Role of Endothelial Nitric Oxide Synthase in Isoflurane Conditioning‐Induced Neurovascular Protection in Subarachnoid Hemorrhage

Abstract

Background Delayed cerebral ischemia remains a common and profound risk factor for poor outcome after subarachnoid hemorrhage (SAH). The aim of our current study is to define the role of endothelial nitric oxide synthase (eNOS) in isoflurane conditioning‐induced neurovascular protection after SAH. Methods and Results Ten‐ to 14‐week‐old male wild‐type mice (C57BL/6) as controls and eNOS knockout male mice (strain # 002684) were obtained for the study. Animals underwent either sham surgery, SAH surgery, or SAH with isoflurane conditioning. Anesthetic post conditioning was performed with isoflurane 2% for 1 hour, 1 hour after SAH. Normothermia was maintained with the homeothermic blanket. In a separate cohort, nitric oxide synthase was inhibited by a pan nitric oxide synthase inhibitor, L‐nitroarginine methyl ester. Vasospasm measurement was assessed 72 hours after SAH and neurological function was assessed daily. Isoflurane‐induced changes in the eNOS protein expression were measured. eNOS protein expression was significantly increased by isoflurane conditioning in naïve mice as well as mice subjected to SAH. Vasospasm of the middle cerebral artery and neurological deficits were evident following SAH versus sham surgery, both in wild‐type mice and eNOS knockout mice. Isoflurane conditioning attenuated vasospasm and neurological deficits in wild‐type mice. This delayed cerebral ischemia protection was lost in L‐nitroarginine methyl ester ‐administered mice and eNOS knockout mice. Conclusions Our data indicate isoflurane conditioning provides robust protection against SAH‐induced vasospasm and neurological deficits, and that this delayed cerebral ischemia protection is critically mediated via isoflurane‐induced augmentation of eNOS.

Role of SIRT1 in Isoflurane Conditioning-Induced Neurovascular Protection against Delayed Cerebral Ischemia Secondary to Subarachnoid Hemorrhage

“…Our data also show that brief exposure to 2% isoflurane beginning one hour after SAH induction protects against one key element of DCI-large artery vasospasm, which is consistent with our past results [15]. In addition, we show that isoflurane conditioning protects against another critical component of DCI-microvessel thrombosis.…”

“…This protection was casually linked to endothelial cell-derived HIF-1α [14]. More recently, we have shown that isoflurane conditioning provides strong protection against vasospasminduced DCI and neurological deficits in a mouse model of SAH [15,39] and that this protection is critically dependent on eNOS [15].…”

“…With respect to SAH-induced brain injury, SIRT1 has been linked to two of the most important forms of post-ictal secondary brain injury: EBI [34][35][36] and DCI [22]. In the case of anesthetic conditioning, there is a growing body of preclinical and clinical evidence indicating a strong protective effect of inhalational anesthetics against SAH-induced DCI [15,[37][38][39][40]. Milner et al [14] previously noted that isoflurane conditioning provides robust protection against multiple elements of DCI including improved neurologic outcomes in a mouse model of SAH.…”

“…To address this issue, we and others have applied a therapeutic strategy-conditioning-that leverages endogenous molecular mechanisms to exert a powerful and remarkably pleiotropic protective effect against DCI after SAH. For years, neurons were felt to be the principal target of this response (neuronal conditioning) [8], but multiple lines of evidence now show that glia (glial conditioning) [9] and vessels (vascular conditioning) [10][11][12][13][14][15] are also causally involved. The latter is of special interest for SAH, given the central role vascular deficits (vasospasm, autoregulatory dysfunction, and microvessel thrombi) play in DCI [3][4][5][6][7].…”

“…In fact, work from the same laboratory has also causally linked the DCI protection afforded by hypoxic conditioning to eNOS [29], which suggests that the SIRT1-eNOS cascade is essential to the observed DCI protection. Interestingly, isoflurane conditioning has also been shown to provide strong DCI protection in SAH, and to do so via upregulation of eNOS [15] and HIF-1α [14]. Therefore, it is logical to posit that anesthetic conditioning-induced DCI protection after SAH may also be critically dependent on SIRT1.…”