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

我们的体外研究表明，在培养的视网膜神经元中，一氧化氮(NO)在NMDA受体介导的神经毒性中具有双重作用。也就是说，低浓度的NO通过抑制NMDA通道的活性来保护视网膜神经元，但过量产生NO，与氧自由基相互作用，导致NMDA神经毒性中的视网膜神经元死亡。我们进一步评估了这一发现与体内视网膜缺血的关系。根据我们对猫视网膜的体内微透析研究，在缺血期间大量释放谷氨酸，在再灌流时更显著。MK-801或一氧化氮合酶抑制剂L可显著抑制缺血/再灌流引起的神经节细胞层细胞数和内网层厚度的减少。玻璃体内注射L-NAME可抑制N-甲基-D-天冬氨酸诱导的视网膜神经毒性。非选择性一氧化氮合酶抑制剂L-NAME不仅能抑制神经型一氧化氮合酶(NNOS)的神经毒性作用，也能抑制内皮型一氧化氮合酶(ENOS)的血管扩张作用。因此，我们的发现L-NAME也能抑制N-甲基-D-天冬氨酸诱导的神经毒性，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起主要作用，尽管NO具有潜在的血管扩张剂的作用。因此，通过NMDA受体产生的NO在缺血诱导的视网膜内层损伤中起关键作用。根据我们的分子遗传学研究，在短暂性视网膜缺血后，视网膜内侧可见TUNEL阳性细胞。此外，缺血视网膜DNA琼脂糖凝胶电泳后可见典型的后一种模式，表明存在核小体间DNA片段化。由此可见，短暂性视网膜缺血可诱导视网膜内神经元的凋亡。

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.