Mitochondria – eNOS – estrogen axis provides pathway to protection against stroke
Authors: Rutkai Ibolya , Wunnava Sanjay V. , Merdzo Ivan , Katakam Prasad V. , Busija David W. 
Published: November 2023

Various studies have provided evidence indicating that circulating sex hormones alter mitochondrial characteristics of the cerebral circulation and provide protection against ischemic stroke. We have previously observed a difference in the mitochondrial function of large cerebral arteries from male (M) and female (FM) rats although the underlying mechanisms are not clear. In addition, our laboratory made the novel and important finding that following transient middle cerebral artery occlusion (MCAO), mitochondrial protein mass, mitochondrial‐derived vasodilation of the MCAs, and mitochondrial oxygen consumption (OCR) on the side ipsilateral (ipsi) to MCAO are intact, whereas all variables are severely reduced on the side contralateral (contra) to MCAO. Our aim was to elucidate the specific subcellular effects of estrogen in the cerebral vasculature under normal conditions and following MCAO.Eight to ten week old M and FM Sprague‐Dawley rats as well as age matched ovariectomized (OVX) rats treated with either a 21 day release, 0.5 mg of 17 beta‐estradiol (OVX+E) or placebo (OVX) pellet were used. The rats were exposed to 90 min of MCAO and 48 h of reperfusion and OCR in pM/min/μg protein was measured using the Seahorse Bioscience XFe24 Analyzer. Western blot was used to determine the protein expression levels of the endothelial and neuronal nitric oxide synthase (NOS) and estrogen receptors (ER) alpha (ERα) and beta (ERβ). A Zeiss laser scanning confocal microscope system was used to investigate vascular smooth muscle calcium spark activity (Ca2+ spark/cell/second).Estradiol treatment resulted in a significantly (p<0.05) greater mitochondrial respiration in OVX+E (basal: 147±9, ATP production: 44±4, proton leak: 102±7, and maximal: 212±13) compared with OVX (105±13, 21±4, 50±6, and 138±10, respectively). Cerebrovascular mitochondria primarily express ERβ whereas vascular tissue express more ERa than ERb. Ratios of phosphorylated eNOS (1.43±0.06) and nNOS (1.15±0.2) normalized to total eNOS and nNOS were significantly (p<0.05) elevated in the OVX+E compared with OVX group (0.92±0.06, 0.53±0.19, respectively). Furthermore, we found a smaller infarct size in females compared with males following MCAO possibly due to the effect of estrogen. This resulted in a greater Ca2+ spark activity in female MCAs (0.69±0.05) compared with males (0.49±0.06) under normal condition and following ischemia (female ipsi: 0.43±0.04 and contra: 0.41±0.07; male ipsi: 0.14±0.06 and contra: 0.26±0.04).Our findings provided new information concerning sex‐specific differences in mitochondrial function using a novel approach on freshly isolated cerebral arteries. Our results provide new mechanistic insights regarding the underlying mechanisms of sex‐related differences of cerebral arteries in health and disease.Support or Funding InformationThis work was supported by National Institutes of Health grants (DWB: HL‐077731and HL093554), Louisiana Board of Regents Support Fund‐Research Competitiveness Subprogram (PVK: LEQSF(2014‐17)‐RD‐A‐11), American Heart Association National Center NRCP Scientist Development Grant (PVK: 14SDG20490359), and American Heart Association Post‐Doctoral Fellowship Grant (IR: 15POST23040005). This research was supported in whole or in part by the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program.