Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Neurobiol Dis. 2013 Mar;51:133-43. doi: 10.1016/j.nbd.2012.11.003. Epub 2012 Nov 10.
Treatments that could extend the therapeutic window of opportunity for stroke patients are urgently needed. Early administration of hyperbaric oxygen therapy (HBOT) has been proven neuroprotective in the middle cerebral artery occlusion (MCAo) in rodents. Our aim was to determine: 1) whether delayed HBOT after permanent MCAo (pMCAo) can still convey neuroprotection and restorative cell proliferation, and 2) whether these beneficial effects rely on HBO-induced activation of protein phosphatase-1γ (PP1-γ) leading to a decreased phosphorylation and ubiquitination of CREB and hence its stabilization. The experiments were performed in one hundred thirty-two male Sprague-Dawley rats with the body weight ranging from 240 to 270 g. Permanent MCAo was induced with the intraluminal filament occluding the right middle cerebral artery (MCA). In the first experiment, HBOT (2.5 ATA, 1h daily for 10 days) was started 48 h after pMCAo. Neurobehavioral deficits and infarct size as well as cyclic AMP response element-binding protein (CREB) expression and BrdU-DAB staining in the hippocampus and the peri-infarct region were evaluated on day 14 and day 28 post-MCAo. In the second experiment, HBOT (2.5 ATA, 1h) was started 3h after pMCAo. The effects of CREB siRNA or PP1-γ siRNA on HBO-induced infarct size alterations and target protein expression were studied. HBOT started with 48 h delay reduced infarct size, ameliorated neurobehavioral deficits and increased protein expression of CREB, resulting in increased cell proliferations in the hippocampus and peri-infarct region, on day 14 and day 28 post-MCAo. In the acute experiment pMCAo resulted in cerebral infarction and functional deterioration and reduced brain expression of PP1-γ, which led to increased phosphorylation and ubiquitination of CREB 24h after MCAo. However HBOT administered 3h after ischemia reversed these molecular events and resulted in CREB stabilization, infarct size reduction and neurobehavioral improvement. Gene silencing with CREB siRNA or PP1-γ siRNA reduced acute beneficial effects of HBO. In conclusion, delayed daily HBOT presented as potent neuroprotectant in pMCAo rats, increased CREB expression and signaling activity, and bolstered regenerative type cell proliferation in the injured brain. As shown in the acute experiment these effects of HBO were likely to be mediated by reducing ubiquitin-dependent CREB degradation owing to HBO-induced activation of PP1γ.
治疗方法可以延长中风患者的治疗窗口期，因此迫切需要找到这种治疗方法。高压氧治疗（HBOT）在啮齿动物大脑中动脉闭塞（MCAo）中已被证明具有神经保护作用。我们的目的是确定：1）永久性 MCAo（pMCAo）后延迟的 HBOT 是否仍能提供神经保护和恢复性细胞增殖，2）这些有益效果是否依赖于 HBO 诱导的蛋白磷酸酶-1γ（PP1-γ）的激活，从而导致 CREB 的磷酸化和泛素化减少，从而稳定 CREB。该实验在 132 只体重为 240 至 270g 的雄性 Sprague-Dawley 大鼠中进行。通过腔内细丝阻塞右侧大脑中动脉（MCA）诱导永久性 MCAo。在第一个实验中，HBOT（2.5ATA，每天 1 小时，共 10 天）在 pMCAo 后 48 小时开始。在 MCAo 后第 14 天和第 28 天，评估神经行为缺陷和梗死面积以及海马和梗死周围区域的环磷酸腺苷反应元件结合蛋白（CREB）表达和 BrdU-DAB 染色。在第二个实验中，HBOT（2.5ATA，1h）在 pMCAo 后 3h 开始。研究了 CREB siRNA 或 PP1-γ siRNA 对 HBO 诱导的梗死面积改变和靶蛋白表达的影响。延迟 48 小时开始的 HBOT 可减少梗死面积，改善神经行为缺陷，并增加 CREB 的蛋白表达，从而增加海马和梗死周围区域的细胞增殖，MCAo 后第 14 天和第 28 天。在急性实验中，pMCAo 导致脑梗死和功能恶化，并降低大脑中 PP1-γ 的表达，这导致 MCAo 后 24 小时 CREB 的磷酸化和泛素化增加。然而，缺血后 3 小时给予的 HBO 逆转了这些分子事件，导致 CREB 稳定，梗死面积减小，神经行为改善。用 CREB siRNA 或 PP1-γ siRNA 进行基因沉默会降低 HBO 的急性有益作用。总之，延迟的每日 HBOT 在 pMCAo 大鼠中表现出强大的神经保护作用，增加了 CREB 的表达和信号活性，并增强了受损大脑中的再生型细胞增殖。如急性实验所示，由于 HBO 诱导的 PP1γ 激活，减少了泛素依赖性 CREB 降解，这些 HBO 的作用可能是介导的。
