Department of Ophthalmology, Tohoku University Graduate School of Medicine, Miyagi, Japan.
J Neurosci Res. 2012 Apr;90(4):802-15. doi: 10.1002/jnr.22800. Epub 2011 Nov 8.
Calpain, an intracellular cysteine protease, has been widely reported to be involved in neuronal cell death. The purpose of this study is to investigate the role of calpain activation in axonal damage-induced retinal ganglion cell (RGC) death. Twelve-week-old male calpstatin (an endogenous calpain inhibitor) knockout mice (CAST KO) and wild-type (WT) mice were used in this study. Axonal damage was induced by optic nerve crush (NC) or tubulin destruction induced by leaving a gelatin sponge soaked with vinblastine (VB), a microtubule disassembly chemical, around the optic nerve. Calpain activation was assessed by immunoblot analysis, which indirectly quantified the cleaved α-fodrin, a substrate of calpain. RGCs were retrogradely labeled by injecting a fluorescent tracer, Fluoro-Gold (FG), and the retinas were harvested and flat-mounted retinas prepared. The densities of FG-labeled RGCs harvested from the WT and CAST KO groups were assessed and compared. Additionally, a calpain inhibitor (SNJ-1945, 100 mg/kg/day) was administered orally, and the density of surviving RGCs was compared with that of the vehicle control group. The mean density of surviving RGCs in the CAST KO group was significantly lower than that observed in the WT group, both in NC and in VB. The mean density of surviving RGCs in the SNJ-1945-treated group was significantly higher than that of the control group. The calpain inhibitor SNJ-1945 has a neuroprotective effect against axonal damage-induced RGC death. This pathway may be an important therapeutic target for preventing this axonal damage-induced RGC death, including glaucoma and diabetic optic neuropathy and other CNS diseases that share a common etiology.
钙蛋白酶是一种细胞内半胱氨酸蛋白酶，广泛报道其参与神经元细胞死亡。本研究旨在探讨钙蛋白酶激活在轴突损伤诱导的视网膜神经节细胞（RGC）死亡中的作用。本研究使用 12 周龄雄性钙蛋白酶抑制剂（一种内源性钙蛋白酶抑制剂）敲除小鼠（CAST KO）和野生型（WT）小鼠。通过视神经挤压（NC）或在视神经周围放置含有长春新碱（VB）的明胶海绵（一种微管分解化学物质）诱导轴突损伤。通过免疫印迹分析评估钙蛋白酶激活，间接定量钙蛋白酶的裂解 α-辅肌动蛋白，这是钙蛋白酶的底物。通过注射荧光示踪剂 Fluoro-Gold（FG）逆行标记 RGC，并收获视网膜并制备扁平视网膜。评估并比较从 WT 和 CAST KO 组收获的 FG 标记的 RGC 的密度。此外，给予钙蛋白酶抑制剂（SNJ-1945，100mg/kg/天）口服，并将存活的 RGC 密度与载体对照组进行比较。NC 和 VB 中，CAST KO 组存活的 RGC 密度的平均值明显低于 WT 组。SNJ-1945 处理组存活的 RGC 密度的平均值明显高于对照组。钙蛋白酶抑制剂 SNJ-1945 对轴突损伤诱导的 RGC 死亡具有神经保护作用。该途径可能是预防这种轴突损伤诱导的 RGC 死亡的重要治疗靶点，包括青光眼和糖尿病性视神经病变以及具有共同病因的其他 CNS 疾病。
