| Plasticity change of periphery and central neurons is the key factor that causes the pain, especially neuropathic pain to develop into the chronic and continuous state. Recently plasticity change of spinal dorsal horn neurons after continuous stimulation has become the hot topic of research. Extracellular signal-regulate kinase (ERK), one member of the mitogen-activated protein kinases (MAPK) family, mediates intracellular transduction of diverse signals. It is found that in different pain models (inflammations induced by capsaicin or complete Freund's adjuvant, visceralgia, and electric stimulus) the expression of phosphorylation ERK increases and administration of inhibitor of its upstream kinase MEK can ameliorate model rat's performance of hyperalgesia. It is indicated that the change of activity is related to transmission of noxious stimulation and neural sensitization. NO is the important messenger molecule and neural transmitter in the cell and plays an important role in the development and maintenance of inflammatory pain.Objective Observe the influence of administration of MEK inhibitor U0126 and NOS inhibitor to rat's pain behavior, and the change of ERK activity and NOS in CCD-induced rat's neuropathic pain model. Discuss the cross-talk between ERK and NO signal pathway in transmission of pain signal in rat's spinal dorsal horn in CCD-induced neuropathic model.Material and Method Do the experimental study using wistar rats of 200-250g provided by Laboratory Animal Center of Jilin University. Intrathecal intubation and intrathecal administration are done according to the methods of Yaksh and Rudy. CCD animal model is built with reference to method of song. The changes of thermal pain threshold and motor function are measured by thermal pain stimulator and inclined plate experiment respectively. The positive neuron expression of pERK is observed by immunohistochemistry method. The level of pERK1 and pERK2 in spinal dorsal horn is determined by western blotting. Immune positive neuron of nNOS and iNOS in spinal dorsal horn is observed by immunofluorescence staining and western blotting.Method of Experiment 1: 122 rats are divided into three parts randomly. 32 rats are used in rat's behavioral detection and divided into sham operation group, CCD group, U0126 group and DMSO group randomly with 8 rats in each group. The rest 90 rats are used in the immunohistochemistry staining, cell counting, western blotting and protein quantitative analysis on 5 days, 10 days, 15 days of sham operation group, CCD group, U0126 group and DMSO group. On the day before building CCD model and the continuous three days after CCD U01265μg/10μl is intrathecally injected while the equivalent volume of DMSO is injected to DMSO group served as comparison. Thermal pain threshold is measured continuously for three days before compression of spinal dorsal root ganglion and the average value of three days is the basal level of rat, then from the first day after compression to the 15th day, the changes of thermal pain threshold and motor function are measured once two days. Rat's L4-5 segments of spinal cord are took to do the detection of immunohistochemistry staining and western blotting detection on 5 days, 10 days and 15 days after CCD.Method of Experiment 2: According to the difference of drug administration divide sham operation group and CCD group into L-NAME group, AG group, 7-NI group and 8-Br-cGMP group. 180 rats are divided into two parts randomly. 48 rats are used in behavioral detection. The rest 132 rats are used in the immunohistochemistry staining, cell counting, and western blotting and protein quantitative analysis. This is the same with method one. Rat's L4-5 segments of spinal cord are took to do the detection of immunohistochemistry staining and western blotting detection on 5 days, 10 days and 15 days after CCD.Method of Experiment 3: 108 rats are divided into two parts randomly. 48 rats are used in behavioral detection ,five days after the CCD Model established, according to the different intrathecal injections, these rats were divided into 6 groups: L-NAME,AG,8-Br-cGMP,7-NI,PBS,Cremophor group;the rest 60 of them were taken into Sham operation group, CCD group and according to the different intrathecal injections divided into L-NAME,AG,8-Br-cGMP,7-NI group, take materials to do immunohistochemistry staining, positive neuron counting and western blot analysis after two hours than the intrathecal injections .Method of Experiment 4: 84 rats are divided into two parts randomly and one part of 24 rats is used in behavioral detection. 24 rats are randomly divided into three groups: U0126 group, MDSO group, and model control with 8 rats in each group. The other part of 60 rats is used to do immunofluorescence staining and western blot analysis. They are randomly divided into six sub groups: Sham operation group, model control, groups of 0.5h, 2h, 12h and 24h after injection of U0126 with 10 rats in each group. In each group there are 6 rats used in immunofluorescence staining and 4 rats used in western blot analysis.Result 1: There is no obvious change after injection of U0126 and DMSO in normal rat. Rats of CCD group have hyperalgia at the first day after neurothlipsis and it reaches the peak at 5 day and maintains steadily during the observation. The intrathecal injection of U0126 in advance can obviously ameliorate hyperalgia caused by CCD. Intrathecal injection of U0126 and MDSO has no influence of rat's motor function. CCD causes the significant increase of number of positive neuron in the ipsilateral spinal dorsal horn. Intrathecal injection of U0126 in advance can significantly inhibit the expression of pERK positive neuron induced by CCD. Western blot indicates that there is obvious increase of levels of pERK1 and pERK2 of in CCD rat's spinal dorsal horn. Intrathecal injection of U0126 can obviously inhibit the increase of level