Rogério Fábio, Jordão Hamilton, Vieira André Schwambach, Maria Carla Cristina Judice, Santos de Rezende Alexandre César, Pereira Gonçalo Amarante Guimarães, Langone Francesco
Department of Physiology and Biophysics, State University of Campinas, UNICAMP, 13083-970, Campinas, SP, Brazil.
Brain Res. 2006 Sep 27;1112(1):80-90. doi: 10.1016/j.brainres.2006.07.021. Epub 2006 Aug 7.
Peripheral axotomy in neonatal rats induces neuronal death. We studied the anti-apoptotic protein Bcl-2 and cell death promoter Bax in spinal cord of neonatal rats after sciatic transection and treatment with melatonin, a neuroprotective substance. Pups were unilaterally axotomized at P2 and received melatonin (1 mg/kg; sc) or vehicle 1 h prior to lesion, immediately after, at 1 h, 2 h and then once daily. Rats were sacrificed at 3 h, 6 h, 24 h, 72 h and 5 days postaxotomy. Intact animals were used as controls. Lumbar enlargement was processed for Nissl staining, immunohistochemistry and RT-PCR for Bax or Bcl-2 and TUNEL reaction. Motoneurons (MN) of lesioned (L) and normal (N) sides were counted, and MN survival ratio (MSR=L/N) was calculated. Bax and Bcl-2 showed cytoplasmic immunoreactivity (IR). Bax IR was noticeable in small cells but less evident in MN. In unlesioned pups, some Bax-positive small cells (B+) and TUNEL-positive nuclei (T+) were mainly seen in the dorsal horn. In lesioned animals given vehicle, Bax mRNA levels and numbers of B+ and T+ were increased in comparison with intact controls at 24 h postaxotomy. The basal IR for Bax in MN was not changed by axotomy. Bcl-2 IR was noted in all cells and, like Bcl-2 mRNA, was unaltered after lesion. Melatonin reduced MN loss at 24 h, 72 h and 5 days and T+ at 24 h after lesion but did not interfere with Bax or Bcl-2 expression. These results suggest that (1) sciatic transection at P2 increases Bax mRNA and the amount of B+ and T+ in the lumbar enlargement, (2) Bax IR in immature MN is not altered by axotomy and (3) melatonin protects MN and dorsal horn cells through a mechanism independent of Bax and Bcl-2.
新生大鼠周围神经切断术会导致神经元死亡。我们研究了坐骨神经横断并给予褪黑素（一种神经保护物质）处理后，新生大鼠脊髓中抗凋亡蛋白Bcl-2和细胞死亡促进因子Bax的情况。幼鼠在出生后第2天（P2）进行单侧神经切断，并在损伤前1小时、损伤后立即、1小时、2小时，然后每天一次接受褪黑素（1毫克/千克；皮下注射）或溶剂。大鼠在神经切断术后3小时、6小时、24小时、72小时和5天处死。完整动物用作对照。对腰膨大进行尼氏染色、免疫组织化学以及Bax或Bcl-2的逆转录聚合酶链反应（RT-PCR）和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）反应。对损伤侧（L）和正常侧（N）的运动神经元（MN）进行计数，并计算MN存活率（MSR = L/N）。Bax和Bcl-2表现出细胞质免疫反应性（IR）。Bax IR在小细胞中明显，但在MN中不太明显。在未损伤的幼鼠中，一些Bax阳性小细胞（B+）和TUNEL阳性细胞核（T+）主要见于背角。在给予溶剂的损伤动物中与完整对照相比，神经切断术后24小时Bax mRNA水平以及B+和T+的数量增加。MN中Bax的基础IR不受神经切断术影响。在所有细胞中均观察到Bcl-2 IR，并且与Bcl-2 mRNA一样，损伤后未发生改变。褪黑素减少了损伤后24小时、72小时和5天的MN损失以及损伤后24小时的T+，但不影响Bax或Bcl-2的表达。这些结果表明：（1）P2时坐骨神经横断增加了腰膨大处Bax mRNA以及B+和T+的数量；（2）未成熟MN中的Bax IR不受神经切断术影响；（3）褪黑素通过独立于Bax和Bcl-2的机制保护MN和背角细胞。
