Increased Expression Of HO-1 In Spinal Cord During Formalin Inflammatory Pain And The Role In Promoting Pain

Objective: Recently carbon monoxide (CO) has been recognized to act as a neurotransmitter or neuromodulator in the nervous system.Heme oxygenase(HO) is a microsomal enzymy in the processes of CO production. The HO is widely distributed in the CNS and can catalyze the processes of heme degradation into one molecule of CO,one atom of iron and biliverdin which is subsequently reduced to bilirubin.HO-1 is one isoform of HO. It is normally expressed only in a few cell populations in the CNS, but can be induced by various factors.Our previous studies showed that expressions of nNOS and iNOS in the dorsal horn of the spinal cord were increased during formalin induced inflammatory pain , and the production of NO was increased too. There is a close relationship between NOS/NO and HO/CO systems. It had been reported that nerve injury caused by the deligation of sciatic nerve could induce up-regulation of HO expression in spinal cord and then produce more CO.But It is not been reported that whether the inflammatory pain caused by formaldehyde can induce the change in expression of HO-1 in spinal cord. Study results had been showed that intrathecally administered Zn-P to rat decreased the number of instances of flinching caused by subcutaneous formalin injection into the plantar surface of the right hindpaw in a dose-dependent manner, which indicated that CO played an important role in thenociceptive information transmission and pain related sensation.However there is no final conclusion on the effec of HO-CO on the production of thermal and mechanical hyperalgia.The present study was undertaken to observe the expression of HO-1 in spinal cord during formalin-induced pathological pain, and to further investigate the important role of HO and CO in the processes of producing the thermal and mechanical hyperalgia.1 Formalin inflammatory pain induced the changes of HO-1 expression in rat's spinal cordFourty-two male SD rats (250±20g in weight) were divided randomly into 7 groups: Control group, formalin 6h group,formalin 12h group, formalin 1d group, formalin 2d group, formalin 3d group, formalin 7d group. There were six rats in each group. Rats in control group were directly sacrificed without other treatments. Rats of formalin groups were subcutaneously injected with 0.15ml 5% formalin into the ventral surface of the right hind paw to induce periphery inflammatory pain and the L5 segment of spinal cord was dissected out at the designed ending time point and sectioned in paraffin preparation for HO-1 immunohistochemical staining.The results were as follows: Injection with formalin into the ventral surface of the rat hind paw induced a biphasic spontaneous pain reaction, including flinches, scratching and biting the wounded paw. The first phase pain reaction appeared right after the injection and lasted for several minutes. The second phase appeared ten minutes after the injection. There is a silent period between these two phases. The flinches lasted 1 h or more.The immunohistochemical staining showed that there is rare HO-1immunoreactive cells in the cornu posterius medullae spinalis and central canal of the L5 segment of spinal cord. Comparing with control group,the number of HO-1 immunoreactive cells in theⅠ-Ⅱlayers of L5 cornu posterius medullae spinalis and central canal of F6h group had an obviously increase , the area of HO-1 immunoreactive cells increased. The density of HO-1 immunoreactive cells had no obviously change. In the F1d group, the number and area of HO-1 immunoreactive cells reached the highest point ,the density of HO-1 immunoreactive cells reached the lowest point. Subsequently, the number and area of HO-1 immunoreactive cells reduced, the density of HO-1 immunoreactive cells increased also .but didn't return to normal level at 7th day after formalin injection.2 The effect of HO/CO on spontaneous pain and hyperalgia induced by formalin injection. Forty-five male SD rats (250±20g in weight) were divided randomly into 2 groups:The first group was divided randomly into 5 subgroups, including control group, formalin group,formalin+DMSOgroup, formalin+ZNPP(50μg),formalin+ZNPP(100μg),formalin+ZNPP(300μg).There were five rats in every group.In formalin+DMSOgroup and different dose ZNPP groups , DMSO or ZNPP was injected intrathecaliy beforehand 30 minutes formalin injection subcutaneouly into the plantar surface of the right hindpaw.The weighted pain score had been recorded. 24 hours later, DMSO or ZNPP was injected intrathecaliy again. Thermal withdrawal latency and mechanical withdrawal threshold were determined at 30min after injection.The second group was divided randomly into 4 sub group,including control group,NaOH group, Hemin187.5μg group and Hemin375μg group. There were five rats in every group. NaOH or Hemin was injected intrathecaliy to normal rats. Thermal withdrawal latency and mechanical withdrawal threshold were determined at 30min after injection.The results were as follows: Comparing with F1d group, intrathecal injection of DMSO had no obviously effect on pain related behavior and thermal and mechanical hyperalgia. After the intrathecal injection of ZNPP ,the weighted pain score reduced obviously in a dose-dependent manner comparing with the rats with formalin inflammatory pain. Comparing with control group,the rats of Formalin 1d group had an obviously prolongation in thermal withdrawal latency and mechanical withdrawal threshold in ipsilateral hindpaws,but an adverse effect on opposite side. Intrathecal injection of ZNPP had no obviously cffect on thermal withdrawal latency and mechanical withdrawal threshold in ipsilateral hindpaws comparing with Formalin 1d group.but there was an prolongation in opposite side of hindpaws . With the increase of the dose of ZNPP,the thermal withdrawal latency and mechanical withdrawal threshold of contralateral hindpaws is higher than control group.Comparing with control group, intrathecal injection of NaOH to normal rats had no obviously change on thermal withdrawal latency and mechanical withdrawal threshold.But intrathecal injection of Hemin to normal rats can shorten the thermal withdrawal latency and reduce the mechanical withdrawal threshold on both sides of hindpaws.There was no obviously difference between the two sides of hindpaws.Conclusion: Formalin inflammatory pain induced the expression of HO-1 in the spinal cord of rats, at 1d after injection of formalin, the expression of HO-1 reached the highest point. Intrathecal injection of the HO inhibitor produced prominent inhibition to pain related behavior and production of thermal and mechanical hyperalgesia induced by formalin. Intrathecal injection of HO inductor can induce the production of thermal and mechanical hyperalgesia. In conclusion, HO/CO took part in the processeses of nociceptive information transmission and the production of hyperalgesia.