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Background Recent evidence implicates inflammation as a key driver in delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage (SAH). Inducible nitric oxide synthase (iNOS) is one of the known major mediators of inflammation. We previously showed that an inhalational anesthetic, isoflurane, provides strong protection against delayed cerebral ischemia after SAH. Our current study aims to define the role of iNOS in isoflurane conditioning-induced protection against delayed cerebral ischemia in a mouse model of SAH. Methods and Results The experiments used 10- to 14-week-old male wild-type (C57BL/6) and iNOS global knockout mice. Anesthetic conditioning was initiated 1 hour after SAH with isoflurane 2% for 1 hour. Isoflurane-induced changes in iNOS expression were measured. N-(3-(aminomethyl) benzyl) acetamidine, a highly selective iNOS inhibitor, was injected intraperitoneally immediately after SAH and then daily. Vasospasm, microvessel thrombosis, and neurological assessment was performed. Data were analyzed by 1-way ANOVA and 2-way repeated measures ANOVA followed by Student Newman Keuls comparison test. Statistical significance was set at P <0.05. Isoflurane conditioning downregulated iNOS expression in naïve and SAH mice. N-(3-(aminomethyl) benzyl) acetamidine attenuated large artery vasospasm and microvessel thrombosis and improved neurological deficits in wild-type animals. iNOS knockout mice were significantly resistant to vasospasm, microvessel thrombosis, and neurological deficits induced by SAH. Combining isoflurane with N-(3-(aminomethyl) benzyl) acetamidine did not offer extra protection, nor did treating iNOS knockout mice with isoflurane. Conclusions Isoflurane conditioning-induced delayed cerebral ischemia protection appears to be mediated by downregulating iNOS. iNOS is a potential therapeutic target to improve outcomes after SAH.

We recently reported that isoflurane conditioning provided multifaceted protection against subarachnoid hemorrhage (SAH)-induced delayed cerebral ischemia (DCI), and this protection was through the upregulation of endothelial nitric oxide synthase (eNOS). SIRT1, an NAD-dependent deacetylase, was shown to be one of the critical regulators of eNOS. The aim of our current study is to examine the role of SIRT1 in isoflurane conditioning-induced neurovascular protection against SAH-induced DCI. Mice were divided into four groups: sham, SAH, or SAH with isoflurane conditioning (with and without EX-527). Experimental SAH via endovascular perforation was performed. Anesthetic conditioning was performed with isoflurane 2% for 1 h, 1 h after SAH. EX-527, a selective SIRT1 inhibitor, 10 mg/kg was injected intraperitoneally immediately after SAH in the EX-527 group. SIRT1 mRNA expression and activity levels were measured. Vasospasm, microvessel thrombosis, and neurological outcome were assessed. SIRT1 mRNA expression was downregulated, and no difference in SIRT1 activity was noted after isoflurane exposure. Isoflurane conditioning with and without EX-527 attenuated vasospasm, microvessel thrombosis and improved neurological outcomes. Our data validate our previous findings that isoflurane conditioning provides strong protection against both the macro and micro vascular deficits induced by SAH, but this protection is likely not mediated through the SIRT1 pathway.

Delayed cerebral ischemia (DCI) is identified as one of the significant contributors to poor patient outcome after aneurysmal subarachnoid hemorrhage (SAH). We previously reported that a supratherapeutic dose of isoflurane conditioning (2%) provided robust protection against SAH-induced DCI. The aim of our current study is to compare the efficacy of the supratherapeutic dose of isoflurane to that typically used to establish general anesthesia or sedation. After IRB approval for animal studies, ten to fourteen-week-old wild-type male mice (C57BL/6) were divided into five groups - sham, SAH alone, or SAH with isoflurane conditioning (0.5%, 1%, and 2%). Conditioning was performed with one-hour of isoflurane initiated one-hour after induction of SAH via endovascular perforation technique. Vasospasm measurement in the middle cerebral artery was assessed 72 h after SAH. Neurological assessment was performed at baseline and for next three days after SAH. It was identified that all tested doses of isoflurane conditioning (0.5%, 1%, and 2%) significantly attenuated large artery vasospasm and markedly improved neurological deficits following SAH. No significant differences in neurovascular outcome were noted between the three doses of isoflurane conditioning. Our data show that isoflurane dosing typically used for general anesthesia (1%) or sedation (0.5%) provide similar levels of DCI protection in SAH as that provided by a supratherapeutic dose (2%). This result has important implications for future translational studies. Additional studies examining the therapeutic potential of anesthetic conditioning for SAH are therefore warranted., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Delayed cerebral ischemia (DCI) is the largest treatable cause of poor outcome after aneurysmal subarachnoid hemorrhage (SAH). Nuclear Factor Kappa-light-chain-enhancer of Activated B cells (NF-kB), a transcription factor known to function as a pivotal mediator of inflammation, is upregulated in SAH and is pathologically associated with vasospasm. We previously showed that a brief exposure to isoflurane, an inhalational anesthetic, provided multifaceted protection against DCI after SAH. The aim of our current study is to investigate the role of NF-kB in isoflurane-conditioning-induced neurovascular protection against SAH-induced DCI. Twelve-week-old wild type male mice (C57BL/6) were divided into five groups: sham, SAH, SAH + Pyrrolidine dithiocarbamate (PDTC, a selective NF-kB inhibitor), SAH + isoflurane conditioning, and SAH + PDTC with isoflurane conditioning. Experimental SAH was performed via endovascular perforation. Anesthetic conditioning was performed with isoflurane 2% for 1 h, 1 h after SAH. Three doses of PDTC (100 mg/kg) were injected intraperitoneally. NF-kB and microglial activation and the cellular source of NF-kB after SAH were assessed by immunofluorescence staining. Vasospasm, microvessel thrombosis, and neuroscore were assessed. NF-kB was activated after SAH; it was attenuated by isoflurane conditioning. Microglia was activated and found to be a major source of NF-kB expression after SAH. Isoflurane conditioning attenuated microglial activation and NF-kB expression in microglia after SAH. Isoflurane conditioning and PDTC individually attenuated large artery vasospasm and microvessel thrombosis, leading to improved neurological deficits after SAH. The addition of isoflurane to the PDTC group did not provide any additional DCI protection. These data indicate isoflurane-conditioning-induced DCI protection after SAH is mediated, at least in part, via downregulating the NF-kB pathway. [ABSTRACT FROM AUTHOR]