Anti-nociceptive effects of caloric restriction on neuropathic pain in rats involves silent information regulator 1 Y. Liu, Y. Ni, W. Zhang, Y.E. Sun,

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Anti-nociceptive effects of caloric restriction on neuropathic pain in rats involves silent information regulator 1 Y. Liu, Y. Ni, W. Zhang, Y.E. Sun, M. Jiang, W.J. Gu, Z.L. Ma, X.P. Gu British Journal of Anaesthesia Volume 120, Issue 4, Pages 807-817 (April 2018) DOI: 10.1016/j.bja.2017.09.009 Copyright © 2017 British Journal of Anaesthesia Terms and Conditions

Fig 1 Effects of CR on body weight, Lee's index, and motor activity. Changes in (a) body weight (in grammes) and (b) Lee's index in AL and CR rats during the 10-week dietary treatment (n=16). (c) Locomotor activity in AL and CR rats under non-stressful conditions before and after a 6-week dietary treatment (n=16). Activity is indicated as number of beams broken per minute. Values are expressed as mean (standard deviation). ∗P<0.05 vs group AL. AL, ad libitum; CR, caloric restriction. British Journal of Anaesthesia 2018 120, 807-817DOI: (10.1016/j.bja.2017.09.009) Copyright © 2017 British Journal of Anaesthesia Terms and Conditions

Fig 2 Effect of CR on CCI-induced neuropathic pain and the role of SIRT1. (a) Mechanical allodynia and (b) thermal hyperalgesia before and after surgery (n=8). Values are expressed as mean (standard deviation). (c) Representative image of SIRT1 and β-actin expression by western blotting. (d) Densitometric quantification of SIRT1 protein from western blots. Individual data points and mean are shown (n=5). (e) Mechanical allodynia and (f) thermal hyperalgesia after intrathecal injection of SIRT1 inhibitor, EX 527, on Day 7 after CCI surgery before and after injection (n=8). Values are mean (standard deviation). For group explanations, see text. (a–d) ∗P<0.05 vs group AL+CCI, #P<0.05 vs baseline, +P<0.05 vs group AL+sham, &P<0.05 vs group CR+sham. (e) and (f) ∗P<0.05 vs group AL+CCI+Veh, #P<0.05 vs group CR+CCI+Veh. AL, ad libitum; CCI, chronic constriction injury; CR, caloric restriction; SIRT1, silent information regulator 1. British Journal of Anaesthesia 2018 120, 807-817DOI: (10.1016/j.bja.2017.09.009) Copyright © 2017 British Journal of Anaesthesia Terms and Conditions

Fig 3 Effect of CR on PGC1α expression, PGC1α acetylation, and mitochondrial-derived ROS in ipsilateral spinal-cord dorsal horn on Day 7 after CCI surgery. (a) Representative image of western blots. (b) Representative images of mitochondrial-derived ROS using MitoSOX Red (Invitrogen Molecular Probes, Eugene, OR). (c–h) Densitometric quantification of proteins from western blots. (i) and (j) Mn-SOD (mitochondrial) and Cu/Zn-SOD (cytosolic) activities. (k) Quantification of ROS as mean fluorescence intensity of MitoSOX positive cells in the superficial dorsal horns. Individual data points and mean are shown (n=5). For explanation of treatment groups, see text. ∗P<0.05 vs group AL+CCI, +P<0.05 vs group AL+sham, &P<0.05 vs group CR+sham. AL, ad libitum; CCI, chronic constriction injury; CR, caloric restriction; Cu/Zn-SOD, copper/zinc superoxide dismutase; DRP1, dynamin-related protein 1; Mfn2, mitofusin-2; Mn-SOD, manganese superoxide dismutase; PGC1α, peroxisome-proliferator-activated receptor gamma coactivator 1α; ROS, reactive oxygen species. British Journal of Anaesthesia 2018 120, 807-817DOI: (10.1016/j.bja.2017.09.009) Copyright © 2017 British Journal of Anaesthesia Terms and Conditions

Fig 4 Effect of CR on acetylated NFκB and IL-1β in ipsilateral spinal-cord dorsal horn on Day 7 after CCI surgery. (a) Representative western blots of p300/CREB-binding protein (CBP), and (b) representative western blots of acetylated NFκB p65 and NFκB p65. (c) Representative western blots of NLRP3, pro-caspase-1, mature caspase-1 (p20), pro-IL-1β, and cleaved-IL-1β. (d–m) Densitometric quantification of proteins on western blots. (n) Caspase-1 activity with data expressed as the fold change relative to control. Individual data points and mean are shown (n=5). For explanation of treatment groups, see text. ∗P<0.05 vs group AL+CCI, +P<0.05 vs group AL+sham, &P<0.05 vs group CR+sham. AL, ad libitum; CCI, chronic constriction injury; CR, caloric restriction; IL-1β, interleukin-1β; NFκB, nuclear factor kappa B; NLRP3, nucleotide-binding oligomerization domain-like receptor protein 3. British Journal of Anaesthesia 2018 120, 807-817DOI: (10.1016/j.bja.2017.09.009) Copyright © 2017 British Journal of Anaesthesia Terms and Conditions

Fig 5 Effect of CR on MAPK phosphorylation and glial-cell activation in the ipsilateral spinal-cord dorsal horn by Day 7 after CCI surgery. (a) Representative western blots of p-JNK, p-ERK, and p-p38 expression. (b–d) Densitometric quantification of p-JNK, p-ERK, and p-p38 immunoreactivities on western blots. (e) and (f) Imaging and mean fluorescence intensity of GFAP expression. (g) and (h) Imaging and mean fluorescence intensity of Iba1 expression. Individual data points and mean are shown (n=5). For explanation of treatment groups, see text. ∗P<0.05 vs group AL+CCI, +P<0.05 vs group AL+sham, &P<0.05 vs group CR+sham. AL, ad libitum; CCI, chronic constriction injury; CR, caloric restriction; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; GFAP, glial fibrillary acidic protein; Iba1, ionized calcium binding adapter molecule 1. British Journal of Anaesthesia 2018 120, 807-817DOI: (10.1016/j.bja.2017.09.009) Copyright © 2017 British Journal of Anaesthesia Terms and Conditions

Fig 6 Effect of CR on phosphorylation of PKCγ, NR1, and NR2B in the ipsilateral spinal-cord dorsal horn by Day 7 after CCI surgery. (a) Representative western blots for p-NR2B, p-NR1, and p-PKCγ expression. (b–d) Densitometric quantification of p-NR2B, p-NR1, and p-PKCγ immunoreactivities on western blots. Individual data points and mean (n=5). For explanation of treatment groups, see text. ∗P<0.05 vs group AL+CCI, +P<0.05 vs group AL+sham, &P<0.05 vs group CR+sham. AL, ad libitum; CCI, chronic constriction injury; CR, caloric restriction; PKCγ, protein kinase C-gamma; NR1, N-methyl-D-aspartate receptor subunit 1; NR2B, N-methyl-D-aspartate receptor 2B. British Journal of Anaesthesia 2018 120, 807-817DOI: (10.1016/j.bja.2017.09.009) Copyright © 2017 British Journal of Anaesthesia Terms and Conditions