| Pain is a common symptom clinically, which bring a great of suffering to patients. Trauma or inflammation of tissues can induce the sensitization of peripheral nociceptors(peripheral sensitization) and the sensitization of spinal cord dorsal horn neurons ( central sensitization ) . Nociperception and hyperalagesia were generated from them. The mechanism of hyperalagesia is commonly consideredthat peripheral continually nociceptive information input can result in abundant release of excitatory neurotransmitter from primary ending. Those excitatory neurotransmitters include excitatory amino acids( EAAs) which mainly include glutamic acid (Glu) and aminosuccinic acid (Asp), subtance P (SP) and calcitonin gene-related peptide(CGRP). Those excitatory neurotransmitters combine with their receptors in secondary neurons of the spinal cord dorsal horn and activate the signal transmission process in cells. As a result, NO is increasingly produced and PKC is activated. The two signal transmission materials can induce the nociperception and hyperalagesia through complicated mechanism.The research results of Zeng-jingbo in our laboratory show that formalin injection into the right palm of rats resulted in an enhance in activity of nitrc oxide synthase(NOS) and an excessive production of NO in spinal cord neurons. Intrathecal injection of MK-801, an inhibitor of NMDA receptor, reversed the enhancements in NOS activity and in NO production in spinal cord of rats with formalin inflammatory pain .Also in our laboratory, recently research results show that the inflammatory pain induced by the injection of formalin into the ventral surface of the rats paw induced apoptosis of hippocampal neurons. Intrathecal injection of NMDA receptor antagonist MK-801 inhibited the apoptosis of hippocampal neurons and the expression of apoptosis-related gene in rats with formalin inflammatory pain. These results implied that the release of EAAs in the central neural system and the activation of its receptor induced by nociceptive input participated in the induction of apoptosis of the neurons of spinal cord.The superficial lamella of spinal dorsal horn is an primary gateway which can receive the nociceptive information input from body .It is also an important locum where inputing information can be dealed with effectively. Whether abundant release of the excitatory neurotransmitters and the excessive activation of their receptors can result in neuronal apoptosis in spinal cord during formalin inflammatory pain and whether excessively production of NO induced by nociceptive information input plays an important role in induction of neuronal apoptosis haven't been, reported by domestic and aboard researchers.Therefore, our present study was undertaken to observe the changes in neuronal apoptosis of rats spinal cord induced by formalin inflammatory pain and its time characters. On the basis of this work, we made a further study to explore the effect of NO on the neuronal apoptosis of spinal cord.1 Formalin inflammatory pain induces the apoptosis of spinal horn neuronal neurons in rats.Methods Thirty male SD rats(280±20g in weight)were divided randomly into 6 groups: Control group, injection formalin 1d group, injection formalin 2d group, injection formalin 3d group, injection formalin 4d group, injection formalin 7d group. There were five rats in each group. Rats in control group were directly sacrificed without other treatments. Rats in injection with formalin 1d,2d,3d,4d,7d group were subcutaneously injected with 0.2ml 5% formalin into the ventral surface of the right hind paw to induce periphery inflammatory pain, first count the flinches of the rat per 5 minute in 1 hour to observe their painful reaction ,then sacrificed at different time after formalin injection. Flow cytometry was used to assay the ratio of neuronal apoptosis of L4~5 segment of rat spinal cord.Results The results were as follows: Injection with formalin into the ventral surface of the rat hind paw induced a biphasic spontaneous pain reaction, including vocalization, flinches, scratching and biting the wounded paw. The first phase pain reaction appeared right after the injection and lasted for about 5~10 minutes followed by a short intermission(about 5 min), then the second phase appeared and it can last over 40 minutes.The ratio of neuronal apoptosis of L4~ 5 segment of the rat spinal cord was very low in control group. Compared with control group, the ratio of neuronal apoptosis of spinal cord increased on 1d after formalin injection, peaked on the third day . The ratio of neuronal apoptosis was decreased on the forth day, but it was still higher than that of control group (P<0.05). On the seventh day after formalin injection, the ratio of neuronal apoptosis of spinal cord falled down to normal level.Conclusion: The results indicated that formalin inflammatory pain could induce neuronal apoptosis in L4~5 segment of the rats's spinal cord. The peak of neuronal apoptosis appeared on the third day after formalin injection and recovered to normal level on the seventh day after formalin injection.2 Effect of NO on neuronal apoptosis of spinal cord induced by formalin inflammatory painMethods Seventy male SD rats (280±20g in weight) were divided randomly into 7 groups: control group, L-Arg group(L-Arg), formalin group(F), formalin+NS group(F+NS), formalin+ L-NAME 0.5μmol group(F+ L-NAME 0.5μmol), formalin+ L-NAME 2μmol group(F+ L-NAME 2μmol), and formalin+L-NAME 4μmol group(F+ L-NAME 4μmol). There were ten rats in every groups respectively, five of which were used for observation of the changes in the ratio of neuronal apoptosis of spinal cord, and the other five were used