TRPV4 Signal Pathway In The Mechanism Of Behavioural Hyperalgesia Following Chronic Compression Of The Dorsal Root Ganglion In Rats

BackgroundChronic compression of the dorsal root ganglion (DRG) (the procedure hereafter termed CCD) in animals, a typical model of neuropathic pain, mimics clinical disc herniation and spinal canal stenosis in humans. CCD rats show ipsilateral spontaneous pain, mechanical allodynia, and thermal hyperalgesia. In association with these behavioral effects, an increased excitability of neuronal somata in the compressed ganglion has been shown, as evidenced by spontaneous activity, lowered rheobase and action potential thresholds. Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and cyclic guanosine monophosphate (cGMP)-dependent protein kinase G (PKG) pathways, both second messenger signals, have been shown to participate in maintaining neuronal hyperexcitability and behavioral hyperalgesia in CCD treatment. Moreover, several types of ion channels, such as voltage-gated Na+ and K+, hyperpolarization-activated cation current, and TRPV4 have been reported to mediate detection and transmission of nociceptive stimuli in CCD neurons. However, to date, the detailed mechanisms of nociceptive signal transfer and the correlation between the ion channels and the second messenger signals in behavioral hyperalgesia remain unclear in the CCD model of neuropathic pain in rats.TRPV4 is a polymodal receptor that is activated by hypotonicity, mechanical stimuli, heat, phorbol esters, low pH, citrate, anandamide (AEA) and its LOX metabolite arachidonic acid (AA), and bisandrographolide A (BAA). TRPV4 is inovolved in mechanical hyperalgesia and allodynia, as revealed by enhanced responses to hypotonic or hypertonic test stimuli after sensitization by PGE2. TRPV4 knockdown studies show abnormal osmotic regulation and increased nociceptive threshold to pressure with unchanged response to heat and touch. TRPV4-/-knockout mice also show reduced thermal hyperalgesia in inflammatory pain. Moreover, TRPV4 has been shown to contribute to mechanical and hypotonic hyperalgesia in neuropathic pain models. While it is recognized that TRPV4 contributes to mechanical and hypotonic hyperalgesia in neuropathic and inflammatory pain, TRPV4 is also reported to be involved in thermal hyperalgesia in inflammatory pain. However, whether TRPV4 participates in thermal hyperalgesia in neuropathic pain is unknown.TRPV4, with its gene and protein expression in DRG increased after CCD, contributes to mechanical hyperalgesia and exhibits robust Ca2+entry on exposure to a hypotonic milieu or a phorbol ester,4a-PDD. Nitric oxide synthase (NOS) is Ca2+-dependent and is responsible for the synthesis of NO from L-arginine. NO has been shown to be a key mediator of nociceptive activity in animal pain models. TRPV4 synthetic activator,4a-PDD, and hypoosmotic stimulation could induce NO production in outer hair cells of the guinea pig cochlea, while NO production is inhibited by ruthenium red, an inhibitor of TRPV4. TRPV4, as an osmosensory transducer, may modulate downstream effectors via glutamatergic synapses. NO is also reported to increase glutamate release through cGMP-PKG signaling pathways. Further,4a-PDD and hypoosmotic stimulation induce TRPV4-dependent CGRP release and an increase in NO is associated with up-regulation of CGRP synthesis and release. TRPV4 may thus be associated with NO-cGMP-PKG pathways. Moreover, NO-cGMP, cGMP-protein kinase G (PKG), and NO-cGMP-PKG pathways have been found to contribute to hyperalgesia in neuropathic and inflammatory pain. We therefore hypothesized that the activation of TRPV4 in DRG initiates an influx of Ca2+, stimulating Ca2+-dependent NOS, thereby activating the NO-cGMP-PKG pathway, which in turn enhances the release of glutamate and CGRP. This pathway may contribute to nociceptive response following CCD. In the present study, we evaluated the role of TRPV4-NO-cGMP-PKG pathways in thermal hyperalgesia in the CCD model by examining the effects of TRPV4 knockdown on behavioral responses and on NO production in the DRG.ObjectiveTo investigate the role of TRPV4-NO-cGMP-PKG pathways in thermal hyperalgesia in the CCD model.Methods1. Establish CCD modelRats were randomly divided into CCD and sham groups. In the CCD rats, under pentobarbital sodium anesthesia (Nembutal,50 mg/kg ip), the transverse process and intervertebral foramina of L4 and L5 were exposed unilaterally as previously described. A stainless steel L-shaped rod (0.63-mm diameter