| BackgroundPain is one of the most common clinical symptoms, it can harm the body as a warning, which is an important signal of human illness. In 1994, the International Association for the Study of Pain(IASP) defined pain as unpleasant sensory and emotional experience which is associated with actual or potential tissue damage.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. The main characteristics of neuropathic pain is spontaneous,hyperalgesia and allodynia. At present, its pathogenesis has not been made clear, and the lack of effectiveand specific treatment means, so look for and find newtarget for the treatment of neuropathic chronic pain andnovel analgesic drug, has been a hot research in neurobiology. Currently, its pathogenesis has not been clear yet. since the lack of effective and specific treatment interventions, seeking and finding a new target and a new analgesic drugs of chronic neuropathic pain has been among the most intensively study. Chronic compression of the DRG (the procedure named CCD) in animals, which mimics clinical disk hernia and spinal canal stenosis in humans, is a typical model of neuropathic pain. The second messenger signals cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and cyclic guanosine monophosphate (cGMP)-dependent protein kinase G (PKG) have been shown to contribute to the maintenance of neuronal hyperexcitability and behavioural hyperalgesia in CCD conditions. Moreover, ion channels, such as voltage-gated Na+ and K+ channels, hyperpolarisation-activated cation current channels, and the transient receptor potential vanilloid 4 (TRPV4) may be involved in the hyperexcitability of DRG neurons after CCD.Members of the transient receptor potential (TRP) family, in addition to the vanilloid receptor TRPV4 in dorsal root ganglia, such as the vanilloid receptor 1 (TRPV1), the vanilloid receptor-like protein 2 (TRPV2), the TRPM8 receptor are expressed in mammalian sensory neurons, and they are thought to function as transducers for thermal and chemical stimuli. Among them, TRPV4 is broadly expressed and is one of the best-characterized TRP channels. It is a polymodally gated TRP channel that can be activated by diverse stimuli including moderate heat, cell swelling, endogenous-chemicals such as anandamide, arachidonic acid and its epoxyeicosatrienoic acid metabolites, as well as by a growing number of exogenous chemical ligands, such as hypotonicity and 4a-phorbol 12,13-didecanoate (4a-PDD).. Mechanosensory responses of colonic serosal and mesenteric afferents were enhanced by a TRPV4 agonist and dramatically reduced by targeted deletion of TRPV4 or by a TRP antagonist. 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. NF-κB, a transcription factor, plays a vital role in the cytokine-induced regulation of genes involved in CNS inflammatory responses. Recent evidence shows that activation of the NF-κB pathway is involved in the transmission and processing of nociceptive information in the spinal cord and DRG of rats with chronic constriction injuries (CCI) Our recent study indicated that TRPV4 enhances pain-related behaviour through a NO-cGMP-PKG cascade that may occur within the DRG in CCD rat. TRPV4 induces NO production through the activation of NF-eB pathway in the DRG. Nitric oxide (NO) has been shown to be involved in the pathophysiological effects in the nucleus pulposus in animal pain models. However, the detailed mechanisms between ion channels (TRPV4) and neurotransmitter (NO) in behavioral hyperalgesia, especially the role of calcium signalling and its association with NF-κB and NO in neuropathic hyperalgesia, are still unknown.Increases in local Ca2+ concentration at the site of injury or in the spinal cord may contribute to the development of neuropathic pain. TRPV4 gating seems to be tightly regulated by intracellular and extracellular calcium concentration. Calcium channels, by regulating Ca2+ influx, mediate numerous cellular processes and functions, including excitation-contraction coupling in muscle cells, excitation-secretion coupling in epithelial and immune cells, neurotransmitter release in certain neurons. Injury to the nerve can produce changes in dorsal horn function and pain. This facilitated processing