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Increased Expression of HO-1 on Spinal Cord during Formalin Inflammatory Pain and the Role in Neuronal Apoptosis

Author: SongYanHui
Tutor: LiQingJun
School: Hebei Medical University
Course: Pathology and Pathophysiology
Keywords: formalin pain neuron apoptosis spinal cord HO-1 ZnppIX
CLC: R402
Type: Master's thesis
Year: 2009
Downloads: 16
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Abstract

Objective: Endogenous carbon monoxide is another important gaseous signal molecule and neurotransmitter after the nitric oxide (NO) be found. It has an important biological function in the physiological and pathological processes of mammals. Heme oxygenase (HO) is rate-limiting enzyme of heme metabolism, widely distributed in many tissues, and it 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 a subtype of HO, normal expression in the nervous system at low, but can be induced by a variety of factors.Our previous studies showed that formalin inflammatory pain can induce the expression of NO in the spinal dorsal horn. Further studies found that NO participated the procedure of the apoptosis-induced by formalin inflammatory pain in spinal neurons. It had been reported that HO / CO system was involved in spinal nociceptive information transmission process, ligation of sciatic nerve injury can induce an increase in HO-1 expression in spinal cord, at the came time, CO is increased. But it has not been reported that whether the formalin inflammatory pain can induce the change in expression of HO-1 and the role of HO-1 expression in the formaldehyde-induced neuron apoptosis of spinal cord.The present study was undertaken to observe the expression of HO-1 in spinal cord during formalin-induced pathological pain, and then intrathecal injection with HO-1 inhibitor zinc protoporphyrinⅨ(ZnppⅨ) ,to investigate the role of HO-1 expression change in the processes of spinal neurons apoptosis-induced by formalin inflammatory pain.1. Formalin inflammatory pain induced the changes of HO-1 expression in rat’s spinal cordTwenty-five male Sprague-Dawley rats (260~280g in weight) were divided randomly into 5 groups: namely normal control group (Control group), formalin 6h group (F6h), formalin 12h group (F12h), formalin 24h group (F24h) , formalin 72h group (F72h), five animals in each group. Control group were directly sacrificed without other treatment. Formalin animals in each group were observed spontaneous pain response after plantar injection with formaldehyde, and then decapitated at different time points .The L5 lumbar spinal dorsal horns was dissected out. Western blotting methods were used to detect the HO-1 protein expression of the left and right spinal dorsal horn.The results showed that: the rats instantly appeared flinches, scratching and biting the wounded pawior, such as injection of adequate spontaneous pain behavior after injection of formalin .The response of spontaneous pain was double-phase. It lasted for more than 1h, and the injection site appeared red, swollen and so obvious inflammatory response.Western blotting results showed that, The HO-1 protein bands of left and right spinal dorsal horn of rats in control group were considerably smaller in area, dyeing was shallow, indicating that there was a certain amount of basic expression of HO-1 protein in normal rats. Compared with Control group, HO-1 protein expression were significantly up-regulated at different time points after plantar injection of formaldehyde, HO-1 protein bands were widened. The ratio of HO-1 andβ-actin integral optical density (integrated optical density, IOD) was increased; The HO-1 increased expression was most significantly in the rats of F24h group, reaching peak and than recoverd. But the HO-1 expression in the rats of F72h group was still higher than the control group. At the came time points, There were not significant difference in HO-1 protein expression in the left and the right sides of the spinal dorsal horn2. The effect of HO/CO on the formaldehyde-induced inflammatory pain in rat spinal cord neuron apoptosisThirty male Sprague-Dawley rats (260~280g in weight) were divided randomly into 5 groups: control group, formalin group (F), formalin+DMSO group (F+DMSO), formalin+50μg ZnppIX group (F+50μgZ), formalin+100μgZnppIX group (F+100μgZ). There were six rats in every group respectively. DMSO or ZnppIX was injected intrathecally before 30 minutes formalin injection into the ventral surface of the right hind paw to induce periphery inflammatory pain .The L5 segment of spinal cord was dissected out at 72h after formalin injection to detecte neuron apoptosis rate of spinal cord by flow cytometry.The results showed that: Compared with formalin group, the intrathecal injection of DMSO had no significant effect on spontaneous pain responses of the rats with formalin inflammatory pain. But intrathecal injection of 50μgZnppIX and 100μgZnppIX both induced an decrease in degree of the spontaneous pain responses induced by formalin injection, and the degree of decrease in spontaneous pain responses in the rats of F +100μgZnppIX group was more obvious than the rats of 50μgZnppIX group.Flow cytometry results showed that: the ratio of neuron apoptosis of spinal cord (L4~5segment) was very low in the rats of control group. Compared with control group, the ratio of neuron apoptosis of spinal cord was increased in the rats of formalin group. Compared with formalin group, the ratio of neuron apoptosis has no significant difference in the rats of F+DMSO group. Compared with formalin group, the ratio of neuron apoptosis of spinal cord was no significant difference in formalin+50μg ZnppIX group also, but increasing ZnppIX dose, intrathecal injection of 100μgZnppIX induced an increase in the ratio of neuron apoptosis of spinal cord in the rats with formalin inflammatory pain. The results showed that HO / CO system can inhibit the process of neuronal apoptosis of spinal cord induced by formalin inflammatory pain.Conclusions: Formalin inflammatory pain could induce the expression of H0-1 on the spinal cord of rats. HO-1 expression reached the highest point at 24 hours after injection of formalin. Intrathecal injection of HO inhibitor ZnppIX can inhibit the degree of spontaneous pain responses induced by formalin injection, and induce an increase in the ratio of neuron apoptosis of spinal cord in the rats with formalin inflammatory pain. The results showed that HO / CO system can inhibit the process of neuronal apoptosis in spinal cord induced by formalin inflammatory pain.

Increased Expression of HO-1 on Spinal Cord during Formalin Inflammatory Pain and the Role in Neuronal Apoptosis

Abstract

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