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In this in vivo and in vitro study, we aimed to investigate whether isoflurane preconditioning-induced neuronal protection is mediated by reactive oxygen species (ROS) signaling at the reperfusion stage. In the in vivo study, Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO) and in the in vitro study, rat pheochromocytoma (PC12) cells were subjected to oxygen glucose deprivation (OGD). Isoflurane preconditioning was carried out prior to MCAO or OGD and the ROS scavenger, N-2-mercaptopropiopylglycine (2-MPG), was administered at the start of reperfusion. Infarct volume, neurological severity score, and TUNEL staining were analyzed in the in vivo study and cell viability, Bcl-2/Bax ratio, cleaved caspase 3/caspase 3 ratio, and ROS fluorescence intensity were measured in the in vitro study. In the in vivo study, infarct volume, neurological severity score, and TUNEL-positive cell count were significantly decreased with preconditioning but were abrogated by administration of 2-MPG. In the in vitro study, cell viability and Bcl-2/Bax ratio were significantly increased with preconditioning, and cleaved caspase-3/caspase-3 ratio and ROS fluorescence intensity were significantly decreased. Administration of 2-MPG for 10 min abrogated this preconditioning effect, but it did not abolish the protection when administered for 60 min of reperfusion. Isoflurane preconditioning-induced protection was abolished by ROS scavengers at the start of reperfusion, indicating that ROS signaling can mediate the isoflurane preconditioning effect, which suggests that the time window can be important., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Isoflurane has either neuroprotective or neurotoxic effects. High-dose oxygen is frequently used throughout the perioperative period. We hypothesized that hyperoxia will affect cell viability of rat pheochromocytoma (PC12) cells that were exposed to isoflurane and reactive oxygen species (ROS) may be involved. PC12 cells were exposed to 1.2% or 2.4% isoflurane for 6 or 24h respectively, and cell viability was evaluated. To investigate the effects of hyperoxia, PC12 cells were treated with 21%, 50%, or 95% oxygen and 2.4% isoflurane for 6h, and cell viability, TUNEL staining, ROS production, and expression of B-cell lymphoma 2 (BCL-2), BCL2-associated X protein (BAX), caspase-3 and beta-site APP cleaving enzyme (BACE) were measured. ROS involvement was evaluated using the ROS scavenger 2-mercaptopropiopylglycine (MPG). The viability of cells exposed to 2.4% isoflurane was lower than that of cells exposed to 1.2% isoflurane. Prolonged exposure (6h vs. 24h) to 2.4% isoflurane resulted in a profound reduction in cell viability. Treatment with 95% (but not 50%) oxygen enhanced the decrease in cell viability induced by 2.4% isoflurane alone. Levels of ROS, Bax, caspase-3 and BACE were increased, whereas expression of Bcl-2 was decreased, in cells treated with 95% oxygen plus 2.4% isoflurane compared with the control and 2.4% isoflurane plus air groups. MPG attenuated the effects of oxygen and isoflurane. In conclusion, isoflurane affects cell viability in a dose- and time-dependent manner. This effect is augmented by hyperoxia and may involve ROS, the mitochondrial apoptotic signaling pathway, and β-amyloid protein., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Although anesthetic-induced inhibition of lipopolysaccharide (LPS)-induced lung injury has been recognized, the underlying mechanism is obscure. Some studies suggest that reactive oxygen species (ROS) by isoflurane play a crucial role for anesthetic-induced protective effects on the brain or the heart; however, it still remains controversial. In this study, we examined the role of isoflurane-derived ROS in isoflurane-induced inhibition of lung injury and nuclear factor κB (NFκB) activation in LPS-challenged rat lungs., Methods: Male Sprague-Dawley rats were subjected to inhalation of 1.0 minimum alveolar concentration of isoflurane for 60 minutes, and intratracheal LPS 0.1 mg was administered 60 minutes later. In some cases, ROS scavenger, 2-mercaptopropinyl glycine or N-acetylcysteine was given 30 minutes before isoflurane. ROS generation was measured by fluorometer before LPS challenge and 4 hours after. Isoflurane's preconditioning effect was assessed by histologic examination, protein content, neutrophil recruitment, and determination of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 levels in bronchoalveolar lavage fluid and lung tissue. Western blotting measured phosphorylation of inhibitory κB α (ser 32/36), NFκB p65, and inducible nitric oxide synthase (iNOS). TNF-α and IL-6 mRNA expression and immunofluorescence staining for iNOS were also assessed., Results: Isoflurane preconditioning reduced inflammatory lung injury and TNF-α, IL-1β, and IL-6 release in the lung. Isoflurane upregulated ROS generation before LPS but inhibited a ROS burst after LPS challenge. ROS scavenger administration before isoflurane abolished the isoflurane preconditioning effect as well as isoflurane-induced inhibition of phosphorylation of inhibitory κBα, NFκB p65, iNOS activation, and mRNA expression of TNF-α and IL-6 in acute LPS-challenged lungs., Conclusions: This study suggests a crucial role of upregulated ROS generation by isoflurane for modification of inflammatory pathways by isoflurane preconditioning in acute inflammation of the lung.

Background: Preexposure of brain to isoflurane, a commonly used anesthetic, induces ischemic tolerance. This phenomenon is called isoflurane preconditioning. However, it is not known whether isoflurane application after ischemia provides neuroprotection., Methods: Corticostriatal slices (400 microm) freshly prepared from adult male Sprague-Dawley rats were subjected to a 15-min oxygen-glucose deprivation (OGD; to simulate ischemia in vitro). Isoflurane was applied after OGD. Brain slices were harvested 2 h after OGD for measuring 2,3,5-triphenyltetrazolium chloride (TTC) conversion to quantify cell injury. Adult male Sprague-Dawley rats were also subjected to middle cerebral arterial occlusion for 90 min and then treated with or without 2% isoflurane for 60 min started at the onset of reperfusion. The infarct volumes, neurologic deficit scores, and performance on rotarod were evaluated at 24 h after the onset of reperfusion., Results: Isoflurane applied immediately after the 15-min OGD for 30 min dose-dependently reversed the OGD-induced decrease of TTC conversion. The TTC conversion was 34 +/- 16% and 58 +/- 28% of the control, respectively, for OGD alone and OGD plus 2% isoflurane (P < 0.05, n = 12). Application of 2% isoflurane for 30 min started at 10 min after the OGD also reduced the OGD-decreased TTC conversion. The presence of 0.3 microm glibenclamide, a general adenosine 5'-triphosphate-sensitive potassium channel blocker, or 500 microm 5-hydroxydecanoic acid, a mitochondrial adenosine 5'-triphosphate-sensitive potassium channel blocker, during the application of 2% isoflurane abolished the isoflurane preservation of TTC conversion. Application of isoflurane during reperfusion also improved neurologic outcome after brain ischemia., Conclusions: The results suggest that isoflurane administrated after OGD or brain ischemia provides neuroprotection. Mitochondrial adenosine 5'-triphosphate-sensitive potassium channels may be involved in this protection.