Inhibiting NADPH oxidase protects against long-term memory impairment induced by neonatal sevoflurane exposure in mice Z. Sun, M. Satomoto, Y.U. Adachi,

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Inhibiting NADPH oxidase protects against long-term memory impairment induced by neonatal sevoflurane exposure in mice Z. Sun, M. Satomoto, Y.U. Adachi, H. Kinoshita, K. Makita British Journal of Anaesthesia Volume 117, Issue 1, Pages 80-86 (July 2016) DOI: 10.1093/bja/aew064 Copyright © 2016 The Author(s) Terms and Conditions

Fig 1 Concentrations of superoxide and lipid peroxidation in the neonatal mouse brain. Representative images of DHE fluorescence in the brain slice (scale bar=200 μm) showing concentrations of superoxide in the (A) NA, (b) Apocynin, (c) SEVO and (d) Apocynin+SEVO groups. (e) Cumulative data showing concentrations of superoxide in the brain after 6 h sevoflurane exposure (*P<0.05, NA vs SEVO, n=5 each). (f) Representative image and quantitative data showing 4-HNE in the brain after 6 h of sevoflurane exposure (*P<0.05, NA vs SEVO, n=5 each). British Journal of Anaesthesia 2016 117, 80-86DOI: (10.1093/bja/aew064) Copyright © 2016 The Author(s) Terms and Conditions

Fig 2 Expression of NADPH oxidase subunits in the neonatal mouse brain. Representative images and relative expression concentrations of (a) p22phox and (b) gp91phox 6 h after sevoflurane exposure (*P<0.05, NA vs SEVO, n=5 each). British Journal of Anaesthesia 2016 117, 80-86DOI: (10.1093/bja/aew064) Copyright © 2016 The Author(s) Terms and Conditions

Fig 3 Representative image and relative concentrations of cytochrome C in the brain 6 h after sevoflurane exposure (*P<0.05, NA vs SEVO, n=5 each). British Journal of Anaesthesia 2016 117, 80-86DOI: (10.1093/bja/aew064) Copyright © 2016 The Author(s) Terms and Conditions

Fig 4 Representative images showing caspase-3 activation in the retrosplenial cortex of the brain (scale bar=80 μm). Black dots indicate cleaved caspase-3-positive cells in (a) NA, (b) SEVO and (c) Apocynin+SEVO groups. (e) Representative image and relative cumulative concentrations of cleaved caspase-3 in 6 h after sevoflurane exposure (*P<0.05, NA vs SEVO, n=5 each). (e) Representative image showing expression of the apoptosis marker, PARP, in the retrosplenial cortex of the brain after sevoflurane exposure. British Journal of Anaesthesia 2016 117, 80-86DOI: (10.1093/bja/aew064) Copyright © 2016 The Author(s) Terms and Conditions

Fig 5 Long-term memory evaluation using fear conditioning tests in adult mice with or without a 6 h period of neonatal sevoflurane exposure. (a) Freezing response in the contextual test (*P<0.05, NA vs SEVO; NA: n=14; Apocynin: n=12; SEVO: n=12; Apocynin+SEVO: n=11). (b) Freezing response in the cued test (*P<0.05, NA vs SEVO; NA: n=13; Apocynin: n=12; SEVO: n=10; Apocynin+SEVO: n=9). Mean and sd shown. British Journal of Anaesthesia 2016 117, 80-86DOI: (10.1093/bja/aew064) Copyright © 2016 The Author(s) Terms and Conditions