Antisense oligodeoxynucleotides against dynamin-related protein 1 reduce remifentanil-induced hyperalgesia by modulating spinal N-methyl-D-aspartate receptor expression in rats

Abstract

Background: Spinal N-methyl-D-aspartate (NMDA) receptor activation is attributed to remifentanil-induced hyperalgesia (RIH). However, the specific mechanism and subsequent treatment is still unknown. Previous studies have shown that the dynamin-related protein 1 (DRP1)-mitochondria-reactive oxygen species (ROS) pathway plays an important role in neuropathic pain. This study examined whether antisense oligodeoxynucleotides against DRP1 (AS-DRP1) could reverse RIH.

Methods: The authors first measured changes in paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) at 24 hours before remifentanil infusion and 4, 8, 24, and 48 hours after infusion. The expression levels of DRP1 and NR₂B were measured after behavioral testing using Western blotting. In addition, DRP1 expression was knocked down by intrathecal administration of AS-DRP1 to investigate the effects of DRP1 on RIH. The behavioral testing, the expression levels of spinal DRP1 and NR₂B, and dorsal mitochondrial superoxide were measured. Changes in mitochondrial morphology were assessed using electron microscopy.

Results: After remifentanil exposure, upregulation of spinal DRP1 and NR₂B was observed along with a reduction in PWMT and PWTL. In addition, AS-DRP1 improved RIH-induced PWTL and PWMT (P < 0.001 and P < 0.001) and reduced remifentanil-mediated enhancement of spinal DRP1 and NR₂B expression (P = 0.020 and P = 0.022). More importantly, AS-DRP1 reversed RIH-induced mitochondrial fission (P = 0.020) and mitochondrial superoxide upregulation (P = 0.031).

Conclusions: These results indicate that AS-DRP1 could modulate NMDA receptor expression to prevent RIH through the DRP1-mitochondria-ROS pathway.

Keywords: Analgesia; Dynamins; Hyperalgesia; Mitochondria; Neuralgia; Oligodeoxyribonucleotides; Reactive Oxygen Species; Receptors, N-Methyl-D-Aspartate; Remifentanil; Superoxides.

Fig. 1

(A, B) Flowgram of the experimental procedure. (A) Experiment 1: Behavioral changes and protein expression in remifentanil-induced hyperalgesia rats. (B) Experiment 2: The effect of antisense oligodeoxynucleotides against dynamin-related protein 1 in remifentanil-induced hyperalgesia rats. MM: mismatch oligodeoxynucleotides to dynamin-related protein 1, AS: antisense oligodeoxynucleotides against dynamin-related protein 1.

Fig. 2

(A, B) Changes in PWTL and PWMT after RIH. PWTL (A) and PWMT (B) were measured in rats at 24 hour before (baseline) and at 4, 8, 24, and 48 hour post-remifentanil infusion (mean ± standard deviation, n = 6 per group, one-way ANOVA). PWTL: paw withdrawal thermal latency, PWMT: paw withdrawal mechanical threshold, RIH: remifentanil-induced hyperalgesia. ***P < 0.001 compared with baseline. ^###P < 0.001 compared with group control.

Fig. 3

(A, B) Western blot bands show the expression levels of NR₂B and DRP1 over time. (A) Expression levels of NR₂B and DRP1 at different time points. (B) The fold change in the density of NR₂B and DRP1 (mean ± standard deviation, n = 6). 4H, 8H, 24H, and 48H represent animal groups of 4, 8, 24 and 48 hours after remifentanil infusion, respectively. DRP1: dynamin-related protein 1. *P < 0.05, **P < 0.01 compared with the group control.

Fig. 4

(A, B) The effect of the AS on hyperalgesia induced by remifentanil. PWTL (A) and PWMT (B) were measured in rats at 24 hour before (baseline) and at 4, 8, 24, and 48 hour post-remifentanil infusion (mean ± standard deviation, n = 15). C: group control, R: group remifentanil, MM: group mismatch oligodeoxynucleotides to dynamin-related protein 1, AS: group antisense oligodeoxynucleotides against dynamin-related protein 1, PWTL: paw withdrawal thermal latency, PWMT: paw withdrawal mechanical threshold. ^***P < 0.001 compared with the group control. ^###P < 0.001 compared with the group AS.

Fig. 5

(A, B) The expression levels of dorsal DRP1 and NR₂B were measured after various treatments in remifentanil-induced hyperalgesia rats. (A) Expression levels of NR₂B and DRP1 in groups after various treatments. (B) The fold change in the density of NR₂B and DRP1 (mean ± standard deviation, n = 6). C: group control, R: group remifentanil, MM: group mismatch oligodeoxynucleotides to dynamin-related protein 1, AS: group antisense oligodeoxynucleotides against dynamin-related protein 1. *P < 0.05 compared with the group control. **P < 0.01 compared with the group control. ^#P < 0.05 compared with the group AS.

Fig. 6

The antisense oligodeoxynucleotides against DRP1 suppressed NR₂B expression in remifentanil-induced hyperalgesia. Representative fluorescence immunohistochemistry images for NR₂B levels in groups A–D: (A) Group control, (B) Group remifentanil, (C) Group MM, (D) Group AS. The selected field was analyzed by a confocal microscope (magnification: ×400). (E) Statistical assessment of positive cells post fluorescence immunostaining in the spinal dorsal horn. DAPI was used to stain the nuclei (mean ± standard deviation, n = 3). The arrows represented NR₂B positive neurons. Scale bars = 25 μm. C: group control, R: group remifentanil, MM: group mismatch oligodeoxynucleotides to dynamin-related protein 1, AS: group antisense oligodeoxynucleotides against dynamin-related protein 1. ^**P < 0.01 compared with the group control, ^#P < 0.05 compared with the group AS.

Fig. 7

Influence of intrathecal the antisense oligodeoxynucleotides against dynamin-related protein 1 on mitochondrial number. (A–D) A: representative image of mitochondria in the group control, B: group remifentanil, C: group MM, D: group AS. (E) Number of mitochondria (mean ± standard deviation, n = 3). The arrows represented mitochondria. Scale bars = 500 nm. C: group control, R: group remifentanil, MM: group mismatch oligodeoxynucleotides to dynamin-related protein 1, AS: group antisense oligodeoxynucleotides against dynamin-related protein 1. ^**P < 0.01 compared with the group C, ^#P < 0.05 compared with the group AS.

Fig. 8

The impact of intrathecal the antisense oligodeoxynucleotides against dynamin-related protein 1 on mitochondrial superoxide in the spinal dorsal horn. Representative MitoSox red images of mitochondrial superoxide in the group control, group remifentanil, group MM and group AS are shown in A, B, C, and D. (E) The number patterns of MitoSox-positive cells are shown in different groups (mean ± standard deviation, n = 3). The arrows represented MitoSox positive neurons. Scale bars = 20 μm. C: group control, R: group remifentanil, MM: group mismatch oligodeoxynucleotides to dynamin-related protein 1, AS: group antisense oligodeoxynucleotides against dynamin-related protein 1. ***P < 0.001 compared with the group control, ^#P < 0.05 compared with the group AS.