and 4-mm length) was inserted into each foramen, one at L4 and the other at the L5 level, to compress the DRG. The muscle and skin layers were then sutured. Penicillin was injected to prevent infection. The sham group underwent the same surgical procedure as described, but without the insertion of the rods. The animals did not show any autotomy, nor was there a complete loss of sensation following the surgery.2. Antisense oligodeoxynucleotide treatmentTo determine the effects of antisense oligodeoxynucleotide (ODN) treatment on CCD-induced thermal hyperalgesia and nitrite production in the DRG, CCD and sham rats were treated with a spinal intrathecal administration of TRPV4 antisense ODN and mismatch ODN, respectively. Each group was sub-divided into control group, TRPV4 antisense ODN group (AS group), and mismatch ODN group (MM group). The TRPV4 antisense ODN sequence,5'-CATCACCAGGATCTGCCATACTG-3' (Invitrogen, Carlsbad, CA, USA) and the mismatch ODN sequence, 5'-CAACAGGAGGTTCAGGCAAACTG-3'(Invitrogen) were designed as described previously. ODN was reconstituted in nuclease-free 0.9%NaCl (10μg/μl) and administered into the spinal intrathecal space at a dose of 40μg, once a day for 7 days until the animals were sacrificed or treated with drugs. As described previously, rats were anesthetized with 2.5% isoflurane inhalation anesthetic (97.5% O2), a 30-gauge needle was inserted into the subarachnoid space on the midline between the L4 and L5 vertebrae and 20μl ODN injected at 1μl/s, using a micro-syringe.3. Behavioral TestingThermal hyperalgesia was assessed using paw withdrawal latencies to radiant heat (BME-410A, Biomedical Engineering Institute of the Chinese Academy of Medical Sciences), as described previously. Animals were acclimated to the cage with a 6-mm thick glass floor, and the temperature of the glass was measured and maintained at 26±0.5℃. Then, the radiant heat source beneath the glass floor was focused on the plantar surface of the ipsilateral hind paw when in contact with the glass floor. The paw withdrawal latencies per animal were obtained five times, with an intervening interval of 5 min. The intensity was pre-calibrated to give a baseline latency of approximately 10 s and the cutoff time was set at 20 s to avoid tissue damage. During this time, the rats initially demonstrated exploratory behavior, but subsequently stopped exploring and stood quietly with occasional bouts of grooming. The rats were tested on each of 2 successive days before surgery. Postoperative tests were conducted 2 h before chemicals treatment on the 7th day after surgery. Rats not demonstrating hyperalgesia were excluded from further study (less than 5%). Additional tests were conducted 1,2,4,8, and 24h after injection of chemicals or saline (2.5%DMSO) into the subarachnoid space on the midline between the L4 and L5 vertebrae on the 7th day after surgery. For experiments investigating the effect of treatment with TRPV4 antisense ODN on nociceptive thresholds, behavioral testing was performed 12 h after the last ODN injection. All behavioral tests were conducted under blind conditions.4. Western blotting The levels of TRPV4 protein expression in different groups were assessed using Western blotting.5. Nitrite Production AssayThe level of nitrite as a measure of NO production in DRG was determined with modified Griess reagent. A nitrite detection kit (Beyotime Biotech Inc., Jiangsu, People's Republic of China) was used according to instructions provided by the manufacturer.Results1. Effects of antagonist and antisense ODN of TRPV4 on CCD-induced thermal hyperalgesia and levels of NO derivative nitrite in DRGCCD produced a clear-cut hyperalgesia compared to the sham rats (F(1,112)=175.858, P<0.001). The levels of NO derivative nitrite in DRG were significantly increased at 7 days in the CCD group when compared with the sham group (P<0.001). There were no significant changes in the levels of nitrite in the sham group (P=0.663).The intrathecal administration of the non-specific inhibitor of TRPV4 ruthenium red in concentrations of 0.1-1 nmol (both P<0.001) produced a dose-dependent reduction of the thermal hyperalgesia in CCD rats, when compared with saline group, but ruthenium red in a concentration of 0.01 nmol did not (P=0.497). The significant reduction of the thermal hyperalgesia was observed at 1 h, peaked at 4 h, and lasted for about 8 h post-injection. There were no significant differences between groups at 24 h after administration (all P>0.05). Intrathecal injection of