Pain-related behaviors of right hind limb in BCP mice.

<p>NSF (A) and PWMT (B) was assessed in the sham and BCP model groups (n = 8) before animal modeling (day 0), and at days 3, 5, 7, 10, and 14 after model establishment. Compared with before modeling, * <i>P</i> < 0.05; compared with the sham group at corresponding time point, ^# <i>P</i> < 0.05.</p

Effects of MrgC agonist and anti-MrgC on expression levels of MrgC, Gi, and NR2B in spinal cord of BCP mice.

<p>(A) The expression levels of MrgC, Gi, and NR2B in the spinal cord were detected with Western blot analysis, in sham mice, model control (Veh) mice, and model mice administered with BAM8-22 or anti-MrgC, at days 1, 3, and 7 after drug administration. (B-D) Statistical analyses of MrgC (B), Gi (C), and NR2B (D) expression levels after drug administration as indicated by Western blot analysis (n = 6). (E) Expression of MrgC (red) and NR2B (green) in the spinal cord were detected with immunohistochemistry in the sham mice, model control (vehicle) mice, and model mice treated with BAM8-22 or anti-MrgC (×200). (F-G) Statistical analyses of MrgC (F) and NR2B (G) expression levels after drug administration as indicated by immunohistochemistry (n = 6). Compared with the vehicle group, ^& <i>P</i> < 0.05; compared with the former time point, ^$ <i>P</i> < 0.05.</p

Expression levels of MrgC, Gi, and NR2B in spinal cord of BCP mice.

<p>(A) Expression levels of MrgC, Gi, and NR2B in the spinal cord of the sham mice and BCP mice were detected with Western blot analysis before animal modeling (day 0; D0), and at days 3, 5, 7, 10, and 14 (D3-D14) after model establishment. (B-D) Statistical analyses of MrgC (B), Gi (C), and NR2B (D) in the spinal cord of sham and BCP mice (n = 6). Compared with before modeling, * <i>P</i> < 0.05; compared with the sham group, ^# <i>P</i> < 0.05.</p

Data_Sheet_1_Inhibition of the integrated stress response reverses oxidative stress damage-induced postoperative cognitive dysfunction.docx

Postoperative cognitive dysfunction (POCD) is a common complication following anesthesia and surgery that might lead to a decline in learning and memory. Oxidative stress damage is one of the pathogenic mechanisms underlying POCD. Recent studies had shown that the integrated stress response (ISR) is closely related to oxidative stress. The core response of the ISR is phosphorylation of eIF2α. Various cellular stress stimuli trigger activation of eIF2α kinases, thus causing phosphorylation of eIF2α. ISR is associated with many neurodegenerative diseases; however, the relationship between POCD and ISR has not been defined. In the present study, the tibias in 4-month-old male C57BL/6 mice were fractured under isoflurane anesthesia to establish the POCD animal model. Cognitive function was assessed by fear conditioning tests and the Y-maze from 3 to 14 days post-surgery. Western blot was used to determine the levels of PeIF2α, eIF2α, ATF4, GADD34, CHOP, BDNF, proBDNF, and p-NR2B expression. The levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) were measured to determine oxidative stress in hippocampal tissues. After tibial fracture surgery in mice, the hippocampus had increased levels of PeIF2α, ATF4, GADD34, and CHOP protein, ROS-positive cells, and average fluorescence intensity, SOD activity was decreased, and the MDA level was increased. The ISR inhibitor, ISRIB, reduced the levels of PeIF2α, ATF4, GADD34, and CHOP protein, and alleviated oxidative stress in the hippocampus of POCD mice. Moreover, ISRIB ameliorated cognitive dysfunction in POCD mice. Our findings suggested that targeting ISR may represent an effective approach to combat POCD.</p

NR2B mRNA relative expression in rats hippocampus.

<p>RT-PCR images for NR2B mRNA extracted from the hippocampus of every group at 1 (D1), 3 (D3), 5 (D5), or 7 (D7) day postoperation. NR2B mRNA values were first normalized with GAPDH of the same sample, and then expressed as mean relative values compared with the sham group (set to 1). ** P<0.01 vs sham group at the same time point; # P<0.05, ## P<0.01 vs HIR group at the same time point.</p

Expression of NR2B protein in rats hippocampus.

<p>A,Werstern blot images of NR2B protein extracted from the hippocampus of every group at 1 (D1), 3 (D3), 5 (D5), or 7 (D7) day afte operation. B, Statistical analysis of the relative optical density normalized to housekeeping gene β-actin (mean±SD,N = 3). * P<0.05, ** P<0.01 vs sham group at the same time point; # P<0.05, ## P<0.01 vs HIR group at the same time point; ★ P<0.01 vs HIR+60sen group at the same time point.</p

NR2B expression in the CA1 area of hippocampus in rats.

<p>Representative photomicrographs of the CA1 area of hippocampus illustrating expression of NR2B from sham group (A) at day 1 postoperation, or HIR group tested at day 1 (B), or 7 (C) postoperation or from HIR+15 (D), HIR+30 (E), or HIR+60 (F) groups tested at day 7 postoperation.</p

Weston blot and statistical analysis showed the effects of intrathecal injection of spironolactone on the expression and phosphorylation of NMDAR in the spinal dorsal horn (n = 3/each).

<p>The expression and phosphorylation of NMDA receptor NR1 subunit (t-NR1 and p-NR1) were significantly up-regulated after CCD surgery compared with Sham group, ^*<i>p</i><0.01 (A and B). Intrathecal delivery of spironolactone (3 µg) twice a day from Days 2 to 4 subsequent to CCD surgery significantly inhibited the expression of t-NR1 and p-NR1 on Day 4 and Day 7 after CCD surgery, ^#<i>p</i><0.05, ^##<i>p</i><0.01 (A and C).</p

Weston blot and statistical analysis showed the effects of intrathecal injection of spironolactone on production of proinflammatory cytokines in the DRG (n = 3/each).

<p>After CCD surgery, the expression of IL-1β, IL-6 and TNF-α were significantly up-regulated compared with Sham group, ^*<i>P</i><0.01 (A and B). Intrathecal injection of spironolactone (3 µg) twice a day from Days 2 to 4 subsequent to CCD surgery significantly reduced the production of IL-1β and TNF-α (A and C) compared with vehicle treated CCD rats, ^#<i>P</i><0.05, ^##<i>P</i><0.01. Spironolactone has no effects on the production of IL-6 compared with vehicle treated CCD rats, <i>P</i>>0.05 (A and C).</p

Weston blot and statistical analysis showed the effects of intrathecal injection of spironolactone on production of proinflammatory cytokines in the spinal dorsal horn (n = 3/each).

<p>After CCD surgery, the expression of IL-1β, IL-6 and TNF-α were significantly up-regulated compared with Sham group, ^*<i>P</i><0.01 (A and B). Intrathecal injection of spironolactone (3 µg) twice a day from Days 2 to 4 subsequent to CCD surgery significantly reduced the production of IL-1β and TNF-α (A and C) compared with vehicle treated CCD rats, ^#<i>P</i><0.05, ^##<i>P</i><0.01. Spironolactone has no effects on the production of IL-6 compared with vehicle treated CCD rats, <i>P</i>>0.05 (A and C).</p