The Role Of TRPV4in Mediating The Ectopic Discharges Following Chronic Compression Of Dorsal Root Ganglion In Rats

BackgroundThe low back pain is a serious problem for human health in modern society, it not only caused great suffering to patients job and life, but also caused serious influence to social productivity. DRG neuron is the primary afferent neuron that carries the sensory signals to spinal cord, it plays an important role in the peripheral mechanism of the low back pain. Radicular neuralgia is the most common pathogenesis of the low back pian, which caused by the chronic compression of dorsal root ganglion with some noxious stimulation. A chronic compression of dorsal root ganglion may produce spontaneous pain, mechanical allodynia and thermal hyperalgesia, which well increase spontaneous discharges and reduce action potential and current perception threshold. Injured DRG can become a source both of ectopic spontaneous discharges and abnormal activity, in the absence of peripheral receptor activation, evoked by sympathetic stimulation and/or endogenous chemicals.DRG neurons can transmit polymodal sensations, such as temperature and mechanical sensations. Many ion channels on DRG neurons play a potential role in mechanical nociception. Among these channels, the TRPV4channel is a polymodal receptor, which plays an important role in nociceptive responses to hypotonic and mechanical stimuli. Our preliminary study found that the chronic mechanical compression can induce the up-regulation of mRNA and protein expression of TRPV4without affecting other TRPs. Meanwhile, the mechanical pressure also enhances the function of the TRPV4channels. Another study found that TRPV4is an outwardly rectifying cation channel, calcium and sodium ion can be permeated. Activated TRPV4receptor can increase the ectopic discharges by increasing calcium and sodium ion concentration.From the above, we speculate that TRPV4may be associated with the ectopic discharges of injured DRG. CCD model was prepared by inserted two stainless steel rods into L4and L5DRG. After CCD motor function, mechanical withdrawal threshold and thermal withdrawal latency were measured. We has shown that the neuroethology changes of the rats and the morphological changes in the DRG neurons as time goes on. To investigate the role of transient receptor potential vanilloid4(TRPV4) in mediating DRG ectopic spontaneous discharge following chronic compression of dorsal root ganglion(CCD). The ectopic discharges of the normal DRG and the injured DRG were recorded by the nerve fibers electrophysiology in vivo.ObjectiveTo investigate the role of transient receptor potential vanilloid4(TRPV4) in mediating DRG ectopic spontaneous discharge following chronic compression of dorsal root ganglion(CCD).Method1. Recording the ectopic discharges1.1CCD surgeryForty-five healthy adult male Wistar rats were randomly divided into seven groups, of which5were normal group,10were CCD group,10were ruthenium red group and the20were phorbol group. Chronic compression of dorsal root ganglion (CCD) model was established by insert a "U" shape stainless rod into the right intervertebral foramina of the lumber5under pentobarbital sodium anesthesia.1.2The ectopic discharges of the normal DRG and the injured DRG were recorded by the nerve fibers electrophysiology in vivo3-8days post-CCD.2. Neuroethology measurement and morphological changes2.1CCD surgeryThirty-five healthy adult male Wistar rats were randomly divided into seven groups, of which5were un-operated control group,15were experiment model group and the rest15were sham-operation group. CCD model was established by insert a "U" shape stainless rod into the right intervertebral foramina of the lumber4and5. The sham-operation group’s surgical procedure was identical to that of the experiment model group but without inserting a stainless rod into the dorsal root ganglion, and no operation was performed to the un-operated control group.2.2Neuroethology measurement2.2.1Walk gait pattern was assessed as the index of motor function."1" indicates normal gait, without foot deformities."2" indicates normal gait with obvious foot deformities."3" indicates slight gait disturbance with foot-drop."4" indicates serious gait disturbance with myasthenic.2.2.2Mechanical withdrawal threshold(MWT) and thermal withdrawal latency(TWL) in the affected hind limb of the rats were measured pre-CCD and2,4,6,8,10,12,14,16,18,20,22,24,26and28days after CCD.MWT was assessed with a von Frey hair monofilament with logarithmically incremental stiffness. The von Frey hair was applied through the mesh floor to the plantar skin of the hindpaw in an ascending order. The MWT was defined as the lowest force that evoked a brisk withdrawal response to at least three of five stimuli.TWL was measured with paw withdrawal latencies to radiant heat. The radiant heat source beneath the glass floor was focused on the plantar surface of the ipsilateral hindpaw when in contact with the floor. The paw withdrawal latencies per animal were obtained for five times with intervals of5min.2.3We observed the dorsal root ganglion by HE under optical microscope after7d,14d and28d respectively.Result1. The ectopic discharges were present in67%of the fibers recorded from the DRG neurons injured with chronic compression in contrast to4.5%from uninjured DRG neurons, CCD increased significantly the ectopic discharges of the injured DRG neurons(n=5, P<0.01).2. Compared with the CCD group the frequency and amplitude of the ectopic discharges decreased significantly after incubating the damage DRG with TRPs inhibitor100μm RR(n=10, p<0.05).3. Compared with CCD group the frequency and amplitude of ectopic discharges were no significant changes after incubating the injured DRG with TRPV4specific activator1nm and1μm4α-PDD(both P>0.05); Compared with CCD group the frequency and amplitude of ectopic discharges increased significantly after incubating the injured DRG with TRPV4specific activator10μm4α-PDD(n=10, P<0.05).4. Neuroethology measurement4.1The walk gait pattern was normal and no foot deformities were found in all rats pre-and post-CCD. The score was "1". There was no significant difference between control and CCD groups.4.2The MWT and TWL in CCD group were significantly lower than the sham and control group. And both of them was lower in the CCD group than the control group at2,4,6,8,10,12,14,16,18,20,22,24,26and28days post-surgery (P<0.05), with the lowest level at6days post-CCD. There was also a significant difference in MWT and TWL at7,14, and28days post-CCD, and there were obvious hyperemia, edema and inflammation in the dorsal root ganglion.5. HE On post-CCD7days and14days groups, there were obvious edema, hyperemia and inflammation in the dorsal root ganglion. On post-CCD28days group, the degree of hyperemia and edema decreased, but the proliferation of fibrocyte in the endoneurium and epineurium was observed. None of the phenomena was observed in the un-operated control group and sham-operation group.ConclusionThe ectopic discharges of injured DRG can be recorded. TRPV4plays a crucial role in mediating DRG ectopic discharge after chronic compression of dorsal root ganglion(CCD).