Sevoflurane pre-conditioning increases phosphorylation of Erk1/2 and HO-1 expression via inhibition of mPTP in primary rat cortical neurons exposed to.

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Sevoflurane pre-conditioning increases phosphorylation of Erk1/2 and HO-1 expression via inhibition of mPTP in primary rat cortical neurons exposed to OGD/R Li-Min Zhang, Dong-Xue Zhang, Xiao-Chun Zhao, Wen-bo Sun, Xiao-Jing Jiang Journal of the Neurological Sciences Volume 372, Pages 171-177 (January 2017) DOI: 10.1016/j.jns.2016.11.055 Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 1 A review of mechanisms underlying neuroprotection by sevoflurane pre-conditioning. Journal of the Neurological Sciences 2017 372, 171-177DOI: (10.1016/j.jns.2016.11.055) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 2 Experimental protocol. To investigate the effects of atractyloside (a mPTP opener), cyclosporine A (a mPTP opening inhibitor), or PD98059 (an inhibitor of Erk1/2 phosphorylation) on sevoflurane pre-conditioning or OGD/R, atractyloside (50μM/L), cyclosporine A (50μM/L), or PD98059 (30μM/L) was administered 5min, 5min, or 1h before sevoflurane pre-conditioning and OGD/R, respectively. Anesthetic pre-conditioning was elicited by sevoflurane administration (2%) for 30min before OGD/R. Cell samples were obtained 60min after reperfusion. Vehicle control: neurons without sevoflurane or OGD/R treatment; OGD/R: neurons incubated with OGD for 90min, and reperfusion for 60min; SEVO+OGD/R: neurons incubated with 2% sevoflurane for 1h, exposed to OGD for 90min, and reperfusion for 60min; CSA+OGD/R: neurons incubated with cyclosporine for 5min, exposed to OGD for 90min, and reperfusion for 60min; ATR+SEVO+OGD/R: neuronal cultures were added into atractyloside for 5min prior to treatment with sevoflurane, incubated with sevoflurane for 1h, exposed to OGD for 90min, and reperfusion for 60min; PD+SEVO+OGD/R: neuronal cultures were added into PD98059 for 1h prior to treatment with sevoflurane, incubated with sevoflurane for 1h, exposed to OGD for 90min, and reperfusion for 60min; SEVO: neurons incubated with 2% sevoflurane for 1h; CSA: neurons incubated with cyclosporine for 5min; ATR: neurons incubated with atractyloside for 5min; PD: neuronal cultures were added into PD98059 for 1h; ATR+OGD/R: neuronal cultures were added into atractyloside for 5min prior to treatment with OGD, incubated with OGD for 90min, and reperfusion for 60min; PD+OGD/R: neuronal cultures were added into PD98059 for 1h prior to treatment with OGD, incubated with OGD for 90min, and reperfusion for 60min. Journal of the Neurological Sciences 2017 372, 171-177DOI: (10.1016/j.jns.2016.11.055) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 3 Opening of mPTP or inhibition of Erk1/2 phosphorylation reduced sevoflurane pre-conditioning induced increase of primary rat cortical neuronal viability after oxygen-glucose deprivation and resuscitation (OGD/R) treatment. (A) TUNEL-positive cells (TUNEL, green; DAPI, blue) increased after OGD/R indicating increased neuronal apoptosis, but the value was decreased after sevoflurane pre-conditioning treatment, while atractyloside or PD98059 reversed it. (B and C) Vehicle control, OGD/R, SEVO+OGD/R, CSA+OGD/R, ATR+SEVO+OGD/R, PD+SEVO+OGD/R, SEVO, CSA, ATR, PD, ATR+OGD/R and PD+OGD/R denotation were described previously. Data are presented as mean±SD (n=12/group). Comparisons between groups were using one-way ANOVA with Bonferroni-Dunn tests as post-hoc procedure. aP<0.05 vs. Vehicle control. bP<0.05 vs. OGD/R. cP<0.05 vs. SEVO+OGD/R. dP<0.05 vs. OGD/R. Journal of the