| Objective: At present, many studies have approved that neuropathic pain or inflammatory pain which were caused by inflammation of peripheral tissue could induce the production of thermal and mechanical hyperalgesia. The characteristics of hyperalgesia were lower pain threshold and/or overreact to the stimulation of normal pain, which is the abnormal phenomenon of skin sensation, also known as pain sensitization. The mechanism of hyperalgesia was related to peripheral sensitization and sensitization of spinal cord. The changes of metabolism in spinal cord neurons, the changes in neuronal excitability induced by abnormal metabolism and expansion in receptive field may play an important role in the generation of hyperalgesia.Some studies have confirmed that ligation of sciatic nerve and inflammatory pain can induce neuronal apoptosis in the spinal cord recently. In our laboratory,we found that the ratio of neuronal apoptosis of spinal cord was significantly increased after plantar injection of formalin 24h, which reached the peak in the third day in the model of formalin inflammatory pain.All of the results lead to consider that maybe there is a relationship between neuronal apoptosis of spinal cord and hyperalgesia. We don't know that whether neuronal apoptosis is involved in the formation of hyperalgesia? At present, some studied about the relationship of neuronal apoptosis in the spinal cord and hyperalgesia have been reported, but the results were not consistent. The results of study exhibited that the severity degree in neuronal apoptosis of spinal cord induced by sciatic nerve was consistent with the severity degrees of thermal and mechanical hyperalgesia, whether or not there is a causal relationship between them can not be confirm. The researchers considered that maybe they are two manifestations which both were caused by neuropathic pain. Some researchers who used antiapoptotic factor, recombinant human erythropoietins, as a tool drug to observe the relationship between neuronal apoptosis and hyperalgesia. They found that Epo could significantly reduce the neuronal apoptosis in the spinal cord which induced by inflammatory pain, and significantly weaken the degree of mechanical hyperalgesia, therefore they considered that neuronal apoptosis was involved in the formation of hyperalgesia. In short, It not be determined that whether the neuronal apoptosis which induced by naturopathic pain and inflammatory pain was involved in the formation of hyperalgesia because of fewer evidence.Our present study was undertaken to clarify whether the neuronal apoptosis which induced by formalin inflammatory pain was involved in the generation of hyperalgesia. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) were adopted to detect the neuronal apoptosis. Intrathecal injection of Ac-DEVD-CHO , a inhibitor of Caspase-3, to investigate the effects of inhibiting neuronal apoptosis on thermal withdrawal latency and mechanical withdrawal threshold during formalin inflammatory pain to explore the role of neuronal apoptosis in the formation of hyperalgesia.Methods: one hundred and twenty six male SD rats (260-280g in weight) were divided randomly into two groupsEighty four rats in the first group were used to observe the effect of caspase-3 inhibitor on the neuronal apoptosis of spinal cord. The rats were divided randomly into seven subgroups: control group, formalin group (F), formalin+NS group (F+NS), formalin+0.5 ng Ac-DEVD-CHO group (F+0.5 ngAC), formalin + 0.6 ng Ac-DEVD-CHO group (F+0.6 ng AC), formalin+0.75 ng Ac-DEVD-CHO group (F+0.75 ng AC), formalin+1 ng Ac-DEVD-CHO group (F+1 ng AC). There were twelve rats in each group. Rats in control group were directly sacrificed without other treatments; NS or Ac-DEVD-CHO were injected intrathecally into rats of F+NS or F+Ac-DEVD-CHO groups before 30 minutes formalin injection subcutaneously into the plantar surface of the right hindpaw. The L4-5 segment of spinal cord was dissected out at 72h after formalin injection. In each group, six specimens were used to detect the ratio of neuronal apoptosis by flow cytometry and six specimens were used to detect the degree of neuronal apoptosis by TUNEL.There were forty two rats in the second group, which were used to observe the effect of inhibitor of Caspase-3 on thermal withdrawal latency and mechanical withdrawal threshold .The rats were also divided randomly into seven subgroups, including control group, formalin group (F), formalin+NS group (F+NS), formalin+0.5 ng Ac-DEVD-CHO group (F+0.5 ng AC), formalin+0.6 ng Ac-DEVD-CHO group (F+0.6 ng AC), formalin+0.75 ng Ac-DEVD-CHO group (F+0.75 ng AC), formalin+1 ng Ac-DEVD-CHO group (F+1 ng AC). There were six rats in each group. NS or Ac-DEVD-CHO was injected intrathecally into the rats of F+NS or F+Ac-DEVD-CHO groups before 30 minutes formalin injection subcutaneously into the plantar surface of the right hindpaw. Thermal withdrawal latency and mechanical withdrawal threshold were determined at 1d,4d,7d,10d,14d after formalin injection.Results:1 The effect of Caspase-3 inhibitor on the ratio of neuronal apoptosis of spinal cord.Flow cytometry results showed that: the ratio of neuronal