Mali Raghuveer S, Cheng Mei, Chintala Shravan K
Eye Research Institute of Oakland University, Rochester, MI 48309, USA.
Invest Ophthalmol Vis Sci. 2005 Jun;46(6):2125-32. doi: 10.1167/iovs.04-1376.
Membrane depolarization and subsequent synaptic release of l-glutamate have been implicated in ischemic retinal damage. However, the mechanisms that lead to ischemia-induced retinal damage are poorly understood. In this study, KCl, a classic membrane depolarizing agent, was injected into the vitreous humor, and the role of matrix metalloproteinase (MMP)-9 in KCl-induced retinal damage was investigated.
Normal adult CD-1 mice were treated with KCl by intravitreal injection. MMP activity in retinal protein extracts was determined by gelatin zymography. Tissue localization of MMP-9 in the retina was determined by immunohistochemistry. MMP-9, MMP-2, tissue inhibitor of MMP (TIMP)-1, TIMP-2, Bax, and BCl-2 proteins in retinal extracts were determined by Western blot analysis. Apoptotic cell death in the retina was determined by TUNEL assays. Retinal damage was assessed by immunolocalization studies with antibodies against neurofilament-light (NF-L) and calretinin.
Depolarizing concentrations of KCl induced a dose- and time-related upregulation in MMP-9 activity and protein in the retina. KCl-mediated MMP-9 upregulation was associated with an increase in proapoptotic protein Bax and apoptotic death of cells in the ganglion cell (GCL) and inner nuclear layer (INL), and subsequent loss of NF-L-positive ganglion cells and calretinin-positive amacrine cells. Intravitreal injection of KCl along with an N-methyl-d-aspartate (NMDA)-type glutamate receptor antagonist, MK-801, and a non-NMDA-type glutamate receptor antagonist, NBQX, resulted in a reduction in KCl-mediated MMP-9 upregulation in the retina. Furthermore, a synthetic MMP inhibitor inhibited KCl-mediated MMP-9 upregulation, which led to a significant attenuation of KCl-induced retinal damage.
These results suggest that upregulation of MMP-9, in part, plays a causative role in KCl-induced retinal damage.
膜去极化及随后L-谷氨酸的突触释放与缺血性视网膜损伤有关。然而，导致缺血性视网膜损伤的机制仍知之甚少。在本研究中，将经典的膜去极化剂氯化钾注入玻璃体内，研究基质金属蛋白酶（MMP）-9在氯化钾诱导的视网膜损伤中的作用。
通过玻璃体内注射氯化钾处理正常成年CD-1小鼠。用明胶酶谱法测定视网膜蛋白提取物中的MMP活性。通过免疫组织化学确定MMP-9在视网膜中的组织定位。通过蛋白质印迹分析测定视网膜提取物中的MMP-9、MMP-2、MMP组织抑制剂（TIMP）-1、TIMP-2、Bax和Bcl-2蛋白。通过TUNEL检测确定视网膜中的凋亡细胞死亡。用抗神经丝轻链（NF-L）和钙视网膜蛋白的抗体进行免疫定位研究来评估视网膜损伤。
去极化浓度的氯化钾诱导视网膜中MMP-9活性和蛋白呈剂量和时间依赖性上调。氯化钾介导的MMP-9上调与促凋亡蛋白Bax的增加以及神经节细胞层（GCL）和内核层（INL）中细胞的凋亡死亡有关，随后NF-L阳性神经节细胞和钙视网膜蛋白阳性无长突细胞丢失。玻璃体内注射氯化钾并联合N-甲基-D-天冬氨酸（NMDA）型谷氨酸受体拮抗剂MK-801和非NMDA型谷氨酸受体拮抗剂NBQX，可导致视网膜中氯化钾介导的MMP-9上调减少。此外，一种合成的MMP抑制剂可抑制氯化钾介导的MMP-9上调，从而显著减轻氯化钾诱导的视网膜损伤。
这些结果表明，MMP-9的上调部分地在氯化钾诱导的视网膜损伤中起因果作用。
