Genetic knockout and pharmacologic inhibition of neuronal nitric oxide synthase attenuate nerve injury-induced mechanical hypersensitivity in mice

Abstract

Neuronal nitric oxide synthase (nNOS) is a key enzyme for nitric oxide production in neuronal tissues and contributes to the spinal central sensitization in inflammatory pain. However, the role of nNOS in neuropathic pain remains unclear. The present study combined a genetic strategy with a pharmacologic approach to examine the effects of genetic knockout and pharmacologic inhibition of nNOS on neuropathic pain induced by unilateral fifth lumbar spinal nerve injury in mice. In contrast to wildtype mice, nNOS knockout mice failed to display nerve injury-induced mechanical hypersensitivity. Furthermore, either intraperitoneal (100 mg/kg) or intrathecal (30 microg/5 microl) administration of L-NG-nitro-arginine methyl ester, a nonspecific NOS inhibitor, significantly reversed nerve injury-induced mechanical hypersensitivity on day 7 post-nerve injury in wildtype mice. Intrathecal injection of 7-nitroindazole (8.15 microg/5 microl), a selective nNOS inhibitor, also dramatically attenuated nerve injury-induced mechanical hypersensitivity. Western blot analysis showed that the expression of nNOS protein was significantly increased in ipsilateral L5 dorsal root ganglion but not in ipsilateral L5 lumbar spinal cord on day 7 post-nerve injury. The expression of inducible NOS and endothelial NOS proteins was not markedly altered after nerve injury in either the dorsal root ganglion or spinal cord. Our findings suggest that nNOS, especially in the dorsal root ganglion, may participate in the development and/or maintenance of mechanical hypersensitivity after nerve injury.

Figure 1

Effect of targeted disruption of the nNOS gene on nerve injury-induced neuropathic pain. L₅ spinal nerve injury produced a significant increase in paw withdrawal frequencies in response to 0.24 mN (low intensity) (A) and 4.33 mN (moderate intensity) (B) mechanical stimuli on the ipsilateral (ipsi) side in wildtype (WT) mice. The nNOS knockout (KO) mice displayed impaired mechanical pain hypersensitivity on the ipsilateral side (A, B). No significant changes in paw withdrawal frequencies were seen on the contralateral (contral) side after L₅ spinal nerve injury in either WT or nNOS KO mice. **P < 0.01 vs corresponding time points in the WT mice.

Figure 2

Effect of intraperitoneal (i.p.) injection of L-NAME or D-NAME on mechanical pain hypersensitivity on day 7 after L₅ spinal nerve injury in WT mice. Paw withdrawal responses to 0.24 mN (A and C) and 4.33 mN (B and D) mechanical stimuli on the ipsilateral (A and B) and contralateral (C and D) sides. *P < 0.05, **P < 0.01 vs the corresponding baseline. ##P < 0.01 vs the post-drug, D-NAME-treated group.

Figure 3

Effect of intrathecal (i.th.) injection of L-NAME or D-NAME on mechanical pain hypersensitivity on day 7 after L₅ spinal nerve injury in WT mice. Paw withdrawal responses to 0.24 mN (A and C) and 4.33 mN (B and D) mechanical stimuli on the ipsilateral (A and B) and contralateral (C and D) sides. **P < 0.01 vs the corresponding baseline. ##P < 0.01 vs the post-drug, D-NAME-treated group.

Figure 4

Effect of intrathecal injection of 7-NI on mechanical pain hypersensitivity on day 7 after L₅ spinal nerve injury in WT mice. Paw withdrawal responses to 0.24 mN (A and C) and 4.33 mN (B and D) mechanical stimuli on the ipsilateral (A and B) and contralateral (C and D) sides. *P < 0.05, **P < 0.01 vs the corresponding baseline. ##P < 0.01 vs the post-drug, vehicle-treated group.

Figure 5

Expression of nNOS, eNOS, and iNOS in the ipsilateral (I) and contralateral (C) L₅ dorsal root ganglia in naïve WT mice and WT mice 7 days after sham surgery or L₅ spinal nerve injury. (A) Representative examples of Western blots. β-actin was used as a loading control. (B-D) Statistical summary of the densitometric analysis expressed relative to the contralateral side in naïve mice. *P < 0.05 vs the corresponding side in naïve mice or the sham-operated group.

Figure 6

Expression of nNOS, eNOS, and iNOS in the ipsilateral (I) and contralateral (C) dorsal horn of L₅ spinal cord in naïve WT mice and WT mice 7 days after sham surgery or L₅ spinal nerve injury. (A) Representative examples of Western blots. β-actin was used as a loading control. (B-D) Statistical summary of the densitometric analysis expressed relative to the contralateral side in naïve mice.