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Cerebral arterioles preparation and PECAM-1 expression in C57BL/6J and ApoE-/- mice
Abderrahmane Bengrine1, Charlene Da Silveira, Ziad A Massy
1INSERM ERI-12 (EA4292) and University of Picardie Jules Verne, Amiens, France.
Abstract:
Exploration of molecular mechanisms involved in alterations of cerebral blood vessels in pathology such as chronic kidney disease implies cerebral vessel isolation for proteins and mRNAs quantification. We describe an updated method to isolate brain microvessels from wild type (WT) mice at 14 weeks of age. The quality of cerebral arterioles preparation was determined by measuring the enzymatic activity of gamma-glutamyl transpeptidase, an enzyme that is especially expressed in cerebral microvessels, and the quantitative expression (Western Blot) of platelet endothelial cell adhesion molecule-1 (PECAM-1), alpha-actin and gamma-enolase as markers of respectively endothelial cells, smooth muscle cells and neurons. We then assessed PECAM-1 expression in 14 and 20 weeks old ApoE(-/-) and WT mice. PECAM-1 expression was increased in 14 weeks old ApoE(-/-) mice compared to age matched WT mice. This difference in PECAM-1 expression disappeared at 20 weeks of age. These findings indicate that the present method of mice cerebral arterioles isolation is a valid method. Moreover, PECAM-1 expression, a marker of endothelial cells, changes with age in cerebral arterioles of ApoE(-/-) mice.
Insights
Researchers developed a new method to isolate brain microvessels for studying diseases like chronic kidney disease. This technique helps analyze proteins and mRNAs, revealing changes in endothelial cells in ApoE(-/-) mice.
Area of Science:
- Neuroscience
- Vascular Biology
- Biochemistry
Background:
- Alterations in cerebral blood vessels are implicated in pathologies like chronic kidney disease.
- Studying these alterations requires reliable methods for isolating cerebral microvessels for molecular analysis.
- Existing methods may require optimization for accurate protein and mRNA quantification.
Purpose of the Study:
- To describe an updated and validated method for isolating brain microvessels from mice.
- To assess the quality of the isolated cerebral arterioles using specific enzymatic and protein markers.
- To investigate changes in endothelial cell markers in a mouse model of atherosclerosis.
Main Methods:
- Isolation of brain microvessels from wild-type (WT) mice.
- Enzymatic activity assay for gamma-glutamyl transpeptidase to confirm microvessel preparation quality.
- Western Blot analysis for platelet endothelial cell adhesion molecule-1 (PECAM-1), alpha-actin, and gamma-enolase.
- Comparative assessment of PECAM-1 expression in 14- and 20-week-old ApoE(-/-) and WT mice.
Main Results:
- The updated method successfully isolated cerebral arterioles, validated by gamma-glutamyl transpeptidase activity.
- Platelet endothelial cell adhesion molecule-1 (PECAM-1) expression was significantly increased in 14-week-old ApoE(-/-) mice compared to age-matched WT mice.
- This difference in PECAM-1 expression diminished by 20 weeks of age in ApoE(-/-) mice.
Conclusions:
- The described method provides a valid approach for isolating mouse cerebral arterioles for molecular studies.
- Platelet endothelial cell adhesion molecule-1 (PECAM-1) expression in cerebral arterioles exhibits age-dependent changes in ApoE(-/-) mice.
- This methodology facilitates research into the molecular mechanisms underlying cerebral vascular pathology.
