Department of Cardiovascular Surgery, Kyushu University Graduate School of Medicine, Fukuoka, Japan.
Department of Clinical Research, National Hospital Organisation Sendai Medical Centre, Sendai, Japan.
Eur J Vasc Endovasc Surg. 2020 May;59(5):824-833. doi: 10.1016/j.ejvs.2019.12.004. Epub 2019 Dec 26.
Necroptosis, a form of regulated necrosis, might be a potential mechanism of delayed paraplegia; therefore, its role in transient spinal cord ischaemia was investigated by immunohistochemical analysis of necroptosis related protein receptor interacting protein kinase (RIP) 1, RIP3, and cellular inhibitor of apoptosis protein (cIAP) 1/2.
This study used rabbit normothermic (n = 24) and hypothermic (n = 24) transient spinal cord ischaemia models and sham controls (n = 6). Neurological function was assessed according to a modified Tarlov score at 8 h, 1, 2, and 7 days after reperfusion (n = 6 each). Morphological changes in the spinal cord were examined using haematoxylin and eosin staining in the sham, 2, and 7 day groups. Western blot and histochemical analyses of RIP1, RIP3, and cIAP1/2, and double label fluorescent immunocytochemical studies of RIP3 and cIAP1/2 were performed at 8 h, 1, and 2 days after reperfusion (n = 6 each).
There were significant differences in neurological function between the normothermic and hypothermic groups (median scores 0 and 5 at 7 days, p = .023). In the normothermic group, most motor neurons were lost seven days after reperfusion (p = .046 compared with sham), but they were preserved in the hypothermic group. Western blot analysis revealed the upregulation of RIP1, RIP3, and cIAP1/2 at 8 h in the normothermic group (RIP1, p = .032; RIP3, p < .001; cIAP1/2, p = .041 compared with sham), and the overexpression of RIP3 was prolonged for two days. In the hypothermic group, the expression of these proteins was not observed. The double label fluorescent immunocytochemical study revealed the induction of RIP3 and cIAP1/2 in the same motor neurons.
These data suggest that transient normothermic ischaemia induces necroptosis, a potential factor in delayed motor neuron death, and that hypothermia may inhibit necroptosis.
细胞坏死是一种受调控的细胞死亡形式，可能是迟发性截瘫的潜在机制；因此，本研究通过免疫组化分析坏死相关蛋白受体相互作用蛋白激酶（RIP）1、RIP3 和细胞凋亡抑制蛋白（cIAP）1/2，探讨其在短暂性脊髓缺血中的作用。
本研究采用兔常温（n=24）和低温（n=24）短暂性脊髓缺血模型和假手术对照（n=6）。再灌注后 8 h、1、2 和 7 天，根据改良 Tarlov 评分评估神经功能（n=6）。假手术、2 天和 7 天组采用苏木精和伊红染色观察脊髓形态变化。再灌注后 8 h、1 和 2 天，采用 RIP1、RIP3 和 cIAP1/2 的 Western blot 和组织化学分析，以及 RIP3 和 cIAP1/2 的双标荧光免疫细胞化学研究（n=6）。
常温组和低温组神经功能存在显著差异（7 天的中位数评分分别为 0 和 5，p=0.023）。常温组再灌注 7 天后，大部分运动神经元丢失（与假手术组相比，p=0.046），但低温组保留。Western blot 分析显示，常温组再灌注 8 h 时 RIP1、RIP3 和 cIAP1/2 上调（RIP1，p=0.032；RIP3，p<0.001；cIAP1/2，p=0.041 与假手术组相比），RIP3 的过表达持续两天。低温组未观察到这些蛋白的表达。双标荧光免疫细胞化学研究显示，RIP3 和 cIAP1/2 诱导相同的运动神经元。
这些数据表明，短暂的常温缺血诱导了潜在的迟发性运动神经元死亡因素坏死，而低温可能抑制了坏死。