Neurological Sciences 2017 372, 171-177DOI: (10.1016/j.jns.2016.11.055) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 4 Inhibition of Erk1/2 phosphorylation reduced sevoflurane pre-conditioning induced decrease of oxidative stress in primary rat cortical neuron culture after oxygen-glucose deprivation and resuscitation (OGD/R) treatment. (A) Representative western blot of HO-1. (B) Optical density (OD) value of HO-1 evaluated by western blot analysis. (C and D) MDA level revealed that the oxidative stress of primary cultured cortical neurons exposed to OGD and 60min reperfusion was increased, but SOD was decreased. Sevoflurane pre-conditioning partially restored oxidative stress, while atractyloside or PD98059 reversed it. Vehicle control, OGD/R, SEVO+OGD/R, CSA+OGD/R, ATR+SEVO+OGD/R, PD+SEVO+OGD/R, SEVO, CSA, ATR, PD, ATR+OGD/R and PD+OGD/R denotation were described previously. Data are presented as mean±SD (n=6/group). Comparisons between groups were using one-way ANOVA with Bonferroni-Dunn tests as post-hoc procedure. aP<0.05 vs. Vehicle control. bP<0.05 vs. OGD/R. cP<0.05 vs. SEVO+OGD/R. dP<0.05 vs. OGD/R. Journal of the Neurological Sciences 2017 372, 171-177DOI: (10.1016/j.jns.2016.11.055) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 5 Inhibition of Erk1/2 phosphorylation reduced sevoflurane pre-conditioning induced changes of primary rat cortical neuronal mitochondrial membrane potential after oxygen-glucose deprivation and resuscitation (OGD/R) treatment by JC-1 fluorescence staining assay. (A) Ratio of red/green fluorescence represented by flow cytometry decreased after OGD/R indicating decreased mitochondrial membrane potential, but the value was increased after sevoflurane pre-conditioning treatment, while PD98059 reversed it. Vehicle control, OGD/R, SEVO+OGD/R, CSA+OGD/R, ATR+SEVO+OGD/R, PD+SEVO+OGD/R, SEVO, CSA, ATR, PD, ATR+OGD/R and PD+OGD/R denotation were described previously. Data are presented as mean±SD (n=6/group). Comparisons between groups were using one-way ANOVA with Bonferroni-Dunn tests as post-hoc procedure. aP<0.05 vs. Vehicle control. bP<0.05 vs. OGD/R. cP<0.05 vs. SEVO+OGD/R. dP<0.05 vs. OGD/R. Journal of the Neurological Sciences 2017 372, 171-177DOI: (10.1016/j.jns.2016.11.055) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 6 Changes of phosphorylated Erk1/2 in mitochondria and opening of mitochondrial permeability transition pore in primary rat cortical neuron cultures caused by indicated stimuli. (A) Representative western blot of phosphorylated Erk1/2 (p-Erk) and total Erk1/2 (t-Erk) in mitochondria. (B) Optical density (OD) value of phosphorylated Erk1/2 evaluated by western blot analysis. Data are presented as mean±SD (n=6/group). aP<0.05 vs. Vehicle control. bP<0.05 vs. OGD/R. cP<0.05 vs. SEVO+OGD/R. dP<0.05 vs. OGD/R. (C) Relative normalized fluorescent unit represented by calein decreased after OGD/R indicating increased opening of mPTP, but the value was increased after sevoflurane pre-conditioning treatment, while PD98059 reversed it. Data are presented as mean±SD (n=6/group). Comparisons between groups were using one-way ANOVA with Bonferroni-Dunn tests as post-hoc procedure. aP<0.05 vs. Vehicle control. bP<0.05 vs. OGD/R. cP<0.05 vs. SEVO+OGD/R. dP<0.05 vs. ATR+SEVO+OGD/R. eP<0.05 vs. OGD/R. fP<0.05 vs. ATR+OGD/R. Vehicle control, OGD/R, SEVO+OGD/R, CSA+OGD/R, ATR+SEVO+OGD/R, PD+SEVO+OGD/R, SEVO, CSA, ATR, PD, ATR+OGD/R and PD+OGD/R denotation were described previously. Journal of the Neurological Sciences 2017 372, 171-177DOI: (10.1016/j.jns.2016.11.055) Copyright © 2016 Elsevier B.V. Terms and Conditions