Quantitative Neuroproteomics of an In Vivo Rodent Model of Focal Cerebral Ischemia/Reperfusion Injury Reveals a Temporal Regulation of Novel Pathophysiological Molecular Markers

Cerebral ischemia or stroke, an acute neurological injury lacking an effective therapy, is the second leading cause of death globally. The unmet need in stroke research is to identify viable targets and to understand their interplay during the temporal evolution of ischemia/reperfusion (I/R) injury. Here we report a temporal signature of the ischemic hemisphere revealed by the isobaric tag for relative and absolute quantification (iTRAQ)-based 2D-LC–MS/MS strategy in an in vivo middle cerebral artery occlusion (MCAO) model of focal cerebral I/R injury. To recapitulate clinical stroke, two hours of MCAO was followed by 0, 4, and 24 h of reperfusion to capture ischemia with an acute and subacute durations of reperfusion injury. The subsequent iTRAQ experiment identified 2242 proteins from the ischemic hemisphere with <1.0% false discovery rate. Data mining revealed that (1) about 2.7% of detected proteins were temporally perturbed having an involvement in the energy metabolism (Pygb, Atp5b), glutamate excitotoxicity (Slc1a3, Glud1), neuro-inflammation (Tf, C3, Alb), and cerebral plasticity (Gfap, Vim, Gap43); (2) astrocytes participated actively in the neurometabolic coupling underlining the importance of a cerebro-protective rather than a neuro-protective approach; and (3) hyper-acute yet progressive opening of the blood brain barrier (BBB), accompanied by stimulation of an innate immune response and late activation of a regenerative response, which provides an extended therapeutic window for intervention. Several regulated proteins (Caskin1, Shank3, Kpnb1, Uchl1, Mtap6, Epb4.1l1, Apba1, and Ube1x) novel in the context of stroke were also discovered. In conclusion, our result supports a dynamic multitarget therapy rather than the traditional approach of a unilateral and sustained modulation of a single target to address the phasic regulation of an ischemic proteome.

脑缺血即脑卒中，是一种缺乏有效治疗手段的急性神经损伤，已成为全球第二大死亡原因。脑卒中研究领域尚未被满足的临床需求，是识别可行的治疗靶点，并阐明缺血再灌注（I/R）损伤随时间进展过程中各靶点的相互作用。本研究基于同重同位素相对与绝对定量（iTRAQ）的二维液相色谱-串联质谱（2D-LC–MS/MS）技术，在局灶性脑I/R损伤的体内大脑中动脉闭塞（MCAO）模型中，揭示了缺血侧大脑半球的时间特征谱。为模拟临床脑卒中的病理过程，我们采用大脑中动脉闭塞2小时后分别再灌注0、4、24小时的建模方案，以捕捉急性与亚急性再灌注损伤阶段的缺血状态。后续的iTRAQ实验在缺血侧大脑半球中共鉴定出2242种蛋白质，错误发现率低于1.0%。数据挖掘结果显示：(1) 约2.7%的检测蛋白呈现时间动态扰动，分别参与能量代谢（Pygb、Atp5b）、谷氨酸兴奋性毒性（Slc1a3、Glud1）、神经炎症（Tf、C3、Alb）以及脑可塑性调节（Gfap、Vim、Gap43）；(2) 星形胶质细胞积极参与神经代谢耦合，这凸显了脑保护而非单纯神经保护策略的重要性；(3) 血脑屏障（BBB）在超急性期即出现进行性开放，伴随先天免疫应答的激活与再生应答的晚期激活，这为干预治疗提供了更宽泛的时间窗口。本研究还发现了数个在脑卒中研究背景下尚未被报道的调控蛋白：Caskin1、Shank3、Kpnb1、Uchl1、Mtap6、Epb4.1l1、Apba1及Ube1x。综上，本研究结果支持采用动态多靶点治疗策略，而非传统的单一靶点持续单向调控方案，以应对缺血蛋白质组的阶段性调控特征。