Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48 percent by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469 percent). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20 percent), reaching the greatest increase (60 percent) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Evaluation of antivenoms in the neutralization of hyperalgesia and edema induced by Bothrops jararaca and Bothrops asper snake venoms

Neutralization of hyperalgesia induced by Bothrops jararaca and B. asper venoms was studied in rats using bothropic antivenom produced at Instituto Butantan (AVIB, 1 ml neutralizes 5 mg B. jararaca venom) and polyvalent antivenom produced at Instituto Clodomiro Picado (AVCP, 1 ml neutralizes 2.5 mg B. aspar venom). The intraplantar injection of B. jararaca and B. asper venoms caused hyperalgesia, which peaked 1 and 2 h after injection, respectively. Both venoms also induced edema with a similar time course. When neutralization assays involving the independent injection of venom and antivenom were performed, the hyperalgesia induced by B. jararaca venom was neutralized only when bothropic antivenom was administered iv 15 min before venom injection, whereas edema was neutralized when antivenom was injected 15 min or immediately before venom injection. On the other hand, polyvalent antivenom did not interfere with hyperalgesia or edema induced by B. asper venom, even when administered prior to envenomation. The lack of neutralization of hyperalgesia and edema induced by B. asper venom is not attributable to the absence of neutralizing antibodies in the antivenom, since neutralization was achieved in assays involving preincubation of venom and antivenom. Cross-neutralization of AVCP or AVIB against B. jararaca and B. asper venoms, respectively, was also evaluated. Only bothropic antivenom partially neutralized hyperalgesia induced by B. asper venom in preincubation experiments. The present data suggest that hyperalgesia and edema induced by Bothrops venoms are poorly neutralized by commercial antivenoms even when antibodies are administered immediately after envenomation