TRPV4 In Spinal Cord Dorsal Horn Takes Part In The Neuropathic Pain Mechanism In Chronic Compression Of Dorsal Root Ganglion Model Of Rat

BackgroundAs the high rate of recurrent and refractory, neuropathic pain (NP)had become a worldwide problem for human health. The emergence of animal models provide basis for neuropathic pain research. The chronic compression of the dorsal root ganglion(CCD) model was widely accepted by researchers. It can simulate the hyperalgesia and spontaneous pain symptoms of root neuralgia made by stenosis of intervertebral foramen.Transient receptor potential family are one of the biggest ion channels. Its family members TRPV1 and TRPV4 can take part in the sensitive process induced by pathological factors as a sensory conduction apparatus. Now we know that TRPV1 expressed in Ⅰ and Ⅱ layer of the spinal cord dorsal horn and may participate in the pathological process of neuropathic pain. And research finding that TRPV1 expression increased for two weeks in spinal cord dorsal horn which consistent with the changes of mechanical hyperalgesia in chronic constriction injury animal model. New findings indicate that TRPV1 in spinal cord dorsal horn may participate in the process of resin toxin neuropathic pain model. Giving intrathecal injection of TRPV1 inhibitors advance not only downregulated its expression but also delayed the heat hyperalgesia at the same time. All the studies show that TRPV1 play an important role in neuropathic pain mechanism.As we all know, the spinal cord dorsal horn is the aggregation of sensory afferent. Peripheral nociceptive sensory information must be integrated in spinal cord dorsal horn and then uploading to the brain.And the download modulation information from the brain should be processed by the spinal cord dorsal horn. The significance of Anatomical structure and location which determined the role of spinal cord dorsal horn in the transduction process of pain.TRPV4 usually distributed in DRG、skin nerve terminals and free nerve endings in nervous system in present reports. And our previous research found that TRPV4 can take part in the pathological processes of mechanical hyperalgesia in DRG neurons and we also found that TRPV4 may play the role by NO-cGMP-PKG signaling pathway. But the hyperalgesia only partly relieved when giving TRPV4 inhibitors in DRG neurons. The research indicated that there may have other mechanisms in CCD neuropathic pain. We also find that TRPV4 positive expression increased in spinal cord dorsal horn in CCD rats through immunofluorescence technique. TRPV1 and TRPV4 are all TRP members which biological activity highly unified because of its similar amino acid sequence. Therefore, we suspect that the spinal cord dorsal horn TRPV4 can take part in the CCD neuropathic pain.ObjectiveTo investigate TRPV4 expression and the its protein and gene alternation in spinal cord dorsal horn in chronic compression of the dorsal root ganglion model of rats, demonstrate the role of TRPV4 in neuropathic pain.Method1. CCD modelSixty healthy adult male Wistar rats were randomly divided into three groups,that were blank control group,CCD group and CCD+RR group(n=20).CCD model was made by insert a "U" shape stainless rod which diameter and length were 0.63mm and 4mm,into the intervertebral foramina lumber 4 and 5. We can see a suddenly contraction of the ipsilateral hind leg muscle which indicating a success of the inserting process. The blank control group are the same as CCD except to inserting a "U" shape stainless rod.CCD+RR group were giving the intrathecal injection of TRPV4 inhibitors Ruthenium red at seven days after CCD surgery.2.Mechanical stimulation pain threshold measurementMechanical stimulation pain threshold were measured at pre-CCD and intrathecal injection before and two hours later seven days after CCD surgery. Method are the following:All group rats will adapt to the environment in the red box thirty minutes before measurement begining. Stimulating the lateral plantar skin vertically between the third and fourth toes of rats through the grid layer of the glass box using the BME-404 electronic mechanical stimulation equipment. A withdrawal response or licking feet phenomenon were positive performence. And then recording the stimulus number. The process of measurement will conduct five times with intervals of 5 minutes and taking the average value for data analysis.3.TRPV4 gene and protein expression in spinal cord dorsal hornAll group rats died after mechanical stimulation pain threshold measurement at seven days after CCD surgery. Using RT-PCR and Western Blot detect the changes of TRPV4 gene and protein in spinal cord dorsal horn of rats.Result1. TRPV4 expression in spinal cord dorsal hornThe results of Western-Blot and RT-PCR show that TRPV4 protein and gene expressed in blank control group which indicating a certain expression of TRPV4 in spinal cord dorsal horn in rats.2. TRPV4 expression changes in spinal cord dorsal horn after CCD modelThe results of Western-Blot and RT-PCR show that the expression of TRPV4 protein and gene increased significantly in ipsilateral spinal cord dorsal horn of rats in CCD group compared with blank control group seven days after CCD surgery(P< 0.05).3.The influence of Ruthenium red(RR) on mechanical stimulation pain threshold of CCD ratsThe results show that the ipsilateral mechanical stimulation pain threshold increased significantly of rats in CCD+RR group cpmpared with CCD group two hours after the intrathecal injection of TRPV4 inhibitors(P<0.001).4.The influence of Ruthenium Red(RR) on TRPV4 expression in ipsilateral spinal cord dorsal horn of CCD rats.The results of Western-Blot and RT-PCR show that the expression of TRPV4 protein and gene decreased significantly in ipsilateral spinal cord dorsal horn of rats in CCD+RR group compared with CCD group two hours after the intrathecal injection of TRPV4 inhibitors (P<0.05).ConclusionTRPV4 protein can express in spinal cord dorsal horn; The express of TRPV4 upregulated significantly after CCD surgery and the ruthenium red can inhibit TRPV4 expression and upregulate the mechanical stimulation pain threshold at the same time. Our research showed that TRPV4 can participates in central sensitization mechanism of neuropathic pain.