Sam Rothberg Ophthalmic Molecular Biology Laboratory, Goldschleger Eye Institute, Sheba Medical Center, 52621 Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Israel.
Exp Eye Res. 2010 Aug;91(2):127-34. doi: 10.1016/j.exer.2009.11.014. Epub 2009 Nov 29.
In central nervous system injury, the secondary degeneration process is known to play a major role in determining the final extent of impairment. Here, we investigated the mechanism of retinal ganglion cell (RGC) death in secondary degeneration of the optic nerve using a unique model that allows morphological separation between primary and secondary degeneration. A partial transection model was applied unilaterally in 110 Wistar rat eyes. The rate of apoptosis was evaluated in primary and secondary degeneration over a period of 6 months using the Hoechst staining technique. The involvement of caspase 3 and members of the Bcl-2 family (Bax, Bad, Bcl-2 and Bcl-xl) was evaluated at multiple time points for 6 months after the injury by immunohistochemistry and RT-PCR. We found that in secondary degeneration of the optic nerve, RGCs died by apoptosis from day 3-6 months following the injury, peaking at 3 months (16.3% +/- 2.5% apoptotic cells, p < 0.01). Both primary and secondary degeneration of the optic nerve resulted in caspase 3 activation, which was longer and more intense in the former. Similarly, both primary and secondary degeneration led to significant (p < 0.05) downregulation of the pro-survival genes Bcl-2 and Bcl-x-L and up-regulation of the pro-apoptotic genes Bax and Bad (p < 0.05), with a suggested delay in secondary degeneration. Thus, secondary degeneration of the optic nerve leads to RGC apoptosis over long periods in a similar mechanism as in primary degeneration.
在中枢神经系统损伤中，继发性变性过程被认为在决定最终损伤程度方面起着重要作用。在这里，我们使用一种独特的模型研究了视神经继发性变性中视网膜神经节细胞（RGC）死亡的机制，该模型允许对原发性和继发性变性进行形态学分离。在 110 只 Wistar 大鼠的单侧眼应用部分横断模型。在 6 个月的时间内，使用 Hoechst 染色技术评估原发性和继发性变性中的细胞凋亡率。通过免疫组织化学和 RT-PCR 在损伤后 6 个月的多个时间点评估半胱氨酸天冬氨酸蛋白酶 3（caspase 3）和 Bcl-2 家族成员（Bax、Bad、Bcl-2 和 Bcl-xl）的参与。我们发现，在视神经的继发性变性中，RGC 从损伤后第 3 天至 6 个月通过细胞凋亡死亡，在 3 个月时达到高峰（16.3% +/- 2.5%凋亡细胞，p < 0.01）。视神经的原发性和继发性变性均导致 caspase 3 激活，前者的激活时间更长且更强烈。同样，原发性和继发性变性均导致促生存基因 Bcl-2 和 Bcl-x-L 的显著下调（p < 0.05）和促凋亡基因 Bax 和 Bad 的上调（p < 0.05），提示继发性变性延迟。因此，视神经的继发性变性在很长一段时间内通过与原发性变性相似的机制导致 RGC 凋亡。
