Estudo da modulação endógena por óxido nítrico na dor aguda e hiperalgésica inflamatória

Abstract

Nitric oxide exerts complex and site-specific modulatory effects on pain signaling pathways. This study evaluated the influence of nitric oxide and nitric oxide synthase inhibition on primary nociceptive neurons in vitro and on nociceptive behavior and inflammatory hyperalgesia in vivo. Cultured rat dorsal root ganglion neurons were exposed to the nitric oxide donor Sodium Nitroprusside (10 μM, 100 μM, 1 mM), and membrane depolarization was assessed via DiBAC4(3) fluorescence. The oxide nitric synthase inhibitor L-NAME was also tested for its impact on capsaicin-induced depolarization and glutamatergic via. Sodium Nitroprusside induced significant depolarization of nociceptive neurons only at 1 mM (0.18 ± 0.02 vs. -0.03 ± 0.005, p < 0.0001), with no effect on capsaicin-induced depolarization. L-NAME alone did not alter resting membrane potential, but potentiated capsaicin-induced depolarization (0.60 ± 0.06 vs. -0.37 ± 0.03, p < 0.001), suggesting a tonic inhibitory role of endogenous nitric oxide. NMDA induced significant depolarization (0.15 ± 0.04 vs. -0.03 ± 0.03, p = 0.03), an effect inhibited by co-administration with L-NAME, indicating nitric oxide-dependent glutamatergic signaling. Intracellular nitric oxide levels were assessed in cultured rat dorsal root ganglion neurons and satellite glial cells using the fluorescent indicator DAF-FM. Cells were exposed to the oxide nitric synthase inhibitor L-NAME and the glutamatergic agonist NMDA to evaluate the contribution of endogenous nitric oxide production to glutamatergic signaling. DAF-FM fluorescence confirmed that L-NAME reduced nitric oxide levels in neurons (-0.44 ± 0.09 vs. 0.11 ± 0.01, p < 0.0001) and satellite glial cells (-0.5 ± 0.09 vs. 0.7 ± 0.01, p < 0.0001). NMDA increased NO levels in neurons (0.11 ± 0.02 vs. -0.72 ± 0.02, p = 0.007) and in SGCs (0.08 ± 0.04 vs. -0.7 ± 0.02, p < 0.0001), and this response was inhibited in the presence of L-NAME. In vivo, L-NAME was administered through intraperitoneal, intraplantar, intrathecal and intraganglionic injections prior to nociceptive stimuli with capsaicin or to carrageenan-induced inflammation. Behavioral nociceptive responses and mechanical hypersensitivity thresholds were measured to assess acute pain and hyperalgesia. Systemic administration of L-NAME increased acute nociceptive responses (64.1 ± 5.3 vs. 40.6 ± 4.7, p < 0.01) with no effect on inflammatory hyperalgesia. Local administration reduced both acute nociception (49.8 ± 2.0 vs. 82.7 ± 4.1, p < 0.0001) and hyperalgesia, both at 1h30 (41.5 ± 3.0 vs. 17.4 ± 2.5, p < 0.002) and 3h (44.9 ± 0.8 vs. 32.9 ± 1.9, p < 0.0001). Spinal administration enhanced acute nociceptive responses (50.7 ± 2.8 vs. 36.2 ± 3.5, p < 0.008) and reduced hyperalgesia (53.3 ± 11.1 vs. 34.0 ± 5.4, p < 0.0001). Intraganglionic injection did not affect acute nociception but reduced inflammatory hyperalgesia (44.0 ± 0.8 vs. 32.8 ± 1.8, p < 0.003), indicating that ganglionic nitric oxide signaling contributes to the maintenance of mechanical hypersensitivity. These data support a dual and compartmentalized role for nitric oxide in pain modulation, acting facilitatively at peripheral sites and tonically inhibiting nociceptive signaling centrally. Understanding the spatially distinct actions of nitric oxide provides insight into its complex involvement in pain processing and highlights the importance of targeted approaches for therapeutic modulation of nitric oxide pathways.