Sevoflurane preconditioning ameliorates traumatic spinal cord injury through caveolin-3-dependent cyclooxygenase-2 inhibition

Abstract

Acute traumatic spinal cord injury (tSCI) results in a lifetime of paralysis associated with a host of medical complications. The developing secondary complications of tSCI may result in further chronic neurodegenerative diseases. Sevoflurane preconditioning (SF-PreCon) has shown guaranteed protective effects in myocardial or cerebral ischemic/reperfusion injury. However, the role of SF-PreCon in tSCI still remains to be elucidated. Here, we found that SF-PreCon ameliorated the developing secondary complications through reducing the apoptosis rate and the secretion of inflammatory cytokines in injured spinal cord tissues, and therefore enhancing the recovery after tSCI. Notably, we demonstrated that SF-PreCon ameliorates tSCI through inhibiting Cycloxygenase-2 (COX-2). Importantly, we verified that SF-PreCon inhibits the expression of COX-2 and reduces the apoptosis rate after tSCI via the induction of Caveolin-3 (Cav-3). Taken together, our results suggest that SF-PreCon ameliorates tSCI via Cav-3-dependent COX-2 inhibition and provide an economical and practical method against the secondary injury after tSCI.

Keywords: COX-2; Cav-3; anaesthesia; sevoflurane preconditioning; traumatic spinal cord injury.

Figure 1. SF-PreCon efficiently improves the neurological assessment scores and attenuates the apoptosis after tSCI

(A) Schematic illustration of SF-PreCon protocol used in this experiment. (B) Comparison of the mean CBS score in the five groups. Data are mean ± SEM; ^** P < 0.01 versus the Sham group; ^# P < 0.05 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group. (C) Representative slides of TUNEL staining of the spinal cord sections. Magnification = ×100. (D) Percentage of TUNEL-positive cell in C were calculated. Data are mean ± SEM; ^** P < 0.01 versus the Sham group; ^# P < 0.05 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group. (E) Representative slides of Cleaved Caspase 3 staining of the spinal cord sections. Magnification = ×100. (F) Percentage of Cleaved Caspase 3-positive cell in E were calculated. Data are mean ± SEM; ^** P < 0.01 versus the Sham group; ^# P < 0.05 or ^## P < 0.01 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group.

Figure 2. SF-PreCon significantly inhibits the secretion of inflammatory cytokines in the injured spinal cord tissues after tSCI

ELISA of cytokines IL-1α (A), IL-1β (B), IL-6 (C), and TNF-α (D) in tSCI rats with or without 1.0 MAC SF-PreCon. Data are mean ± SEM; ^* P < 0.05 versus the Sham group; ^# P < 0.05 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group.

Figure 3. SF-PreCon attenuates the secondary injury after tSCI through the inhibition of COX-2 expression

(A) Real-time PCR and western blotting assay for COX-2 mRNA and protein levels in tSCI rats with or without 1.0 MAC SF-PreCon. Data are mean ± SEM; ^* P < 0.05 versus the Sham group; ^# P < 0.05 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group. (B) Representative slides of TUNEL staining of the spinal cord sections with or without NS-398 treatment. Magnification = ×100. (C) Percentage of TUNEL-positive cell in B were calculated. Data are mean ± SEM; ^** P < 0.01 versus the Sham group; ^# P < 0.05 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group. (D-G) ELISA of cytokines IL-1α (D), IL-1β (E), IL-6 (F), and TNF-α (G) in tSCI rats with or without NS-398 treatment. Data are mean ± SEM; ^* P < 0.05 or ^** P < 0.01 versus the Sham group; ^# P < 0.05 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group. (H) The mean CBS score in tSCI rats was improved by NS-398 treatment. Data are mean ± SEM; ^** P < 0.05 versus the Sham group; ^# P < 0.05 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group.

Figure 4. SF-PreCon attenuates tSCI through Cav-3 dependent COX-2 inhibition

(A) Real-time PCR and western blotting assay for Cav-3 mRNA and protein levels in tSCI rats with or without 1.0 MAC SF-PreCon. Data are mean ± SEM; ^* P < 0.05 versus the Sham group; ^# P < 0.05 versus the Vehicle group; unpaired two-tailed Student's t-test. n = 5 per group. (B) Real-time PCR assay for COX-2 and Cav-3 mRNA levels after Cav-3 knockdown with 1.0 MAC SF-PreCon. Data are mean ± SEM; ^* P < 0.05 versus the Sham group; ^# P < 0.05 versus the Vehicle group; $ P < 0.05 or $$ P < 0.01 versus the shCtrl group; unpaired two-tailed Student's t-test. n = 5 per group. (C) Western Blotting assay for COX-2 and Cav-3 protein levels after Cav-3 knockdown with 1.0 MAC SF-PreCon. (D) Representative slides of TUNEL staining of the spinal cord sections after Cav-3 knockdown with 1.0 MAC SF-PreCon. Magnification = ×100. (E) Percentage of TUNEL-positive cell in D were calculated. Data are mean ± SEM; ^** P < 0.01 versus the Sham group; ^# P < 0.05 versus the Vehicle group; $ P < 0.05 versus the shCtrl group; unpaired two-tailed Student's t-test. n = 5 per group.