Late preconditioning with isoflurane in cultured rat cortical neurones

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Late preconditioning with isoflurane in cultured rat cortical neurones T. Kaneko, K. Yokoyama, K. Makita British Journal of Anaesthesia Volume 95, Issue 5, Pages 662-668 (November 2005) DOI: 10.1093/bja/aei228 Copyright © 2005 British Journal of Anaesthesia Terms and Conditions

Fig 1 Experimental protocol showing times of exposure to preconditioning stimuli and oxygen–glucose deprivation (OGD). This was referred to as ischaemic preconditioning (IPC) with OGD for 30 min. Cultures were replaced with medium containing 500 µM 5-hydroxydecanoate (5-HD) 5 min before OGD for 120 min in the 5-HD, IPC+5-HD and Diaz+5-HD groups, and 5 min before exposure to isoflurane 1.4% for 180 min in the ISO+5-HD group. BSS, balanced salt solution containing glucose; Diaz, diazoxide; ISO, isoflurane. British Journal of Anaesthesia 2005 95, 662-668DOI: (10.1093/bja/aei228) Copyright © 2005 British Journal of Anaesthesia Terms and Conditions

Fig 2 Lactate dehydrogenase (LDH) activity expressed as a percentage of total LDH (determined for each experiment by assaying the supernatant of sister cultures after 24 h of exposure to Triton X-100 1%). The small amount of LDH present in the medium of sister cultures without lethal oxygen–glucose deprivation (OGD) was subtracted from the levels in experimental conditions. The data were obtained from three independent experiments with at least eight wells per condition. Data are presented as mean (sd). *P<0.05 compared with the control group. #P<0.05 compared with the IPC+5-HD, Diaz+5-HD and ISO+5-HD groups. 5-HD, 5-hydroxydecanoate; IPC, ischaemic preconditioning; Diaz, diazoxide; ISO, isoflurane. British Journal of Anaesthesia 2005 95, 662-668DOI: (10.1093/bja/aei228) Copyright © 2005 British Journal of Anaesthesia Terms and Conditions

Fig 3 Neuronal viability expressed as a percentage of the sample cultured without lethal oxygen-glucose deprivation (OGD). The data were obtained from three independent experiments with at least eight wells per condition. Data are presented as mean (sd). *P<0.05 compared with the control group. #P<0.05 compared with IPC+5-HD, Diaz+5-HD and ISO+5-HD groups. 5-HD, 5-hydroxydecanoate; IPC, ischaemic preconditioning; Diaz, diazoxide; ISO, isoflurane. British Journal of Anaesthesia 2005 95, 662-668DOI: (10.1093/bja/aei228) Copyright © 2005 British Journal of Anaesthesia Terms and Conditions

Fig 4 Preconditioning-induced changes in β-actin, Kir6.2 and EAAC1 mRNA in cortical neurones. (a) Gels showing quantitative RT-PCR products. (b), mRNA abundance quantified by integrating the volume of bands from six separate experiments. Values are the means (sd) of the fold change compared with control. *P<0.05 compared with the control group. IPC, ischaemic preconditioning; Diaz, diazoxide; ISO, isoflurane. British Journal of Anaesthesia 2005 95, 662-668DOI: (10.1093/bja/aei228) Copyright © 2005 British Journal of Anaesthesia Terms and Conditions

Fig 5 Western blot analysis of Kir6.2 and EAAC1 in cortical neurones. (a) Representative Western blot showing the preconditioning effect on expression of Kir6.2 and EAAC1 proteins. (b) Protein abundance quantified by integrating the volume of bands from four separate experiments. The immunoreactivity of each sample was normalized to the amount of β-actin in the lysate fraction of that sample from the same gel. Values are the means (sd) of the fold change compared with control. *P<0.05 compared with the control group. IPC, ischaemic preconditioning; Diaz, diazoxide; ISO, isoflurane. British Journal of Anaesthesia 2005 95, 662-668DOI: (10.1093/bja/aei228) Copyright © 2005 British Journal of Anaesthesia Terms and Conditions