apoptosis of L4-5 segment of spinal cord was very low in control group. Compared with control group, the ratio of neuronal apoptosis of spinal cord was obviously increased in formalin group. Compared with formalin group, the ratio of neuronal apoptosis has no significant difference in the rats of F+NS group, which showed that intrathecal injection of NS didn't significantly effect on neuronal apoptosis of spinal cord. Compared with formalin group, the ratio of neuronal apoptosis of spinal cord has no significant difference in formalin+0.5 ng AC group, flowing the increase of Ac-DEVD-CHO doses, the ratio of neuronal apoptosis of spinal cord were significantly reduced in the rats of F+ 0.6 ng AC group, F+0.75 ng AC group and F+1 ng AC group, among them, 0.75 ng AC is the most effective dose in reduction of neuronal apoptosis. 2 The effect of Caspase-3 inhibitor on the index of neuronal apoptosis of spinal cord.The results of TUNEL detection show that there were brown granules in the apoptotic cells, apoptotic cells were irregular shape; In the spinal dorsal horn of rats in control group, apoptotic cells was fewer. Compared with control group, the number of apoptotic cells significantly increased in the spinal dorsal horn in formalin group, the ratio of apoptotic cells and total cell (apoptotic index) was significantly increased. Compared with formalin group, intrathecal injection of NS or 0.5 ng AC did not induce obviously change in apoptotic index; but increasing Ac-DEVD-CHO doses, comparing with formalin group, the number of apoptotic cells were significantly decreased in F+0.6 ng AC group, F+0.75 ng AC group and F+1 ng AC group, apoptotic index was significantly reduced, among them, 0.75 ng AC is the most effective dose in the reduction of apoptotic index.3 The effect of Caspase-3 inhibitor on the thermal withdrawal latency and mechanical withdrawal threshold during formalin inflammatory pain.Compared with control group, the rats of formalin group which were subcutaneously injected with 5% formalin into the ventral surface of the right hind paw had an obviously prolongation in thermal withdrawal latency and mechanical withdrawal threshold in the injected area at 1d,4d,7d,10d after formalin injection, which showed that lags in response for the stimulation of thermal and mechanism occurred in the injected area. 14d returned to normal; but an adverse effect in opposite side at 1d,4d,7d,10d,14d after formalin injection, which showed that thermal and mechanical hyperalgesia occurred in the contralateral hindpaws in the formalin group. Compared with formalin group, intrathecal injection of NS had no significantly differences on thermal withdrawal latency and mechanical withdrawal threshold at every time points. Compared with formalin group, intrathecal injection of different doses of Ac-DEVD-CHO had no obviously effect on thermal withdrawal latency and mechanical withdrawal threshold in the injected area, but there have different changes on thermal withdrawal latency and mechanical withdrawal threshold in contralateral hindpaws as the increase of the dose of Ac-DEVD-CHO. Compared with formalin group, at 1d after injection, intrathecal injection of every doses of Ac-DEVD-CHO all have no obviously effect on thermal withdrawal latency and mechanical withdrawal threshold in contralateral hindpaws. At every time points Intrathecal injection of 0.5 ng AC had no obviously effect on thermal withdrawal latency and mechanical withdrawal threshold of contralateral hindpaws comparing with formalin group. Intrathecal injection of 0.6 ng AC, the thermal withdrawal latency and mechanical withdrawal threshold of contralateral hindpaws were all higher than formalin group at 4 d after 5 % formalin injection subcutaneously into the plantar surface of the right hindpaw,but had no significantly effect on thermal withdrawal latency and mechanical withdrawal threshold at 7d, 10d and 14d after formalin injection. Intrathecal injection of 0.75 ng AC and 1 ng AC, the thermal withdrawal latency and mechanical withdrawal threshold of contralateral hindpaws were all higher than formalin group in the 4d, 7d and 10d after 5 % formalin injection subcutaneously into the plantar surface of the right hindpaw, which showed that thermal and mechanical hyperalgesia were reduced by inhibiting the neuronal apoptosis with intrathecal injection of Caspase-3 inhibitor. Compared with formalin group, all of the F+ Ac-DEVD-CHO groups had no obviously effect on the thermal withdrawal latency and mechanical withdrawal threshold in contralateral hindpaws at the 14d after 5 % formalin injection subcutaneously into the plantar surface of the right hindpaw.Conclusion: Neuronal apoptosis of spinal cord and the hyperalgesia of thermal and mechanical can be induced by formalin inflammatory pain, intrathecal injection of certain dose of Caspase-3 inhibitor could obviously inhibited the neuronal apoptosis of spinal cord which induced by formalin inflammatory pain and relieved the extent of thermal and mechanical hyperalgesia, which tipped that the pathway of neuronal apoptosis might be involved in the formation of hyperalgesia.
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