Chronic Diseases Research Center (CEDOC), Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal.
PLoS One. 2012;7(8):e42632. doi: 10.1371/journal.pone.0042632. Epub 2012 Aug 28.
Perinatal hypoxia-ischemia is a major cause of acute mortality in newborns and cognitive and motor impairments in children. Cerebral hypoxia-ischemia leads to excitotoxicity and necrotic and apoptotic cell death, in which mitochondria play a major role. Increased resistance against major damage can be achieved by preconditioning triggered by subtle insults. CO, a toxic molecule that is also generated endogenously, may have a role in preconditioning as low doses can protect against inflammation and apoptosis. In this study, the role of CO-induced preconditioning on neurons was addressed in vitro and in vivo. The effect of 1 h of CO treatment on neuronal death (plasmatic membrane permeabilization and chromatin condensation) and bcl-2 expression was studied in cerebellar granule cells undergoing to glutamate-induced apoptosis. CO's role was studied in vivo in the Rice-Vannucci model of neonatal hypoxia-ischemia (common carotid artery ligature +75 min at 8% oxygen). Apoptotic cells, assessed by Nissl staining were counted with a stereological approach and cleaved caspase 3-positive profiles in the hippocampus were assessed. Apoptotic hallmarks were analyzed in hippocampal extracts by Western Blot. CO inhibited excitotoxicity-induced cell death and increased Bcl-2 mRNA in primary cultures of neurons. In vivo, CO prevented hypoxia-ischemia induced apoptosis in the hippocampus, limited cytochrome c released from mitochondria and reduced activation of caspase-3. Still, Bcl-2 protein levels were higher in hippocampus of CO pre-treated rat pups. Our results show that CO preconditioning elicits a molecular cascade that limits neuronal apoptosis. This could represent an innovative therapeutic strategy for high-risk cerebral hypoxia-ischemia patients, in particular neonates.
围产期缺氧缺血是新生儿急性死亡和儿童认知及运动障碍的主要原因。脑缺氧缺血可导致兴奋性毒性和坏死及凋亡性细胞死亡，其中线粒体起着主要作用。通过轻微损伤触发的预处理，可以增加对主要损伤的抵抗力。CO 是一种内源性产生的有毒分子，可能在预处理中发挥作用，因为低剂量 CO 可以预防炎症和细胞凋亡。在这项研究中，我们在体外和体内研究了 CO 诱导预处理对神经元的作用。在经历谷氨酸诱导的凋亡的小脑颗粒细胞中，研究了 1 小时 CO 处理对神经元死亡（质膜通透性和染色质浓缩）和 bcl-2 表达的影响。在新生儿缺氧缺血的 Rice-Vannucci 模型（颈总动脉结扎+8%氧气 75 分钟）中研究了 CO 在体内的作用。通过立体学方法计数尼氏染色评估的凋亡细胞，并评估海马体中裂解的 caspase 3 阳性谱。通过 Western Blot 分析海马体提取物中的凋亡特征。CO 抑制兴奋性毒性诱导的细胞死亡并增加神经元原代培养物中的 Bcl-2 mRNA。在体内，CO 可防止海马体缺氧缺血诱导的细胞凋亡，限制从线粒体释放的细胞色素 c 并减少 caspase-3 的激活。然而，CO 预处理大鼠幼仔海马体中的 Bcl-2 蛋白水平更高。我们的结果表明，CO 预处理可引发限制神经元凋亡的分子级联反应。对于高危脑缺氧缺血患者，特别是新生儿，这可能代表一种创新的治疗策略。