ruthenium red (1 nmol) significantly decreased the concentration of NO metabolites nitrite in DRG in CCD rats when compared to saline (P<0.001). The reduction of nitrite in DRG peaked at 4 h and was positively associated with the changes of thermal responses (r=0.997, P<0.05) after ruthenium red (1 nmol) injection at 7 days post-surgery.Western blotting showed that, TRPV4 expression in DRG 7 days following compression was significantly (P<0.001) inhibited by intrathecal injection of antisense ODN, but not inhibited by mismatch ODN treatment, as compared to controls.The PWLs in CCD rats were similar at baseline among groups (all P>0.05), but after intrathecal ODN treatment for 7 days, the PWL was decreased significantly in the CCD and MM groups when compared with the baseline (both P<0.001). In contrast, thermal hyperalgesia was partly reversed in the AS group (P=0.059) compared with baseline. There were no significant changes in the PWL following intrathecal AS or MM in sham rats (data not shown). In CCD rats, the antisense ODN, but not the mismatch ODN, significantly (P<0.001) inhibited the nitrite production when compared with the vehicle group.2. Effects of L-NAME on CCD-induced thermal hyperalgesia and levels of NO derivative nitrite in DRGIntrathecal pretreatment with L-NAME (a non-specific NOS inhibitor,30-300 nmol) significantly (P<0.001) and dose-dependently inhibited CCD-induced thermal hyperalgesia from 1 h to 8 h with the peak inhibitory effect at 4 h post-injection. Moreover, the level of NO derivative nitrite in the DRG of CCD-treated rats was significantly (F(2,120)=201.725, P<0.001) suppressed by intrathecal pretreatment with L-NAME (300 nmol). The level of nitrite began to decrease 1 h post-injection and reached a minimum 4 h post-injection, followed by a gradual recovery, and the changes in nitrite were not significantly different from the saline group at 24 h post-injection (P>0.05). Moreover, intrathecal pretreatment with D-NAME (an inactive isomer of L-NAME,300 nmol) had no effects on thermal hyperalgesia or on the concentration of nitrite at any time following DRG compression (both P>0.05).3. Effects of ODQ and Rp-8-pCPT-cGMPS on CCD-induced thermal hyperalgesiaIntrathecal pretreatment with ODQ (50-100 nmol), or Rp-8-pCPT-cGMPS (25-50 nmol) significantly (both P<0.01) and dose-dependently increased the PWL in CCD-treated rats 1,2,4 and 8 h post-injection when compared with vehicle treatment.4. Effects of 4a-PDD on the suppressive effects of L-NAME on CCD-induced thermal hyperalgesia and nitrite production4a-PDD (the TRPV4 agonist,1 nmol) attenuated the suppressive effects of L-NAME (300 nmol) on CCD-induced thermal hyperalgesia (P<0.001), compared to L-NAME (300 nmol) alone. Moreover, the decreased level of nitrite induced by intrathecal injection of L-NAME (300 nmol) was also attenuated by concomitant administration of 4a-PDD (1 nmol) 1 h post-injection (P<0.05).ConclusionsThis study demonstrates that the TRPV4-NO-cGMP-PKG pathway is involved in thermal hyperalgesia in CCD rats. BackgroundSeveral lines of evidence indicate a role for nitric oxide (NO) as a mediator of pathological nociception. NO, acting as an inter-and intracellular messenger molecule in the peripheral and central nervous system, plays a pivotal role in the development and maintenance of hyperalgesia.Three isoforms of NO synthase (NOS), including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS), mediate NO synthesis from L-arginine in the presence of oxygen. These NOS isoforms'expression can be upregulated in nervous tissues under pathological conditions, suggesting that the pathophysiologic functions of NO in the nervous system may be regulated by altering expression and activity of NOS isoforms. Neuronal NOS is expressed in the neurons and produces predominantly NO in neuronal tissues. The contribution of nNOS-synthesized NO to nociceptive processing has been characterized in several neuropathic pain models. In recent years iNOS has become of significant interest in the pathophysiology of neuropathic pain. iNOS expressed in neuronal tissues is thought to be involved in mechanisms of hyperalgesia with some authors showing a specific role in the pain hypersensitivity associated with models of spinal cord injury. Increased nNOS and iNOS exoression in neuronal tissues have also been observed in the chronic constriction injury (CCI) model and spinal nerve ligation, suggesting their role in the development of neuropathic pain, lines of evidence have demonstrated that nNOS