of pERK induced by CCD.Result 2: There is no obvious change of rat's paw withdrawal latency before and after operation in sham operation group. In CCD group on the first day after operation paw withdrawal latency obviously shortens. Compared with CCD group, in 7-NI group paw withdrawal latency of obviously extends from the first day to 11th day after operation. In L-NAME group paw withdrawal latency of obviously extends from the first day to 7th day after operation. In AG group paw withdrawal latency of obviously extends from the first day to 5th day after operation. In 8-Br-cGMP group on the first day after operation paw withdrawal latency obviously shortens and there is no obvious difference from the third day. Intrathecal injection of NOS inhibitor or activator has no obvious influence to rat's motor function.At 5 day after operation in sham operation group there is few nNOS and iNOS immune positive neurons in injured side spinal dorsal horn. Chronic compression injury causes the obvious increase of nNOS and iNOS immune positive neurons. Compared with CCD group, the number of immune positive neurons in injured side spinal dorsal horn obviously decreases in groups of L-NAME, AG and 7-NI. Western blot indicates that compared with sham operation, CCD induces the obvious increase of level of nNOS and iNOS in rat's spinal dorsal horn nerves. Intrathecal injection of nonselective nNOS inhibitor L-NAME and selective nNOS inhibitor 7-NI can obviously inhibit expression of nNOS in spinal dorsal horn of CCD rat while selective iNOS inhibitor has no obvious influence to expression of nNOS. Similarly, intrathecal application of nonselective iNOS inhibitor L-NAME and selective inhibitor AG can obviously inhibit expression of iNOS while selective nNOS inhibitor has no obvious influence to expression of iNOS. 8-br-cGMP can increase expression of nNOS and iNOS in spinal dorsal horn of CCD rat.Result 3: At postoperative 5 day obvious hyperalgesia is produced in rear leg of all the model rats in the experiment. Compared with before intrathecal injection, after injection there is no obvious change of pain behavior of rats of both PBS group and Cremophor group at each point of time. Compared with PBS group, there is no obvious change of thermal paw withdrawal latency in operative side of rat of L-NAME group, AG group and 7-NI group at 12h and 24h and there is obvious extension of thermal paw withdrawal latency in operative side of rat of L-NAME group, AG group and 7-NI group at 30min, 2h and 6h. Compared with PBS group, there is obvious shortening of thermal paw withdrawal latency in operative side of rat of 8-Br-cGMP group at 30min and 2h.The result of immunohistochemistry indicates that at 5 day of CCD there is obvious increase of pERK immunoreactions positive neuron in rat's spinal dorsal horn of operative side; in L-NAME group, AG group and 7-NI group the number of pERK immunoreactions positive neuron in rat's spinal dorsal horn of operative side obviously decreases at 2h after intrathecal injection; and in 8-Br-cGMP group the number of pERK immunoreactions positive neuron in rat's spinal dorsal horn of operative side increases at 2h after intrathecal injection. Western blot indicates that CCD causes the increase of pERK level in rat's spinal dorsal horn neuron and compared with CCD group and 8-Br-cGMP group, pERK content in dorsal horn neuron of L-NAME group, AG group and 7-NI group obviously decreases.Result 4: At postoperative 5 day, in sham operation group there is very few nNOS immune positive neurons in the injured side of spinal dorsal horn of rat and chronic compression injury causes the obvious increase of nNOS immune positive neurons. Compared with sham operation group, in model control the number of neuronal nitric oxide synthase immunoreaction positive cell in injured side of spinal dorsal horn obviously increases. Compared with model control, at 0.5, 2 and 12h after injection of U0126 the number of neuronal nitric oxide synthase immunoreaction positive cell in injured side of spinal dorsal horn obviously decreases. At 24h after injection of U0126 there is no obvious change in the number of neuronal nitric oxide synthase immunoreaction positive cell in injured side of spinal dorsal horn. Western blot indicates that chronic compression injury causes the increase of expression level of nNOS in spinal dorsal horn. Compared with sham operation group, in model control expression level of neuronal nitric oxide synthase immunoreaction positive cell in injured side of spinal dorsal horn obviously increases in 12h group after injection of U0126 and in 24h group after injection of U0126. Compared with model control, expression level of neuronal nitric oxide synthase immunoreaction positive cell in injured side of spinal dorsal horn obviously decreases in 0.5h group, 2h group and 12h group after injection of U0126 while expression level of neuronal nitric oxide synthase immunoreaction positive cell in injured side of spinal dorsal horn does not have obvious change in 24h group after injection of U0126.Conclusion1. CCD can obviously increase expression of pERK, NOS positive neuron in superficial layer of spinal dorsal horn in injured side.2. While intrathecal injection ERK signal conduction pathway inhibitor U0126 and NOS inhibitor ameliorate rat's pain behavior, they can also obviously inhibit expression of pERK1/2 and NOS in spinal dorsal horn of injured side.3. Rat's pain behavior is in line with the expression of pERK, nNOS and iNOS of spinal dorsal horn in time course.4. In regulation of pain signal of spinal cord level, the change of activity of ERK signal pathway in spinal dorsal horn neuron can influence expression of neuronal nitric oxide synthase in spinal dorsal horn. Extracellular signal regulated kinase is involved and mediated the function of nitric oxide in neuropathic pain.
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