| Neuropathic pain syndromes—pain after a lesion or disease of the peripheral or central nervous system—are cliniacally characterized by spontaneous pain allodynia(pain response tonormally innocuous stimuli) and hyperalgesia(aggravated pain evoked by noxious stimuli). Neuropathic pain is common in clinical practice, and it greatly impairs the quality of life of patient. Most patients fall into threre broad classes:peripheral focal and multifocal nerve lesions(traumatic, ischemic or inflammatory), peripheral generalized polyneuropathies(toxifc metabolic, hereditary or inflammatory), and CNS lesions(e.g. stroke, multiple sclerosis, spinal cord injury). Neuropathic pain is still a chanllange to clinical doctors, because the underlying mechanisms are unknow, and the effective treatments are still under research. More over, neuropathic pain often response poorly to NSAIDs and opioids. Recent research showed that hydrogen acts as a ROS scaverger by selectively reducing hydroxyl radicals, and hydrogen rich saline(HRS) can also reduces ROS, and the storage and administration of HRS are easier compared to hydrogen. However, the analgesic efficacy of hydrogen on persistent pain is unknown.In a rat model of neuropathic pain induced by spinal nerve ligation(SNL),HRS was administrated intrathecally, administration of saline served as control. The study consists three parts, firstly we tested the effect of single injection of HRS on neuropathic pain, secondly, we evaluated the effect of continuous injection of HRS on neuropathic pain, in the third place, we tested the effect of preemptive injection of HRS on neuropathic pain. The behavior tests contained 50% paw withdraw threashold and paw withdraw latency. Based on the confirmation of analgesia effect of HRS on rat model of neuropathic pain, a pilot study of underlying mechanism was initiated. On the first place,12 days after spinal nerve ligation, all the animals were sacrificed, and fixed with 4% paraformaldehyde, the L5 spinal cord segment was removed. The level of ROS in the spinal dorsal horn was examed by immunostaining of 8-OH-G, the activation of astrocytes and microglias were examed by immunostaining of GFAP and Iba-1 respectively; secondly, changes of inflammatory factors(TNF-αand IL-1β) levels on the ipsilateral side and the contralateral side of the spinal cord were tested using ELISA; thridly, the number of neurons on apoptotic state were examed by Hoechest staining. The main results are as follows:1. Study of animal models:①compared with saline group, single intrathecal injection of HRS significantly reliefed menchanical allodynia and thermal hyperalgesia(p<0.01),2 hrs after single injection, the analgesia effect reached peak,8 hrs after injection, the effect of HRS disappeared.②comparede with salien injection, repeated intrathecal injection of HRS significantly reliefed menchanical allodynia and thermal hyperalgesia(p<0.01), no tolerance effect developed.③preemptive injection of HRS has clear and long-last effect. Therefore, HRS has analgesia effect on neuropathic pain.2. Study of underlying mechanism:①ompared with sham group, level of 8-OH-G in dorsal horn of ipsilateral side of rats.treated with saline was significantly higher(p<0.01), numbers of activated astrocytes and microglias in dorsal horn of ipsilateral side of rats.treated with saline were significantly higher; compared with control group, level of 8-OH-G in dorsal horn of ipsilateral side of rats treated with HRS was significantly lower(p<0.01), numbers of activated astrocytes and microglias in dorsal horn of ipsilateral side of rats treated with HRS were significantly lower(p<0.01). Such changes were not found on the contralateral side.②.results of ELISA showed that levels of proinflammatory factors in dorsal horn of ipsilateral side of rats treated with saline were significantly higher than sham group(p<0.01), intrathecal injection of HRS significantly reversed this change(p<0.01). Such changes were not found on the contralateral side.③compared with sham group, numbers of neurons on apoptotic state in dorsal horn of ipsilateral side of rats.treated with saline were significantly higher; compared with control group, numbers of neurons on apoptotic state in dorsal horn of ipsilateral side of rats treated with HRS were significantly lower(p<0.01).In conclusion, our study demonstrates that HRS significantly reduces mechanical allodynia and thermal hyperalgesia in neuropathic rats. No tolerance effect developed when HRS was administrated repeatedly. Preemptive injection of HRS has clear and long-last effect. Study of underlying mechanisms showed that HRS significantly reduced ROS level in dorsal horn of ipsilateral side, reversed the over-expression of proinflammatory factors in dorsal horn of ipsilateral side.blocked the activation of spinal glial cells, attenuated the apoptosis of neuron in spinal dorsal horn. So we speculate that HRS scavenges ROS in the spinal cord and block the activation of glial cells, blockede the apoptosis of neurons, and reduces over-expression of proinflammatory factors, then reliefs neuropathic pain. |
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