Characterization of vascular protein expression patterns in cerebral ischemia/reperfusion using laser capture microdissection and ICAT‐nanoLC‐MS/MS

Cerebral ischemia rapidly initiates structural and functional changes in brain vessels, including blood-brain barrier disruption, inflammation, and angiogenesis. Molecular events that accompany these changes were investigated in brain microvessels extracted using laser-capture microdissection (LCM) from Sprague-Dawley rats subjected to a 20 min transient global cerebral ischemia followed by 1, 6, or 24 h reperfusion. Proteins extracted from approximately 300 LCM captured microvessels (20-100 microm) 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. Pattern analyses using k-means clustering method classified 57 differentially expressed proteins in 7 distinct dynamic patterns. Protein function was assigned using Panther Classification system. Early reperfusion (1 h) was characterized by down-regulation of ion pumps, nutrient transporters, and cell structure/motility proteins, and up-regulation of transcription factors, signal transduction molecules and proteins involved in carbohydrate metabolism. The up-regulation of inflammatory cytokines and proteins involved in the extracellular matrix remodeling and anti-oxidative defense was observed in late reperfusion (6-24 h). The up-regulation of IL-1beta and TGF-1beta in ischemic brain vessels was confirmed by ELISA, quantitative PCR, and/or immunohistochemistry. A biphasic postischemic (1 and 24 h) BBB opening for (3)H-sucrose was evident in the same model. Differentially expressed proteins identified in brain vessels during reperfusion are likely involved in orchestrating functional vascular responses to ischemia, including the observed BBB disruption.