and iNOS contribute to NO production involved in the maintenance of behavioural hyperalgesia developing after spinal nerve transection and sciatic nerve chronic constriction injury. However, its contribution to behavioural hypersensitivity in other animal models of neuropathic pain still remains unclear.In particular, we have previously demonstrated that the NO production in the DRG is increased and intrathecal injection of non-selective NOS inhibitors such as NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) reduces thermal hypersensitivity in neuropathic pain produced by a chronic compression of DRG (CCD)-an injury that may accompany an intraforaminal stenosis, a laterally herniated disc or other disorders of the spine leading to radicular pain, where the NOS isoforms involved in the maintenance of neuropathic pain in this model have not been evaluated. We also have found that the NO production is mediated by TRPV4, a mechano-and thermo-receptor in the DRG following CCD.The aim of the present study, therefore, was to assess NOS isoforms and TRPV4-dependent NOS isoforms involved in the maintenance of thermal hypersensitivity developing in an animal model of neuropathic pain prepared by CCD.Objective1. To investigate the effects of CCD on the expression levels of mRNA and protein of nNOS and iNOS.2. To determine the role of TRPV4 in CCD-induced nNOS and iNOS overproduction.Methods1. CCD surgerySixty-eight adult male Wistar rats weighing 150-180 g (Shandong University) were randomly divided into sham group, CCD group, CCD+MM group, and CCD+AS group,17 in each group. In CCD rats, under pentobarbital sodium anesthesia, the stainless steel L-shaped rods were inserted into the intervertebral foramina of L4 and L5 as described in part I. Animals were sacrificed at 7 days after surgery or 12 h after the last ODN injection by decapitation.2. Antisense oligodeoxynucleotide treatmentTo determine the effects of antisense oligodeoxynucleotide (ODN) treatment on CCD-induced thermal hyperalgesia and nNOS and iNOS production, CCD+MM and CCD+AS rats were treated with a spinal intrathecal administration of TRPV4 mismatch ODN and antisense ODN, respectively. The method was as described in part I.3. Behavioral TestingThermal hyperalgesia of four group rats were tested on each of 2 successive days before surgery and on the 7th day after surgery or at 12 h after the last ODN injection. The method was as described in partⅠ4. Real-time PCRThe mRNA levels of nNOS and iNOS in the DRG in each group were quantified by real-time reverse-transcriptase polymerase chain reaction (RT PCR) using SYBR Green technology.5. Western blottingWestern blotting was performed to investigate the proteins expression of nNOS and iNOS in the DRG in different groups.Results1. Effects of CCD on gene expression of nNOS and iNOS in the DRGThe PWLs were similar at baseline among groups (all P>0.05), but after intrathecal ODN treatment for 7 days, the PWL was decreased significantly in the CCD and MM groups when compared with the baseline (both P<0.01). The AS ODN treatment significantly (P<0.01) attenuated the thermal hyperalgesia induced by CCD suggesting that the effectivity of TRPV4 AS ODN in interfering TRPV4 protein expression.The gene expression was monitored on the 7th day after surgery or at 12 h after the last ODN injection. CCD significantly increased the gene transcription of nNOS and iNOS to 4.94-fold and 4.44-fold as compared with the sham group (both P<0.01,), respectively.2. Effect of CCD on the protein expression nNOS and iNOS in the DRGResults showed CCD induced a significantly up-regulation in nNOS and iNOS protein levels, which were 4.69-fold and 4.65-fold higher than the sham group, respectively (both P<0.01).3. Effects of antisense ODN of TRPV4 on CCD-induced gene overexpression of nNOS and iNOS in the DRGThe AS ODN significantly decreased gene overexpression of nNOS and iNOS in the DRG following CCD (both P<0.01).4. Effects of antisense ODN of TRPV4 on CCD-induced protein overexpression of nNOS and iNOS in the DRGThe AS ODN induced a significantly down-regulation in nNOS and iNOS protein levels in the DRG following CCD (both P<0.01).Conclusions1. It was shown that CCD in rats increases the levels of nNOS and iNOS.2. TRPV4 plays a crucial role in CCD-induced overproduction of nNOS and iNOS. BackgroundThe dorsal root ganglion (DRG) in the intervertebral foramen has an important role in the pathogenesis of low back pain and sciatica in patients with disc herniation and spinal canal stenosis, because