Tyrosine phosphorylation of the N-Methyl-D-Aspartate receptor 2B subunit in spinal cord contributes to remifentanil-induced postoperative hyperalgesia: the preventive effect of ketamine

Abstract

Background: Experimental and clinical studies showed that intraoperative infusion of remifentanil has been associated with postoperative hyperalgesia. Previous reports suggested that spinal N-methyl-D-aspartate (NMDA) receptors may contribute to the development and maintenance of opioid-induced hyperalgesia. In the present study, we used a rat model of postoperative pain to investigate the role of tyrosine phosphorylation of NMDA receptor 2B (NR2B) subunit in spinal cord in the postoperative hyperalgesia induced by remifentanil and the intervention of pretreatment with ketamine.

Results: Intraoperative infusion of remifentanil (0.04 mg/kg, subcutaneous) significantly enhanced mechanical allodynia and thermal hyperalgesia induced by the plantar incision during the postoperative period (each lasting between 2 h and 48 h), which was attenuated by pretreatment with ketamine (10 mg/kg, subcutaneous). Correlated with the pain behavior changes, immunocytochemical and western blotting experiments in our study revealed that there was a marked increase in NR2B phosphorylation at Tyr1472 in the superficial dorsal horn after intraoperative infusion of remifentanil, which was attenuated by pretreatment with ketamine.

Conclusions: This study provides direct evidence that tyrosine phosphorylation of the NR2B at Tyr1472 in spinal dosal horn contributes to postoperative hyperalgesia induced by remifentanil and supports the potential therapeutic value of ketamine for improving postoperative hyperalgesia induced by remifentanil.

Figure 1

Effects of remifentanil on PWMT during the postoperative period and the intervention of ketamine. Ketamine (10 mg/kg, 0.1 ml) or saline was subcutaneously injected 30 min before surgery. Under sevoflurane anesthesia, remifentanil (0.04 mg/kg, 0.4 ml) or saline was subcutaneously infused in the absence or presence of the right hind paw incision during a period of 30 min. PWMT was evaluated at 24 h before incision and at 2 h, 6 h, 24 h and 48 h after surgery. Number of rats per group was twelve. Data are expressed as means ± SD. *P < 0.01 vs baseline, ^# P < 0.01 vs group C, ^▽P < 0.01 vs group I, ^△P < 0.01 vs group R.

Figure 2

Effects of remifentanil on PWTL during the postoperative period and the intervention of ketamine. Ketamine (10 mg/kg, 0.1 ml) or saline was subcutaneously injected 30 min before surgery. Under sevoflurane anesthesia, remifentanil (0.04 mg/kg, 0.4 ml) or saline was subcutaneously infused in the absence or presence of the right hind paw incision during a period of 30 min. PWTL was evaluated at 24 h before incision and at 2 h, 6 h, 24 h and 48 h after surgery. Number of rats per group was twelve. Data are expressed as means ± SD. * P < 0.01 vs baseline, ^# P <0.01 vs group C, ^▽P <0.05 vs group I, ^△P < 0.01 vs group R.

Figure 3

Typical photomicrographs representing tyrosine phosphorylation of NR2B immunoreactive neurons in the superficial dorsal horn. The L4-L5 spinal cords for analysis were collected at 48 h after the surgery. In preparations of control group, hardly any tyrosine phosphorylation of NR2B immunoreactive neurons was found in the dorsal horn region (A). In preparations of rats with incision receiving saline or ketamine, moderate tyrosine phosphorylation of NR2B immunoreactive neurons was obtained in the superficial dorsal horn (Laminae I-II) at the L4-L5 spinal cord, ipsilateral to the incision (B and C). The number of tyrosine phosphorylation of NR2B immunoreactive neurons were drastically upregulated in rats receiving infusion of remifentanil (D), which was remarkably inhibited by pretreatment with ketamine (E). Magnification: × 100. Scale bar = 100 μm.

Figure 4

Immunohistochemical analysis. The L4-L5 spinal cords for analysis were collected at 48 h after the surgery. The mean optical density of NR2B tyrosine phosphorylation (py-NR2B) in the superficial dorsal horn (Laminae I-II) at the L4-L5 spinal cord are summarized. Data from five groups, each using six rats. For each rat, the mean optical density of NR2B tyrosine phosphorylation was obtained by averaging the values from six sections. Data are expressed as means ± SD. * P < 0.01 vs group C, ^# P < 0.05 vs group I, ^△P < 0.05 vs group R.

Figure 5

The expression level of tyrosine phosphorylation of NR2B in superficial spinal cord in each group. The L4-L5 spinal cords for analysis were collected at 48 h after the surgery. Proteins were extracted from the dorsal half of the L4-L5 spinal cord.(A) Representative western blot for tyrosine phosphorylation of NR2B (py-NR2B) in the superficial dorsal horn at the L4-L5 spinal cord; (B) quantification of NR2B tyrosine phosphorylation in each group. Data from five groups, each using six rats. Data are expressed as means ± SD.* P < 0.01 vs group C, ^# P < 0.01 vs group I, ^△P < 0.01 vs group R.