may be mediated in part by voltage-sensitive calcium channels. Activation of these channels increases intracellular calcium, thereby mediating transmitter release and activating cascades serving to alter membrane excitability and initiate protein transcription. These effects are consistent with the high levels of expression on primary afferents and dorsal horn neurons of these channels.Five major types of voltage sensing calcium channels (VSCCs) have been identified (L-, T-, N-, P/Q-and Rtypes), with unique electrophysiologic and pharmacologic characteristics. Specifically, the T-type VSCCs, first described in peripheral sensory neurons of the DRG, are activated by subthreshold excitatory postsynaptic potentials (EPSPs), reducing threshold for the generation of action potentials and therefore contributing to the excitability of neuronal membranes. In both rat models of diabetic neuropathy and the chronic constriction nerve injury model of neuropathic pain, T-type channel current density is remarkably increased. Since mibefradil demonstrates between 10-fold and 100-fold selectivity in blocking T-type current over L-type current, indicating considerable selectivity, it was thus employed here to explore the contributions of the T-type VSCC to experimental neuropathic pain.ObjectiveTo investigate the effects of the calcium channels inhibitor (mibefradil dihydrochloride) on paw withdrawal latency and Mechanical withdrawal threshold in the DRG in CCD model.Methods1. Establishemnt of CCD model and intrathecal catheter implantationMale Wistar rats weighing between 200 g-220 g (Shandong University Lab Animal Center, Jinan, China), were used in these experiments. The rats were housed in individual cages with free access to standard laboratory food and tap water for at least 7 days before surgery and kept at 23±1℃ on a 12-h light/dark cycle at 50%-60% relative humidity. Rats were randomly divided into CCD groups and sham groups. In CCD rats under pentobarbital sodium anaesthesia (50 mg/kg, i.p.) The lumbar intervertebral spaces were widened by placing a 20 ml syringe under the abdomen of the rat. A 1 cm longitudinal skin incision was made over LS-L6, lumbar vertebrae but a 1 cm lateral to the midline, a PE-10 (polyethylene) intrathecal catheter was implanted into the subarachnoid space. Then the paraspinal muscles were separated, and the transverse processes and intervertebral foramina of L4 and L5 were unilaterally exposed as previously described. Two stainless steel L-shaped rods (0.63 mm diameter and 4 mm length) were inserted into the intervertebral foramina of L4 and L5 to compress the DRG. The muscle and skin layers were then closed independently. Penicillin was injected q.d. for 3 days to prevent infection. The animals did not exhibit complete loss of sensation or the loss of any autonomy following the surgery. Finally, 10μl of sterile saline was injected, and the end of the catheter was melted to seal the catheter. Only animals with normal motor function were used.2. Chemicals and treatmentAfter 3 days recovery, the rats underwent a lidocaine infusion that determined the functionality and position of the catheter tip in the subarachnoid space, and intrathecal doses of 10μl/day LV-NC and LV-TRPV4 was continuously infused over the course of 3 days. On the 7th day after surgery, rats received an intraperitoneal (i.p.) injection of mibefradil dihydrochloride (a calcium channels inhibitor, sigma, M5441-5MG, USA), in a volume of 0.1 ml/kg. All chemicals were dissolved in saline. Mechanical and thermal paw withdrawal thresholds were assessed to determine the analgesic effects. Each rat was tested in a random order, and experimenters were blind to the treatment of each rat. The i.t. delivery caused no adverse effects in any of the animals that received LV-NC or LV-TRPV4.3. Behavioral testingMechanical withdrawal thresholds (MWTs) and paw withdrawal latency (PWL) of rats were tested for mechanical allodynia and heat hyperalgesia. MWTs and PWLs were measured 1 day before CCD and 4,7,14, and 21 days after the intrathecal administration of lentivirus or saline into the subarachnoid space through the intrathecal catheter. The effect of mibefradil on CCD-induced allodynia was tested at 0,15,30,45 and 60 min after injections.