凝胶电泳后，从缺血视网膜获得典型的后者模式，表明存在核小体间DNA片段化。提示短暂性视网膜缺血可诱导视网膜内层神经元凋亡。

Our in vitro study demonstrated in the cultured retinal neurons that nitric oxide (NO) has dual actions in the NMDA-receptor mediated neurotoxcicity. That is, low concentration of NO protected retinal neurons by inhibiting the NMDA-channel activity but overproduction of NO,interacting with oxygenradicals lead to the death of retinal neurons in NMDA neuroxicity. We further evaluated how this findings were involved in the in vivo retinal ischemia. Based on our in vivo microdialysis study of the cat ratina, large release of glutamate occurred during ischemia and more prominently during reperfusion. Administration of MK-801 or L-NAME,aNO synthase (NOS) inhibitor, into the rat significantly inhibited the ischemia/reperfusioninduced reduction in the cell number of the ganglion cell layr and the thickness of the inner plexiform layr. Intravitreal injection of L-NAME inhibited NMDA-induced neurotoxicity in the retinal. Non-selective NOS inhibitor such as L-NAME inhibits not only neuronal NOS (nNOS) from neurotoxic action but also endothelial NOS (eNOS) from vasodilating action. Therefore, our findings that L-NAME also inhibited NMDA-induced neurotoxicity, indicate a principal role of NO-induced neurotoxicity in the pathogenesis of retinal ischemic injury albeit NO's potential ameliorating action as a vasodilator. It is thus concluded that NO generated through NMDA-receptors play a key role in the ischemia-induced damage in the inner layrs of the retina. According to our molecular genenic study, TUNEL positive cells were found in the inner retina after transient retinal ischemia. Furthermore, typical latter pattern after agarose gel electrophoresis of DNA was obtained from the ischemic retina, indicating the presence of internucleosomal DNA fragmentation. Thus, it is suggested that transient retinal ischemia induces apoptosis of the inner retinal neurons.