primary sensory neurons with their cell bodies are present in this structure. DRG is easily stimulated or compressed in the intraforaminal or subarticular zones by degenerative changes of the lumbar vertebrae. Chronic compression of the dorsal root ganglion (DRG) (the procedure hereafter termed CCD) in animals, which mimics clinical disc herniation and spinal canal stenosis in humans, is considered to be a typical model of neuropathic pain. CCD rats produces ipsilateral cutaneous allodynia that is associated with an increased excitability of neuronal somatas in the compressed ganglion, as evidenced by spontaneous activity and a lower rheobase. But the underlying mechanisms are still not fully elucidated.TRPV4 is a polymodal receptor that is activated by hypotonicity, mechanical stimuli, heat, phorbol esters, low pH, citrate, anandamide (AEA) and its LOX metabolite arachidonic acid (AA), and bisandrographolide A (BAA). TRPV4 is inovolved in mechanical and thermal hyperalgesia in neuropathic and inflammatory pain. TRPV4, with its gene and protein expression increased in DRG after CCD, contributes to mechanical and thermal hyperalgesia. Substance P (SP) and calcitonin gene-related peptide (CGRP), both are well-known pain-related neuropeptide, also participate in the transmission of nociceptive signals and contribute to mechanical and thermal hyperalgesia. It is reported that TRPV4 is co-expressed in some DRG neurones with SP and CGRP. TRPV4 agonists 4a-PDD and hypotonic solutions also stimulate SP and CGRP release from peripheral tissues and dorsal horn of spinal cord in inflammatory pain. The early research provides evidence that release of SP from rat nerve neuroma depends on calcium, and N-and L-type Ca2+channels have been found to contribute to CGRP release from rat skin non-neuropathic nerve endings in vitro. Moreover, the DRG, that contain the cell bodies of sensory nerves that terminate peripherally in blood vessels and in other tissues innervated by the sensory nervous system and that terminate centrally in the dorsal horn of the spinal cord, is a prominent site of SP and CGRP production. So, it is noteworthy to identify that whether TRPV4 is involved in SP and CGRP production in DRG following CCD.Objective1. To investigate the effects of CCD on the expression levels of mRNA and protein of SP and CGRP.2. To determine the role of TRPV4 in CCD-induced SP and CGRP overproduction.Methods1. CCD surgeryThree hundreds adult male Wistar rats weighing 150-180 g (Shandong University) were randomly divided into sham group, CCD group, CCD+MM group, and CCD+AS group,75 in each group, In CCD rats, under pentobarbital sodium anesthesia, the stainless steel L-shaped rods were inserted into the intervertebral foramina of L4 and L5 as described in part I. Animals were sacrificed at 7 days after surgery or 12 h after the last ODN injection by decapitation.2. Antisense oligodeoxynucleotide treatmentTo determine the effects of antisense oligodeoxynucleotide (ODN) treatment on CCD-induced thermal hyperalgesia and SP and CGRP production, CCD+MM and CCD+AS rats were treated with a spinal intrathecal administration of TRPV4 mismatch ODN and antisense ODN, respectively. The method was as described in part I3. Behavioral TestingThermal hyperalgesia of four group rats were tested on each of 2 successive days before surgery and on the 7th day after surgery or at 12 h after the last ODN injection. The method was as described in part I.4. Western blottingWestern blotting was performed to investigate the proteins expression of TRP V4 in the DRG in different groups.5. Real-time PCRThe mRNA levels of SP and CGRP in the DRG in each group were quantified by real-time reverse-transcriptase polymerase chain reaction (RT PCR) using SYBR Green technology.6. ELISA procedure for DRG SP and CGRP contentThe aim of this experiment was to determine whether CCD induces increased SP or CGRP protein expression is related to TRPV4 in the DRGs at 7 days post-injury. The DRGs were collected under isoflurane anesthesia, immediately frozen in liquid nitrogen and stored at-80℃for further examination. Six DRGs from 3 rats dissected as one sample were quickly frozen in liquid nitrogen and stored at-80℃for further examination. Six frozen DRGs from 3 rats as one sample were homogenized in the homogenization buffer (50 mM Tris-HCl,0.1 mM EDTA,0.1 mM EGTA,1 mM phenylmethylsulfonyl fluoride,1 mM leupeptin,2 mM pepstatin A,0.1% 2-mercaptoethanol). The crude homogenate was centrifuged at 4℃for 15 min at 15000 