Abstract
Objective: In order to study the mechanisms associated with neovascularization after indirect bypass (EDAS), we have developed a mice model of synangiosis and cerebral ischemia. Method: Healthy 12-24 week old male C57/BL6J mice were used. The temporalis muscle served as donor for the external carotid artery vessels through a window in the temporalis bone. The distal MCA was ligated after opening the dura. The temporalis muscle was reattached in direct apposition to the ischemic brain surface. Animals were sacrificed at one month and their heads were prepared in block. Brain slices were stained with CD31 (endothelial cell marker), Ki67 (proliferation maker), and DAPI (nuclear marker) antibodies. Confocal laser microscopy was used for immunofluorescent imaging. Standardized relative quantification of immunofluorescence staining was performed using ImageJ following the Arqués protocol. Inferential comparisons among groups were performed with a generalized linear model. Data was nested in the model by animal to account for intrasubject correlation. Results: A total of 12 mice were operated. One subject died after surgery (attrition rate 8%). All other subjects displayed no deficits. The number of DAPI+ cells/image was no different between the synangiosis and the control side. The index CD31/DAPI was significantly higher in the synangiosis side (mean 0.90 ± 1.13 vs. controls: 0.60 ± 0.65, p<0.001). The proliferation index Ki67/DAPI was significantly higher in the synangiosis side (mean 0.21 ± 0.08 vs. controls: 0.12 ± 0.05, p=0.03). There was 8% colocalization of the 3 markers on the synangiosis side and none on the controls. Conclusions: Our model of synangiosis in mice was well tolerated and produced quantifiable neovascularization in the brain as indicated by the neovascularization indexes observed in confocal microscopy. This model of synangiosis is a promising tool for future studies of neovascularization and collateral formation in individuals with ischemia.
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