Proteomic Analysis of Rat Cerebral Cortex in the Subacute to Long-Term Phases of Focal Cerebral Ischemia-Reperfusion Injury

Abstract

Stroke is a leading cause of mortality and disability, and ischemic stroke accounts for more than 80% of the disease occurrence. Timely reperfusion is essential in the treatment of ischemic stroke, but it is known to cause ischemia-reperfusion (I/R) injury and the relevant studies have mostly focused on the acute phase. Here we reported on a global proteomic analysis to investigate the development of cerebral I/R injury in the subacute and long-term phases. A rat model was used, with 2 h-middle cerebral artery occlusion (MCAO) followed with 1, 7, and 14 days of reperfusion. The proteins of cerebral cortex were analyzed by SDS-PAGE, whole-gel slicing, and quantitative LC-MS/MS. Totally 5621 proteins were identified, among which 568, 755, and 492 proteins were detected to have significant dys-regulation in the model groups with 1, 7, and 14 days of reperfusion, respectively, when compared with the corresponding sham groups (n = 4, fold change ≥1.5 or ≤0.67 and p ≤ 0.05). Bioinformatic analysis on the functions and reperfusion time-dependent dys-regulation profiles of the proteins exhibited changes of structures and biological processes in cytoskeleton, synaptic plasticity, energy metabolism, inflammation, and lysosome from subacute to long-term phases of cerebral I/R injury. Disruption of cytoskeleton and synaptic structures, impairment of energy metabolism processes, and acute inflammation responses were the most significant features in the subacute phase. With the elongation of reperfusion time to the long-term phase, a tendency of recovery was detected on cytoskeleton, while inflammation pathways different from the subacute phase were activated. Also, lysosomal structures and functions might be restored. This is the first work reporting the proteome changes that occurred at different time points from the subacute to long-term phases of cerebral I/R injury and we expect it would provide useful information to improve the understanding of the mechanisms involved in the development of cerebral I/R injury and suggest candidates for treatment.

Differential proteomic analysis of mouse cerebrums with high-fat diet (HFD)-induced hyperlipidemia

“…Western blotting of the brain proteins was performed as previously reported ( Wen et al, 2019 ). Briefly, the protein samples were thawed and supplemented to have final concentrations of 4 or 6.15 µg/µL protein, 2% w/v SDS, 100 mM DTT and 12% w/v sucrose in 0.01 M Tris-0.02 M glycine buffer (pH 9.0).…”

1DE-MS Profiling for Proteoform-Correlated Proteomic Analysis, by Combining SDS-PAGE, Whole-Gel Slicing, Quantitative LC–MS/MS, and Reconstruction of Gel Distributions of Several Thousands of Proteins

“…Animal experiments have been previously reported in detail. 1 Briefly, as shown in Figure 1A, a rat model of focal cerebral I/R injury was built by 2 h-MCAO followed with 1, 7, and 14 days of reperfusion. Sham groups were set up for the three reperfusion time points accordingly, with the animals treated with identical surgical procedures except that the occlusion suture was not advanced to block the middle cerebral artery (MCA).…”

“…The procedures have been reported in detail previously, including protein extraction, SDS-PAGE, whole-gel slicing, in-gel digestion, and LC−MS/MS measurement. 1 Briefly, proteins were extracted from the ipsilateral cerebral cortices of the damaged region using denaturing and reducing conditions, successively by bead-beating homogenization, addition of 2% w/v SDS and 0. 1B).…”

“…Briefly, we re-analyzed the data previously obtained by combining SDS-PAGE, whole-gel slicing, and LC−MS/MS with rat cerebral cortex proteins after focal cerebral ischemia-reperfusion (I/R) injury. 1 The rat model was established by 2 h middle cerebral artery occlusion (MCAO) followed with 1, 7, and 14 days of reperfusion, and sham groups were set accordingly. The cerebral cortex proteins (6 groups, 4 rats per group) were separated by SDS-PAGE, and each sample lane was cut into 35 squares, and all the gel pieces were analyzed with standardized procedures of in-gel digestion and LC−MS/MS.…”

Phosphoproteome Analysis Identifies a Synaptotagmin-1-Associated Complex Involved in Ischemic Neuron Injury

“…Recent advances in phosphoproteomic and proteomic technologies offer opportunities to study the global phosphorylated protein and protein landscape of various samples in a single experiment (3)(4)(5)(6). Neurons in the cortex, striatum and hippocampal region are highly vulnerable to cerebral ischemia, and the proteome of the cortex and striatum after cerebral ischemia has been established (7)(8)(9)(10)(11)(12). To further uncover the biological events in the hippocampus involved in cerebral ischemia-induced neuronal injury, we used the mouse hippocampus of a focal cerebral ischemia model and monitored changes in phosphorylated protein abundance using a combination of stable isotope labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS) to systematically profile the phosphoproteome and proteome after acute cerebral ischemia onset.…”