(1). Walking gait pattern was assessed as the index of motor function.’1’ indicates normal gait, withoutfoot deformities.’2’ indicates normal gait with obvious foot deformities.’3’indicates slight gait disturbance with foot-drop.’4’indicates serious gait disturbance with myashtnia. The walk gait pattern was normal and no foot deformities were found in all rats pre-and post-CCD. The score was’1’.(2). Mechanical hyperalgesiaMechanical withdrawal thresholds (MWTs) were measured with von Frey hair monofilaments (BME-403, Biomedical Engineering Institute of Chinese Academy of Medical Sciences) with logarithmically incremental stiffness (0.09-17.30 g), Starting with the lowest filament force, von Frey fibers were pressed against the lateral plantar surface of hindpaws in ascending order with sufficient force to cause slight bending and held for 5 s. A positive response was noted if the paw was immediately withdrawn. The stimulation of the same force was repeated five times at intervals of 5 s. If there were no less than three withdrawal responses to any of the five applications, the next lower force fibers was repeated. If less than three withdrawl responses, the process was tried with the next higher force fibers.(3). Thermal hyperalgesiaThermal hyperalgesia was assessed as paw withdrawal latency (PWL) in response to radiant heat (BME-410C). Rats were first placed in a plastic chamber atop a 6 mm thick glass floor. Then, the radiant heat source beneath the glass floor was aim at the mid plantar surface of hindpaws. The paw withdrawal latencies per animal were obtained during five trials at 5 min intervals. The intensity of the heat source was pre-calibrated to result in a baseline latency of approximately 10 s, and the cutoff time was set to 20 s.4. Data analysisA two-way repeated measures analysis of variance (RM ANOVA) was used to compare the differences in PWL over time between several treatment and control groups.Results1. The effects of lentivirus on CCD-induced hyperalgesia.The 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.Compared to sham group, CCD rats expressed a significant decrease in PWLs (in response to temperature and mechanical stimulus) of the ipsilateral hind paws (P< 0.05). The decrease in PWLs peaked at 7 days post-CCD.MWTs and PWLs were measured 1 day before CCD (baseline) and at 4,7.14, and 21 days after intrathecal administration of lentivirus. The mechanical allodynia and thermal hyperalgesia was prevented by an intrathecal administration LV-TRPV4 (p<0.05). In contrast, administration of LV-NC had no effect on CCD-induced hyperalgesia and allodynia (p>0.05).The MWTs and PWLs of sham rats do not change following either intrathecal LV-TRPV4 or LV-NC(p>0.05).2. The effects of mibefradil on CCD-induced hyperalgesia.PWLs were measured at 0,15,30,45 and 60 min after intraperitoneal (i.p.) injection of mibefradil dihydrochloride on the 7th day after surgery. The intraperitoneal injection of mibefradil (5,10 and 20 mg/kg) exerted a dose-dependent reversal of CCD-induced thermal hyperalgesia when compared with CCD+NS group. The highest dose used in the current study (20 mg/kg, i.p.) elicited a significant reduction of the thermal hyperalgesia at 15 min (P< 0.05), peaked at 30 min (P< 0.05), and antihyperalgesia was still evident at 60 min. Therefore, we chose the time point at 30 min after intraperitoneal injection of mibefradil for all of the following studies.3. Effects of 4a-PDD on the mibefradil-induced suppression of hyperalgesia in CCD rats4α-PDD (the TRPV4 selective agonist, 100μmol/L) attenuated the suppressive effects of mibefradil (20mg/kg) on CCD-induced thermal hyperalgesia when compared to mibefradil (20mg/kg) alone.ConclusionsThis study demonstrates that TRPV4-dependent calcium changes could be involved in TRPV4-NO pathway in CCD-induced thermal hyperalgesia following chronic compression of the dorsal root ganglion in rat.BackgroundThe animal model of neuropathic pain produced by chronic compression of the dorsal root ganglion (DRG) (this procedure is hereafter termed CCD) leads to spontaneous pain, mechanical allodynia, and thermal hyperalgesia. Such behavioural and electrophysiological effects can also be observed in humans with conditions such as intervertebral foraminal stenosis and herniated intervertebral discs (the latter is regarded as a major cause of low back pain). Enhanced activity of Ca2+ current is associated with increased neuronal activity. Mechanosensory responses of colonic serosal and mesenteric afferents were enhanced by a TRPV4 agonist and dramatically reduced by targeted deletion of TRPV4 or by a TRP antagonist. Our recent study indicated that TRPV4 enhances pain-related behaviour through a NO-cGMP-PKG cascade that may occur within the DRG in CCD rat。