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Journal of Molecular Neuroscience

, Volume 61, Issue 3, pp 396–411 | Cite as

Alterations in the Cerebral Microvascular Proteome Expression Profile After Transient Global Cerebral Ischemia in Rat

  • Stine Spray
  • Sara E. Johansson
  • Alistair V. G. Edwards
  • Martin R. Larsen
  • Aneta Radziwon-Balicka
  • Gro K. Povlsen
  • Lars Edvinsson
Article
  • 245 Downloads

Abstract

This study aimed at obtaining an in-depth mapping of expressional changes of the cerebral microvasculature after transient global cerebral ischemia (GCI) and the impact on these GCI-induced expressional changes of post-GCI treatment with a mitogen-activated protein kinase kinase (MEK1/2) inhibitor. GCI was induced in male Wistar rats followed by treatment with either vehicle or the MEK1/2 inhibitor U0126 every 12 h post-GCI. Seventy-two hours after GCI or sham surgery, the cerebral microvasculature was isolated and the protein content analysed with state-of-the-art mass spectrometry. The proteomic profile of the isolated cerebral microvasculature 72 h after GCI (compared to sham) indicated that the main expressional changes could be divided into nine categories: (1) cellular respiration, (2) remodelling of the extracellular matrix, (3) decreased contractile phenotype, (4) clathrin-mediated endocytosis, (5) ribosomal activity, (6) expression of chromatin structure-related proteins, (7) altered synaptic activity, (8) altered G-protein signalling and (9) instability of the membrane potential. Treatment with U0126 partly normalized the expression of one or more of the proteins in all nine categories. Flow cytometry confirmed key findings from the proteome such as upregulation of the extracellular proteins lamininβ2 and nidogen2 (p < 0.05) after GCI. These results provide valuable molecular insight into the broad and complex expressional changes in the cerebral microvasculature after GCI and the effect of early MEK1/2 inhibitor treatment on these changes.

Keywords

Cerebral microvasculature Extracellular matrix remodelling Global cerebral ischemia MEK1/2 inhibitor treatment Proteomics 

Notes

Acknowledgements

We kindly acknowledge the Lundbeck Foundation for financial support (LE—Grant of Excellence and MRL—Junior Group Leader Fellowship).

Supplementary material

12031_2016_875_Fig4_ESM.gif (112 kb)
S1 Fig

Experimental characteristics of flow cytometry experiments. In each experiment we identified the total cell population on a forward scatter versus side scatter dot blot (A), followed by identification of the single-cell population P1 (B). For each antibody in every experiment the antibody specific signal was defined according to the IgG control (C-H). (GIF 111 kb)

12031_2016_875_MOESM1_ESM.tif (21.7 mb)
High resolution image (TIFF 22183 kb)
12031_2016_875_MOESM2_ESM.pdf (116 kb)
S1 Table Cerebral microvascular proteins up regulated both after GCI treated with vehicle and the MEK1/2 inhibitor U0126 (both compared to sham). Accession number and protein name is given along with the number of up regulated peptides the protein identification was based upon and the relative expressional increase of each protein based on the iTRAQ ratios (Log2). (PDF 116 kb)
12031_2016_875_MOESM3_ESM.pdf (98 kb)
S2 Table Cerebral microvascular proteins down regulated both after GCI treated with vehicle and the MEK1/2 inhibitor U0126 (both compared to sham). Accession number and protein name is given along with the number of down regulated peptides the protein identification was based upon and the relative expressional decrease of each protein based on the iTRAQ ratios (Log2). (PDF 98 kb)
12031_2016_875_MOESM4_ESM.pdf (140 kb)
S3 Table Proteins with a unique regulation in the cerebral microvasculature 72 h after GCI (vehicle) compared to sham. Accession number and protein name is given along with the number of up regulated peptides the protein identification was based upon and the relative expressional increase of each protein based on the iTRAQ ratios (Log2). (PDF 139 kb)
12031_2016_875_MOESM5_ESM.pdf (106 kb)
S4 Table Proteins with a unique down regulation in the cerebral microvasculature 72 h after GCI (vehicle) compared to sham. Accession number and protein name is given along with the number of up regulated peptides the protein identification was based upon and the relative expressional decrease of each protein is given based on the iTRAQ ratios (Log2). (PDF 106 kb)
12031_2016_875_MOESM6_ESM.pdf (159 kb)
S5 Table Proteins with a unique up regulation in the cerebral microvasculature 72 h after GCI treated with the MEK1/2 inhibitor U0126 compared to sham. Accession number and protein name is given along with the number of up regulated peptides the protein identification was based upon and the relative expressional increase of each protein based on the iTRAQ ratios (Log2). (PDF 158 kb)
12031_2016_875_MOESM7_ESM.pdf (120 kb)
S6 Table Proteins with a unique down regulation in the cerebral microvasculature 72 h after GCI treated with the MEK1/2 inhibitor U0126 compared to sham. Accession number and protein name is given along with the number of down regulated peptides the protein identification was based upon and the relative expressional decrease of each protein based on the iTRAQ ratios (Log2). (PDF 120 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Clinical Experimental Research, Glostrup Research InstituteRigshospitaletGlostrupDenmark
  2. 2.Department of Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdenseDenmark
  3. 3.Department of Clinical Sciences, Division of Experimental Vascular ResearchLund UniversityLundSweden

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