Premium
Effects of transient ischemia‐reperfusion on in vivo mitochondrial fission and fusion in the murine cerebral vasculature
Author(s) -
Rutkai Ibolya,
Chandra Partha K.,
Cikic Sinisa,
Mostany Ricardo,
Busija David W.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.05938
Subject(s) - ischemia , mitochondrial fission , perfusion , mitochondrion , mitochondrial fusion , cerebral circulation , medicine , biology , pathology , anatomy , microbiology and biotechnology , mitochondrial dna , biochemistry , gene
Mitochondria play important roles in the regulation of cerebral circulation. The effects of ischemia with and without reperfusion on mitochondrial morphology have been well documented in different cell types of the brain. However, the time course of these events, an important determinant of mitochondrial targeting, has been understudied due to technical limitations. The aim of this study was to determine how mitochondrial fission and fusion dynamics are affected by ischemia‐reperfusion, in endothelial cells that form the basis of the neurovascular unit. A thinned skull cranial window was implanted in young, male and female (3–6 month old; n = 5) offspring of B6;129S‐Gt(ROSA)26Sor tm1(CAG‐COX8A/Dendra‐2)Dcc /J and B6.Cg‐Tg(Tek‐cre) 1Ywa/J mice expressing mitoDendra 2 fluorescent protein in endothelial mitochondria. Two‐photon microscopy was used to visualize the brain vasculature, via RhodamineB Dextran labelled blood plasma and mitoDendra2 simultaneously at 875 nm. After 2 imaging sessions taken 60 minutes apart, animals were allowed to recover. Then, mice were exposed to 30 minutes of ischemia via middle cerebral artery occlusion followed by 4 hours of reperfusion. Additional time‐lapse images were taken between the 2 nd and 4 th hour of the re‐perfusion period. Three stacks of 50 images, 1 μm apart, were taken of different regions of interests, containing arteries, veins, and capillaries using a 40X objective and a 1X digital zoom; whereas 3X digital zoom was used to take images for the analyses of mitochondrial fission and fusion events. Ischemia‐reperfusion resulted in a prominent vasoconstriction of the pial vasculature (15% decrease in vessel diameter; n = 66 diameter measurements) as well as the disintegration of green mitochondria signal, suggestive of fission. The in‐depth analysis of time‐lapse images (8–12 stacks of images per channel per imaging session per mouse using a 3X digital zoom) is currently ongoing. Overall, we expect to correlate in vivo mitochondrial changes derived by fission and fusion with vascular events in response to ischemia‐reperfusion in large, penetrating arteries and veins as well as in capillaries. Support or Funding Information AHA 17SDG33410366; U54 GM104940; HL‐093554