非人灵长类动物脑梗死后远隔部位和周围神经继发性损害以及PSD-95抑制剂的保护作用

Our previous studies found that cerebral infarction not only led to necrosis in the ischemic core lesion, but also induced secondary damage in nonischemic areas and even peripheral nerves remote from the primary focus, which hampers the neurological function recovery. The underlying mechanisms are complicated, in which excitotoxicity may be involved. Therefore, the targeted prophylactic and therapeutic strategies were limited. Emerging evidence indicates that PSD-95 is the crucial protein mediating excitotoxicity after cerebral infarction, and applying PSD-95 inhibitor within 3 hours following cerebral ischemia have shown promising neuroprotective effect in cynomolgus monkey models. However, due to the restriction of strict time window, the clinical feasibility was limited. The remote lesions occur several days after cerebral infarction and have clinical feasible therapeutic time window. Therefore, investigating the efficacy of PSD-95 inhibitor in remote lesions after cerebral infarction has great significance. To address these issues, the present project is designed to take advantage of an ischemic stroke model of nonhuman primates that we have developed successfully, and to administrate PSD-95 inhibitor or placebo 24 hours following operation. Secondly, multi-modal magnetic resonance imaging is applied to study the secondary change pattern of the whole brain, electromyography to detect the electrophysiological alteration of peripheral nerves and muscles, and molecular pathology to analyze the underlying mechanisms of the secondary changes mentioned above at 1, 4, and 12 week after onset. Finally, we aim to examine the neuroprotective effect of PSD-95 for improving remote lesions and neurological function after cerebral infarction. Findings from the present study would be of great scientific value and potential clinical significance for neurological function recovery following ischemic stroke.

我们前期研究发现，脑梗死可致远离缺血区的远隔部位甚至周围神经出现继发损害并阻碍神经功能恢复。远隔损害发生机制复杂，兴奋性毒性可能参与其中，但缺乏明确有效的防治手段。新近发现，PSD-95是介导兴奋性毒性的重要物质，脑缺血3小时内应用PSD-95抑制剂在食蟹猴模型中显示出良好的保护作用，但由于严格的时间窗限制，临床可行性差。而远隔损害出现在脑梗死后数天，具备临床可行的干预时间窗，研究PSD-95抑制剂在脑梗死后远隔损害中的作用意义重大。本项目拟采用我们前期研究已成功构建的非人灵长类动物脑梗死模型，术后24小时给予药物或安慰剂，第1、4、12周运用多模态磁共振研究全脑继发性改变模式，同步采用肌电图检测周围神经电生理变化，并联合分子病理学分析上述继发性改变的发生基础，深入探讨PSD-95抑制剂对脑梗死后远隔损害及神经功能的保护作用。研究结果对脑梗死后神经功能恢复将具有重要科学价值和潜在临床意义。

本研究的主要内容是探究食蟹猴脑梗死后脑内多个远隔部位及运动传导通路特别是脊髓和周围神经出现的继发性损害。另外，本研究在食蟹猴脑梗 死后应用 PSD-95 抑制剂阻断谷氨酸介导的兴奋性细胞毒性，进一步观察是否 能减轻这种继发性损害并改善神经功能。首先我们成功复制了适合远隔损害研究的食蟹猴脑梗死模型，术后MRI显示脑皮层梗死灶，12 周时仍遗留较明显对侧肢体瘫痪症状。我们发现食蟹猴脑梗死后脑内丘脑、海马远隔部位出现继发性损害，术后 12 周同侧及对侧丘脑 NeuN+ 细胞数量均明显减少 (P < 0.05)，GFAP+和 Iba-1+细胞数量均明显增多 (P < 0.05)，且其细胞 体积较大，突触增多变粗，呈活化状态。进一步发现食蟹猴脑梗死后脊髓、周围神经、神经肌肉接头等部位出现继发性损害，术后 12 周颈 5 及腰 5 节段对侧脊髓前角 NeuN+神经元数量均明显减少(P < 0.05)，而同侧神经元数量无明显差异(P >0.05)。与假手术组相比，术后 12 周颈 5 及腰 5 节段同侧及对侧脊髓前角 GFAP+和 Iba-1+细胞数量均明显增多，呈活化状态， 对侧增加更为明显(P < 0.05);提示食蟹猴脑梗死后脊髓前角出现神经元丢失、星型胶质细胞和小胶质细胞增生及活化等继发性损害。但我们发现发现了食蟹猴脑梗死后给予PSD-95抑制剂不能减少梗死灶体积、神经功能缺失情况及皮质脊髓束的损害，我们发现给予PSD95抑 制剂的食蟹猴在MCAO后12周脑梗死体积无显著性差异(P >0.05)。PSD95抑制剂组食蟹猴与溶剂组相比脑梗死 神经功能评分无统计学差异(P>0.05)。确定性纤维 追踪分析显示给予PSD抑制剂的食蟹猴与对照组相比，左右侧皮质脊髓束纤 维束数量比例无显著性差异(P>0.05)。在白质微观结构分析方面，梗死侧(左侧)皮质脊髓束上的平均FA值两组无显著性差异 (P>0.05)