Spalding Kirsty L, Dharmarajan Arun M, Harvey Alan R
School of Anatomy and Human Biology, The University of Western Australia, Crawley, WA, Australia.
Eur J Neurosci. 2005 Jan;21(1):33-45. doi: 10.1111/j.1460-9568.2004.03826.x.
In neonatal rats, superior colliculus (SC) ablation results in a massive and rapid increase in retinal ganglion cell (RGC) death that peaks about 24 h post-lesion (PL). Naturally occurring cell death during normal development, and RGC death after axonal injury in neonatal and adult rats, has primarily been ascribed to apoptosis. Given that normal developmental cell death is reported to involve caspase 3 activation, and blocking caspase activity in adults reduces axotomy-induced death, we examined whether blocking caspases in vivo reduces RGC death after neonatal SC lesions. Neither general nor specific caspase inhibitors increased neonatal RGC survival 6 and 24 h PL. These inhibitors were, however, effective in blocking caspases in another well-defined in vitro apoptosis model, the corpus luteum. Caspase 3 protein and mRNA levels in retinas from normal and SC-lesioned neonatal rats were assessed 3, 6 and 24 h after SC removal using immunohistochemistry, western and northern blots and quantitative real-time polymerase chain reaction. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) was used to independently monitor retinal cell death. The polymerase chain reaction data showed a small but insignificant increase in caspase 3 mRNA in retinas 24 h PL. Western blot analysis did not reveal a significant shift to cleaved (activated) caspase 3 protein. There was a small increase in the number of cleaved caspase 3 immunolabelled cells in the ganglion cell layer 24 h PL but this represented only a fraction of the death revealed by TUNEL. Together, these data indicate that, unlike the situation in adults, most lesion-induced RGC death in neonatal rats occurs independently of caspase activation.
在新生大鼠中，上丘（SC）切除导致视网膜神经节细胞（RGC）大量快速死亡，在损伤后约24小时（PL）达到峰值。正常发育过程中自然发生的细胞死亡，以及新生和成年大鼠轴突损伤后的RGC死亡，主要归因于细胞凋亡。鉴于据报道正常发育性细胞死亡涉及半胱天冬酶3激活，并且在成年动物中阻断半胱天冬酶活性可减少轴突切断诱导的死亡，我们研究了在体内阻断半胱天冬酶是否能减少新生大鼠SC损伤后的RGC死亡。无论是通用的还是特异性的半胱天冬酶抑制剂都没有增加新生大鼠RGC在损伤后6小时和24小时的存活率。然而，这些抑制剂在另一个明确的体外凋亡模型——黄体中有效地阻断了半胱天冬酶。使用免疫组织化学、蛋白质印迹、Northern印迹和定量实时聚合酶链反应，在SC切除后3小时、6小时和24小时评估正常和SC损伤的新生大鼠视网膜中的半胱天冬酶3蛋白和mRNA水平。末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）用于独立监测视网膜细胞死亡。聚合酶链反应数据显示，在损伤后24小时，视网膜中的半胱天冬酶3 mRNA有小幅但不显著的增加。蛋白质印迹分析未显示裂解（活化）的半胱天冬酶3蛋白有显著变化。在损伤后24小时，神经节细胞层中裂解的半胱天冬酶3免疫标记细胞数量有小幅增加，但这仅占TUNEL显示的死亡细胞的一小部分。这些数据共同表明，与成年动物的情况不同，新生大鼠中大多数损伤诱导的RGC死亡独立于半胱天冬酶激活而发生。
