Assessing Mitochondrial Respiratory Function in Isolated Mouse Brain Microvessels using Seahorse XFe Analyzer: Role of Neuronal Nitric Oxide Synthase
Authors: Sure Venkata NLR. , Sakamuri Siva SVP. , Sperling Jared A. , Merdzo Ivan , Evans Wesley R. , Rutkai Ibolya , Busija David W. , Katakam Prasad VG. 
Published: June 2021

Microvessels play a critical role in the microcirculation of the organs thus contribute to their function through supplying nutrients and oxygen. They regulate blood flow within the organs, contributes to the blood pressure, and play a critical role in blood coagulation and tissue inflammation. Determining the functional changes in microvasculature helps in understanding physiology and pathophysiology of the microcirculation. Mitochondrial dysfunction is known to play a causative role in vascular diseases. High throughput methods are available to study the mitochondrial respiratory function in the isolated cells from the vasculature, but no methods are available to study mitochondrial function in isolated microvessels. Here we developed a high throughput method to study the mitochondrial respiratory function in the isolated mouse brain microvessels using Seahorse Xfe24 analyzer. We also tested the role of neuronal nitric oxide synthase (nNOS) in regulating mitochondrial respiration of the microvessels.MethodsWe isolated the microvessels from the saline‐perfused mouse brains (3 months of age) by the combination of gradient centrifugation using 17.5% Dextran (W/V, 60kD‐90kD) and filtration. Isolated microvessels (300μm ‐ 40μm) were treated with saline or nNOS inhibitor, N‐[4‐(2‐[(3‐Chlorophenyl) methyl] aminoethyl) phenyl]‐2‐thiophene carboximidamide dihydrochloride (ARL 17477, 1μM) are plated on to seahorse microculture plate. Oxygen consumption rate (OCR) was measured at the basal level and after the injection of oligomycin, FCCP and antimycin/rotenone. Basal and maximal respiration were measured along with ATP production, proton leak, and maximal respiration.ResultsMicrovessels isolated from mouse brain exhibited basal respiration of 71.3±3.6 picomoles of O2/min/μg protein, whereas the maximal respiration calculated from OCR after FCCP injection was 84.0±8.9 picomoles of O2/min/μg protein. Amount of oxygen consumed for the ATP production by mouse brain microvessels was 43.9±3.6 picomoles of O2/min/μg and proton leak calculated from deducting OCR after oligomycin injection from OCR after antimycin injection was 25.1±3.12 picomoles of O2/min/μg. Mouse brain microvessels exhibited non‐mitochondrial respiration of 29.1±2.4 picomoles of O2/min/μg. nNOS inhibitor, ARL treatment increased the maximal respiration by 21.2% (101.8±6.4 vs 84.0±8.9 picomoles of O2/min/μg) and ATP production by 19% (52.3±4.4 vs 43.9±3.6 picomoles of O2/min/μg) in mouse brain microvessels. Basal respiration, proton leak, and non‐mitochondrial respiration were not altered by ARL treatment in the microvessels.ConclusionsWe report a novel high‐throughput method for studying mitochondrial respiration in the isolated mouse brain microvessels. We conclude that neuronal NOS decreases the mitochondrial respiration of the microvasculature thus validating the method.Support or Funding InformationAmerican Heart Association (PVG: 14SDG20490359, IR: 17SDG33410366 and VNS: 16PRE31450006), and National Institute of Health: (PVK: R01NS094834 and DWB: HL‐077731, HL093554).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.