Comparative Proteome Analysis Of Cerebral Microvessels Following Subarachnoid Hemorrage In Rats

ObjectiveTo establish a technical platform of cerebral microvessels isolated bylaser capture microdissection (LCM) and their proteome analyzed bytwo-dimensional gel electrophoresis (2-DE). Further, to investigatedifferentially expressed proteins in cortical microvessels followingsubarachnoid hemorrage in rats by comparative proteomics in order toelucidate the molecular mechanism underlying the microvessel injuryfollowing subarachnoid hemorrage at the protein level.Methods1Induction of SAH model in rat: SAH models were induced by aprechiasmatic blood injection technique.2Analysis of dynamic changes in cortical microvessels after SAH:Blood–brain barrier (BBB) permeability was determined at different timepoints by fluorescence spectrophotometer and fluorescence microscopy,respectively. The ultrastructural changes in BBB were observed withtransmission electron microscope.3Isolation of cortical microvessels by LCM after SAH: Immunohistochemical staining (antibody to CD31) was employed to labelendothelial cells throughout the cerebral microvascular tree. Selectivemicrodissection of labeled cortical microvessels in frozen tissue sections byLCM.4Analysis of differentially expressed proteins in cortical microvesselsat different time points following subarachnoid hemorrage in rats bycomparative proteomics: Firstly, optimal parameters and conditions of2-DE were ascertained, including suitable loading quantity of sample, IEF,staining method etc. Secondly, microvessels proteome at various timepoints post-SAH were separated by2-DE. Next, PDQuest software (7.4)was applied to analyze2-DE images to screen differentially expressedprotein spots. Differentially expressed protein spots were subjected todigest with trypsin, and then identified by matrix assisted laserdesorption/isonization time of flying mass spectrometry (MALDI-TOF-MS)to obtain peptide mass fingerprint (PMF). PMF was used to search theSwiss-Prot or NCBInr database with MASCOT search engine.5Validation of candidate differential proteins: Interesting proteinswere selected and subjected to Western blotting analysis.Results1The subarachnoid blood was widely distributed throughout theanterior cranial fossa base, Willis circle, basal cisternal system and over thehemispheres and cerebellum at6h after SAH induction. 2SAH brought about a significant increase in cortical BBBpermeability. BBB permeability began to increase at24hours after SAH,peaked at36hours, and significantly declined at later observations period,normalized at72hours after SAH. Electron micrograph demonstrated at36hours post-SAH, a notable perivascular edema combined with a collapse ofthe capillary. Astrocytic end feet and mitochondria were swollen. Thelumina of the endothelial cells appeared unsmooth. The tight junctions andthe basal laminas were identified normal.3Preparation methods of PEN tissue sections (including cutting,fixation, immunostaining and dehydration) and microdissection parameterswere optimized before isolation of microvessels by LCM. Approximately32000cortical microvessels from6h group and32500cortical microvesselsfrom36h group were successfully isolated by immuno-LCM.4Based on optimal conditions and parameters of2-DE, the loadingquantity of sample, category of IPG strips and staining methods weredetermined as120μg, nonlinear IPG strip (pH3-10,17cm) and silverstaining, respectively. Compared with SAH6h group, eighty-onedifferentially expressed protein spots were showed in SAH36h group.Among them, forty-eight protein spots were up-regulated markedly, whilethirty-three protein spots were down-regulated significantly. Thirtydifferential proteins were randomly selected and subjected to identify byMALDI-TOF-MS and bioinformatics analysis. Twenty-four differential proteins were successfully identified.5To validate the2-DE results, two interesting proteins, VDAC1andMAPK10, were selected and subjected to Western blotting analysis. Theresults showed that both VDAC1and MAPK10had an increasedabundance in SAH36h group as compared with SAH6h group.Conclusions1The prechiasmatic blood injection model resembles clinical SAH. Itis very reproducible and appropriate for the study on thepathophysiological mechanisms of aneurysmal SAH in the anteriorcirculation.2SAH could induce rapid changes in microvascular function andstructure at36hours. Moreover, microvascular dysfunction may play acrucial role in the development of secondary brain injury and unfavorableoutcome.3LCM technique is a powerful method which could isolate purermicrovessels from complicated brain tissue than other methods, and couldbe applied in the fields of genomics and proteomics.4Twenty-four differentially expressed proteins identified in this studymight be strongly associated with the development of cerebral microvesselinjury following SAH.