Longitudinal examination of mitochondrial dynamics in vivo in cerebral vascular endothelial cells
Authors: Rutkai Ibolya , Evans Wesley R , Bess Nikita , Salter‐Cid Tomas , Cikic Sinisa , Katakam Prasad VG , Mostany Ricardo , Busija David W 
Published: June 2021

Mitochondria are important regulators of vascular function and provide resiliency against stress and disease but in vivo mitochondrial dynamics in the cerebral circulation are unexplored. To remedy this deficit we used multi‐photon microscopy, permanent mitochondrial labeling using mitochondrial Dendra2 fluorescent protein (mitoDendra2 FP) specific to the endothelium, and intravascular Rhodamine dextran to visualize the microcirculation in anesthetized mice. We determined optimal imaging conditions, validated the specificity of mitochondrial labeling in endothelium, developed approaches to data analysis, and explored usefulness of this approach in preliminary studies.Cranial windows were implanted in adult mice and the brain microcirculation and mitoDendra2 FP characteristics were examined in the same animal starting at 5 mo of age and at 2, 4, and 12 wks (Groups I–IV). Stacks of 100 consecutive images were taken at 1 μm increments. Immunohistochemistry and cultured brain microvascular cells confirmed the location of the mitochondrial signal in endothelium. Endothelial expression of mitoDendra2 FP was confirmed in vitro on brain slices using endothelium specific VWF and CD31 antibodies. In addition, we observed an overlapping mitoDendra2 FP and Chromeo mitochondrial staining of cultured brain microvascular endothelial cells. Mitochondrial and vessel area (Ma; Va; respectively; expressed in μm2), their ratio (Ma/Va), and the mean vessel diameter [Vd; expressed in μm] were determined using ImageJ. Mitochondrial and vessel area, as well as the vessel diameters were similar among groups [Ma I: 3,157 ± 488.9*; Ma II: 2,556 ± 512.9; Ma III: 2,174 ± 368.8; Ma IV: 3,178 ± 627.9; n = 24–27/session; * p <0.05 Ma I vs. II–IV; Va I: 16,138 ± 1,503; Va II: 16,627 ± 2,439; Va III: 14,634 ± 1,673; Va IV: 22,056 ± 2,940; n = 24–27/session; Ma/Va I: 0.182 ± 0.013#; Ma/Va II: 0.138 ± 0.009; Ma/Va III: 0.144 ± 0.011; Ma/Va IV: 0.138 ± 0.015; n= 24–27/session; #p <0.05 Ma/Va I vs. IV; Vd I: 5.00 ± 0.05; Vd II: 4.95 ± 0.07; Vd III: 4.84 ± 0.08; Vd IV: 5.00 ± 0.12; n = 8–9/session). In preliminary studies, we also found that exposure of the cerebral vasculature to 30–60 min of ischemia followed by reperfusion led to changes in mitochondrial dynamics including changes in appearance suggesting fragmentation. In addition, the mitoDendra2 FP signal was particularly pronounced in the vasculature of a juvenile mouse, possibly indicating the need for more mitochondrial‐derived ATP to promote blood vessel growth as the brain matures.Our results indicate that mito‐Dendra2 FP signal and vascular parameters were stable and suitable for longitudinal examination of the in vivo cerebral circulation in mice and that these methods are useful in examining the mechanisms of vascular development and remodeling and the responses of the cerebral microcirculation to disease processes.Support or Funding InformationAHA 17SDG33410366, U54 GM104940 and HL‐093554.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.