g. The supernatants was collected and stored at-80℃. After quantification by BCA assay (Beyotime Biotech Inc., Jiangsu, People's Republic of China), all DRG samples were assayed in duplicate using EIA kits for SP (Assay Designs) and for CGRP (Caymen Chemical). Both assays followed the manufacturer's protocols.7. ImmunohistochemistryAt 7 days post-surgery rats were anesthetized with isoflurane and then transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4);the DRGs was immediately removed and post-fixed in 4% paraformaldehyde (PFA) for 7-8 h, then the tissues were treated with dehydration, embedded in paraffin and cut into serial paraffin sections (5μm). The paraffin-embedded sections were heated for 2 h at 120℃,deparaffinized in xylene, and rehydrated through graded ethanol at room temperature. After three rinses in PBS, microwave accentuation was used for 10 minutes; then the sections were washed in PBS,3% H2O2 to eliminate endogenous peroxydase, and blocked with 10% normal goat serum; then sections were incubated overnight at 4℃with primary antibodies, anti-SP polyclonal antibody (1:100, Santa Cruz) or anti-CGRP polyclonal antibody (1:100, Santa Cruz). After the sections were washed, they were incubated with anti-rabbit IgG peroxidase conjugate (1:300, Zhongshan Gold Bridge, Beijing, China) for 1 h at room temperature. After several rinses, peroxidase was revealed by a 3,3'-diaminobenzidine tetrahydrochloride substrate kit (Zhongshan Gold Bridge, Beijing, China). Finally, the sections were weakly counterstained with hematoxylin. In negative controls, the sections were incubated with PBS instead of primary antibody. All images were captured and analyzed by use of a color image analysis system composed of a video camera (Olympus DP71, Olympus Co., Japan), a light microscope (Olympus BX51, Olympus Co.), and Image-Pro Plus 5.0 software (Media Cybernetics Inc., USA). The relative intensity of the histochemical reaction was determined by measuring the integral optical density (IOD) of the histochemical reaction product. The IOD was measured in 5 randomly selected sections from each animal and the average was calculated.Results1. Effects of antisense ODN of TRPV4 on CCD-induced thermal hyperalgesia and protein expression of TRPV4 in DRGBefore surgery, rats in groups exhibited similar baseline thresholds to the noxious thermal stimuli (all P>0.05). After intrathecal ODN treatment for 7 days, CCD and MM rats produced marked thermal allodynia, indicated by significantly decreased thermal withdrawal latency compared with the corresponding baseline (both P<0.01,). In contrast, thermal hyperalgesia was partly reversed in the AS group compared with baseline. There were no significant changes in the PWL following intrathecal AS or MM in sham rats. All rats walked normally post-CCD which indicated that the CCD did not injury the motor behavior. Western blotting showed that, in CCD rats, the antisense ODN, but not the mismatch ODN, significantly (P<0.01) inhibited the TRPV4 protein expression when compared with the control group.2. Effect of antisense ODN of TRPV4 on CCD-induced mRNA expression of SP and CGRPSP and CGRP mRNA levels were determined by quantitative reverse transcription polymerase chain reaction in DRG extracts. Our data showed that the mRNA levels of SP and CGRP were significantly upregulated in CCD and MM groups compared with the sham group (P<0.01). But there were no significant changes in the mRNA levels of SP and CGRP in AS group compared with the sham group. These results indicate that antisense ODN of TRPV4 can regulate the expression of SP and CGRP at the mRNA level following CCD.3. Effect of antisense ODN of TRPV4 on CCD-induced protein expression of SP and CGRPSP and CGRP protein levels in DRG were determined by ELISA and immunohistochemistry. Our data showed that there was a significant increase in SP protein level in CCD or MM group rats compared with that in sham group rats (all P< 0.01), as was CGRP protein level (all P<0.01). SP protein level was lower in AS group than that in CCD or MM group 7 days post-CCD (all P<0.01), as was CGRP protein level (all P<0.01). These results showed that SP or CGRP expression in DRG 7 days following compression was significantly (all P<0.01) inhibited by intrathecal injection of antisense ODN, but not inhibited by mismatch ODN treatment, as compared to controls.Conclusions1. It was shown that CCD in rats increases the levels of SP and CGRP.2. TRPV4 plays a crucial role in CCD-induced overproduction of SP and CGRP.