Introduction
Ischemic stroke causes profound anatomical and functional changes in the neurovascular unit. Molecular effectors of these changes were investigated in laser-capture microdissection (LCM)-extracted ischemic brain vessels using ICAT-based proteomics adapted to small tissue samples.
Methods
A transient 20-min forebrain ischemia followed by 1, 6 or 24 h of reperfusion was induced in Sprague-Dawley rats by bilateral common carotid artery occlusion and hypotension (42–45 mmHg) 1 . Proteins extracted from ∼300 LCM captured microvessels (<100 um) were ICAT-labeled and analyzed by nanoLC-MS. In-house software was used to identify paired ICAT peaks, which were then sequenced by nanoLC-MS/MS. The proteins were clustered using k-means clustering method in Matlab 7.0 software and categorized according to their function using Panther Classification system.
Results
Pattern analyses classified 57 differentially expressed proteins in 7 distinct dynamic patterns (Fig. 1). Early reperfusion was characterized by down-regulation of ion pumps, nutrient transporters and cell structure/motility proteins, and the up-regulation of transcription factors, signal transduction and proteins involved in carbohydrate metabolism. In late reperfusion (6–24 h) up-regulation of inflammatory cytokines, proteins involved in the extracellular matrix remodeling and anti-oxidative defence were observed. The up-regulation of IL-1alpha and TGF-1beta in ischemic brain vessels was confirmed by ELISA and immunohistochemistry.
Conclusions
Observed protein expression profiles in brain vessels after a transient global ischemia can be ‘mapped’ to and possibly causally associated with functional vascular responses previously reported in the same model (2) including a biphasic (1 h and 24 h) BBB opening and vasogenic brain edema. 3.