Pretreatment of the ROS Inhibitor Phenyl-N-tert-butylnitrone Alleviates Sleep Deprivation-Induced Hyperalgesia by Suppressing Microglia Activation and NLRP3 Inflammasome Activity in the Spinal Dorsal Cord

Abstract

Sleep deprivation, a common perioperative period health problem, causes ocular discomfort and affects postsurgical pain. However, the mechanism of sleep deprivation-induced increased pain sensitivity is elusive. This study aims to explore the role of ROS in sleep deprivation (SD)-induced hyperalgesia and the underlying mechanism. A 48-h continuous SD was performed prior to the hind paw incision pain modeling in mice. We measured ROS levels, microglial activation, DNA damage and protein levels of iNOS, NLRP3, p-P65 and P65 in mouse spinal dorsal cord. The involvement of ROS in SD-induced prolongation of postsurgical pain was further confirmed by intrathecal injection of ROS inhibitor, phenyl-N-tert-butylnitrone (PBN). Pretreatment of 48-h SD in mice significantly prolonged postsurgical pain recovery, manifesting as lowered paw withdrawal mechanical threshold and paw withdrawal thermal latency. It caused ROS increase and upregulation of iNOS on both Day 1 and 7 in mouse spinal dorsal cord. In addition, upregulation of NLRP3 and p-P65, microglial activation and DNA damage were observed in mice pretreated with 48-h SD prior to the incision. Notably, intrathecal injection of PBN significantly reversed the harmful effects of SD on postsurgical pain recovery, hyperalgesia, microglial activation and DNA damage via the NF-κB signaling pathway. Collectively, ROS increase is responsible for SD-induced hyperalgesia through activating microglial, triggering DNA damage and enhancing NLRP3 inflammasome activity in the spinal dorsal cord.

Keywords: Chronic postsurgical pain; Microglia activation; NLRP3; Reactive oxygen species; Sleep deprivation.

Fig. 1

Time-dependent changes in paw withdrawal mechanical threshold and thermal latency after continuous 48 h sleep deprivation in mice. A Paw withdrawal thresholds in response to mechanical stimulation during SD (Day -2 and -1) and on the days post-incision (Day 1, 3, 5, 7, 9, and 14). B Paw withdrawal to heat stimulation in corresponding time points. Two-way repeated ANOVA followed by the Bonferroni post hoc test. n = 8 per group. Con vs. I: **p < 0.01, ***p < 0.001; Con vs. SD: ^#p < 0.05, ^##p < 0.01,^###p < 0.001; Con vs. SD + I: ^$$p < 0.01,^$$$p < 0.001; I vs. SD + I: ^&p < 0.05, ^&&p < 0.01,^&&&p < 0.001

Fig. 2

ROS levels and iNOS expression on Day 1 and Day 7. A, B Fluorescence intensity of ROS levels in spinal cord tissue of Con, I, SD, and SD + I group mice on day 1 and 7 post-incision. n = 4 per group. C iNOS expression in spinal cord on day 1 and 7 post-incision measured by western blot. n = 4 per group. D Quantification of iNOS protein levels. Two-way ANOVA for comparisons including more than two groups. Con vs. I: **p < 0.01,***p < 0.001; Con vs. SD: ^##p < 0.01,^###p < 0.001; Con vs. SD + I: ^$$p < 0.01,^$$$p < 0.001; I vs. SD + I: ^&p < 0.05, ^&&p < 0.01

Fig. 3

Effects of preoperative sleep deprivation on the activation of spinal microglia and DNA damage after incisional surgery. A Images of immunostaining for Iba1(microglia marker) and 8-OHdG (DNA damage marker) in lumbar spinal dorsal horn ipsilateral to incision were captured in group Con and group SD + I on Day 7 post-incision. Scale bar = 50 μm. n = 3 per group. B–C. Quantification of Iba1 and 8-OHdG fluorescence intensity, respectively. Two-way ANOVA for comparisons between four groups. Con vs. SD + I: ^$$$p < 0.001, I vs. SD + I: ^&&&p < 0.001

Fig. 4

Activation of the NF-κB/NLRP3 signaling after preoperative sleep deprivation in spinal cord. A NLRP3, p-P65, and P65 protein expression in spinal cord on day 1 and 7 post-incision measured by western blot. n = 4 per group. B–D Quantification of P65, p-P65, and NLRP3 protein levels, respectively. Two-way ANOVA for comparisons including more than two groups. Con vs. I: ***p < 0.001; Con vs. SD: ^##p < 0.01,^###p < 0.001; Con vs. SD + I: ^$$$p < 0.001; I vs. SD + I: ^&&p < 0.01, ^&&&p < 0.001

Fig. 5

Time-dependent changes in paw withdrawal mechanical threshold and thermal latency in SD-exposed followed hind paw incision mice with pretreatment of intrathecal injection PBN (SD + I + PBN) or saline (SD + I + Veh). A Paw withdrawal thresholds in response to mechanical stimulation during SD (Day -2 and -1) and on the days post-incision (Day 1, 3, 5, 7, 9, and 14). B Paw withdrawal to heat stimulation in corresponding time points. One-way ANOVA followed by Bonferroni test was performed. n = 8 per group. SD + I + Veh vs. SD + I + PBN: *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Effect of PBN intrathecal injection on oxidative stress, microglia activation, DNA damage, and NF-κB/NLRP3 activity in spinal dorsal cord of SD-exposed followed hind paw incision mice. A iNOS expression in spinal cord on day 7 post-incision measured by western blot. n = 4 per group. B Quantification of iNOS protein level. C Fluorescence intensity of ROS levels in spinal cord tissue. n = 3 per group. D Images of immunostaining for Iba1and 8-OHdG in lumbar spinal dorsal horn ipsilateral to incision were captured in group SD + I + Veh and group SD + I + PBN on Day 7 post-incision. Scale bar = 50 μm. n = 4 per group. E Quantification of Iba1 and 8-OHdG fluorescence intensity, respectively. F NLRP3, p-P65, and P65 protein expression in spinal cord on day 1 and 7 post-incision measured by western blot. n = 4 per group. G–I Quantification of P65, p-P65, and NLRP3 protein levels, respectively. Two-way ANOVA for comparisons including more than two groups; One-way ANOVA for two group comparisons; SD + I + Veh vs. SD + I + PBN: **p < 0.01