for observation the expression of p53 protein in L4~5 segment of spinal cord dorsal horn. Rats in control group were directly sacrificed without other treatments. Rats in L-arg group were only treated with intrathecal injection of L-Arg (4.7μmol). Pain reaction was observed at first and then rats were sacrificed 12h after intrathecal injection of L-Arg. The rats in other groups were first counted the number of flinches to observe their spontaneous pain reaction just after the right paw injection of formalin, then they were directly sacrificed on the third day after formalin injection. The rats of formalin+NS group,formalin+ L-NAME (0.5μmol) group, formalin+ L-NAME (2μmol) group and formalin+ L-NAME ( 4μmol ) group were intrathecally injected using NS, L-NAME(0.5μmol),L-NAME (2μmol), L-NAME (4μmol) solution 10μl 15 minutes prior to formalin injection. After formalin injection, flinches per 5min in 1 hour was counted immediately to observe the spontaneous pain reaction immediately. Flow cytometry was used to assay the ratio of neuronal apoptosis of L4~5 segment of spinal cord. The immunohistochemistry was used to observe the expression of p53 protein in spinal cord dorsal horn.Results The results were as follows: after simple intrathecal injection of L-Arg , the animals in L-Arg group show a affinis nociceptive behavioural response consisting of fidgets, irritability and bray. In formalin group and formalin+NS group,subcutaneous injection of formalin elicited a characteristic nociceptive behavioural response consisting of licking and biting the injected paw, etc. just as we have describe in the first stage of the article. But there was no significant difference between two groups. Intrathecal injection of NOS inhibitor L-NAME could dose-dependently inhibit nociceptive behavioural response that induced by formalin injection. Manifestation as the spontaneous flinches of pain reflect in the double phase of nociceptive were significantly decreased (P<0.05). There was a transient paralysis and locomotor ataxia in posterior limb of rats in formalin+ L-NAME(4μmol) group.The ratio of neuronal apoptosis of L4~5 segment of rats's spinal cord in control group was fairly low as 4.36±0.469.Compared with control group, the ratio of neuronal apoptosis of rats spinal cord in L-Arg group was increased. Compared with control group, the ratios of neuronal apoptosis of L4~5 segment of spinal cord in formalin group and formalin+NS group were significantly increased (P<0.05) also, but there was no significant difference between the two groups. Contrasted to formalin+NS group, the ratios of neuronal apoptosis of spinal cord of formalin+ L-NAME (0.5μmol),formalin+ L-NAME(2μmol) and formalin+ L-NAME(4μmol) group were significantly decreased (P<0.05). The larger dose of L-NAME , the lower ratio of neuronal apoptosis .There was a little of p53 protein distributed in full-thickness of L4~5 segment of the spinal cord dorsal horn in control group rats. The cell bodies of p53 positive cells were mainly showed ovoid or unregulated shape, some of these positive bodies were surrounded with conspicuous apophysis and fibrilla which corresponded the characteristic of the neuron。There was no difference between two sides of the spinal cord dorsal horn. Compared with control group, there was an increased number of p53 positive neuron in the laminaⅠ~Ⅱof the L4~5 spinal cord dorsal horn of the L-Arg group, the staining of them were deeper , and in the laminaⅢ~Ⅵof the spinal cord dorsal horn, there were still a little of p53 protein distributed as well. Compared with control group, the number and staining degree of p53 immunoreactive neurons in the laminaⅠ~ⅡandⅢ~Ⅵof the L4~5 spinal cord dorsal horn of formalin and formalin+NS groups were significantly increased (P<0.05), there was no significant difference between the expressions of p53 protein of between two groups. The number and staining degree of p53 immunoreactive neurons in the injection ipsilateral spinal cord dorsal horn were higher than the contralateral dorsal horn(P<0.01). Compare with formalin+NS group, the number and staining degree of P53 immunoreactive neurons of formalin+ L-NAME (0.5μmol),formalin+ L-NAME(2μmol) and formalin+ L-NAME(4μmol ) group were significantly decreased ( P<0.05 ) , Decreased degree was positive correlative with the dose of L-NAME.The results indicated that Intrathecal injection of L-Arg could induce a biphasic spontaneous pain reaction in the normal animals and caused an upregulation of the expression of p53 protein and an increase in ratio of neuronal apoptosis of spinal cord. Yet the intrathecal injection of L-NAME could dose-dependently inhibit the spontaneous pain reaction and increases in the ratio of neuronal apoptosis and expression of p53 protein in L4~5 spinal cord induced by formalin inflammatory pain, which indicated that the enhance of the NOS activity as well as execessive production of NO might participate the induction of neuronal apoptosis during formalin inflammatory pain. And in the procedure, one of the important mechanisms in NO inducing spinal neuronal apoptosis was by upregulateing p53 expression.Conclusion: The inflammatory pain induced by injection of formalin into the ventral surface of the rats paw could induce neuronal apoptosis in spinal cord. The ratio of neuronal apoptosis peaked on the third day after the formalin injection. The enhance of the NOS activity as well as execessive production of NO participated the induction of neuronal apoptosis during formalin inflammatory pain. One of the important mechanisms in NO inducing neuronal apoptosis was by upregulateing p53 expression.
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