APOPTOSIS IN TRAUMATIC SPINAL CORD INJURY

本资助申请中提出的研究重点是识别和表征外伤性脊髓损伤（SCI）后凋亡细胞死亡的分子信号事件。尽管最近有证据表明脊髓损伤后神经元和胶质细胞中存在广泛的凋亡，但凋亡途径的分子成分在很大程度上仍然未知。提出的实验将提供凋亡机制中负责启动和执行细胞死亡过程的成分的证据。此外，实验表明，阻断凋亡细胞死亡通路的步骤可在急性脊髓损伤中提供神经保护并促进功能恢复。我们假设钙调磷酸酶介导的BAD去磷酸化是凋亡级联的初始上游事件之一，并与caspase-3激活有关。谷氨酸诱导的Ca2+内流导致钙调磷酸酶激活，高水平的谷氨酸在脊髓损伤后很快释放。因此，我们将研究脊髓鞘内注射选择性谷氨酸受体激动剂是否会导致钙调磷酸酶介导的BAD去磷酸化。此外，我们将证明注射谷氨酸受体拮抗剂可降低caspase-3凋亡级联的激活。最后，在脊髓损伤后观察到的功能缺陷中，凋亡的作用目前尚不清楚。因此，我们将使用钙调神经磷酸酶和caspase-3抑制剂治疗受伤动物，并使用形态学、组织学和行为标准测量凋亡细胞死亡和功能恢复的指标。这些研究的结果将对减少脊髓损伤后继发性神经元和神经胶质细胞死亡和促进功能恢复具有明确的临床意义。

The studies proposed in this grant application focus on identifying and characterizing the molecular signaling events involved in apoptotic cell death following traumatic spinal cord injury (SCI). Despite the recent evidence of widespread apoptosis in neurons and glia after SCI, the molecular components of the apoptotic pathway remain largely unknown. The proposed experiments will provide evidence of components in the apoptotic machinery responsible for initiating and executing this cell death process. In addition, experiments are proposed to demonstrate that blocking steps involved in the apoptotic cell death pathway provides neuroprotection and promotes functional recovery in acute SCI. We hypothesize that calcineurin-mediated BAD dephosphorylation is one of the initial upstream events in the apoptotic cascade and is linked to caspase-3 activation. Glutamate-induced Ca2+ influx leads to calcineurin activation and high levels of glutamate are released soon after SCI. Therefore, we will examine whether intrathecal injections of selective glutamate receptor agonists into uninjured spinal cord lead to calcineurin- mediated BAD dephosphorylation. In addition, we will demonstrate that injections of glutamate receptor antagonists reduce activation of the caspase-3 apoptotic cascade. Finally, the contribution of apoptosis to the functional deficits observed following SCI is not clear at this time. Therefore, we will treat injured animals with inhibitors targeting calcineurin and caspase-3 and use morphological, histological, and behavioral criteria to measure indices of apoptotic cell death and functional recovery. The outcome of these studies will have clear clinical implications for minimizing secondary neuronal and glial cell death and promoting recovery of function following SCI.