TRPV4 induces NO production through the activation of NF-κB pathway in the DRG. Nitric oxide (NO) has been shown to be involved in the pathophysiological effects in the nucleus pulposus in animal pain models. However, the detailed mechanisms between ion channels (TRPV4) and neurotransmitter (NO) in behavioral hyperalgesia, especially the role of calcium signalling and its association with NF-κB and NO in neuropathic hyperalgesia, are still unknown.Increases in intracellular calcium can contribute to depolarizing membrane current; initiate transmitter release by promoting the activation of membrane docking proteins, initiate increased transcription (by activation of kinases), and initiate phosphorylation of membrane proteins (eg, N-methyl-D-asparate [NMDA] and AMPA receptors) that can enhance channel efficacy or activate a variety of intracellular enzymes (eg, phospholipase A2). TRPV4 gating seems to be tightly regulated by intracellular and extracellular calcium concentration. The calcium-permeable TRPV4 ion channel in skin epithelial cells has been found to be identified as critical for translating the UVB stimulus into intracellular signals and also into signals from epithelial skin cell to sensory nerve cell that innervates the skin, causing pain. Five major types of voltage sensing calcium channels (VSCCs) have been identified (L-, T-, N-, P/Q-and Rtypes), with unique electrophysiologic and pharmacologic characteristics. Specifically, T-type VSCCs can lower the threshold for action potentials, promote bursting activity and synaptic excitation; all these actions may enhance the development of pain. In both rat models of diabetic neuropathy and the chronic constriction nerve injury model of neuropathic pain, T-type channel current density is remarkably increased. In the central nervous system, T-type Ca2+channel can induce pacemaker activity and low-threshold nerve impulse. In peripheral nervous system, T-type Ca2+ channels mainly distribute in the small or middle size DRG nerouns. The major subtype of T-type Ca2+channel in DRG nerouns is CaV3.2. Recently,there are studies indicating that malfunction of the T-type Ca2+ channel are related to many kinds of diseases, such as epilepsy, hypertension, heart diseases, diabetes, pain, cancer. T-type Ca2+channel play important roles in both physiological and pathophysiological conditions, and the moleculer mechanisms of the T-type Ca2+channel modulation is the study focus of many groups.Recent study showed that TRPV4 inhibition attenuated Ca2+influx, blocked nuclear factor-kappa B (NF-κB) nuclear translocation, decreased the expression levels of glial cell-released proinflammatory cytokines (e.g. IL-1β and TNF-α) and ultimately protected against infrasound-induced neuronal injury in rats. Activation of the NF-κB pathway is involved in the transmission and processing of nociceptive information in the spinal cord and DRG of rats with chronic constriction injuries (CCI). Accordingly, we hypothesized that Ca2+might be involved in the TRPV4-NO pathway through upregulating NF-κB activity in the CCD model. To test this hypothesis, we investigated the effects of intracellular calcium concentration, regulated by intrathecal injection of TRPV4-targeted siRNA (LV-TRPV4), and mibefradil dihydrochloride, an calcium channels inhibitor, on neuropathic hyperalgesia, NF-κB activityand NO content in the DRG in the CCD model.Objective1. To investigate the effects of TRPV4 siRNA on NF-κB activityand NO content in the DRG in the CCD model.2. To investigate the effects of the calcium channels inhibitor (mibefradil dihydrochloride) on NF-κB activityand NO content in the DRG in the CCD mode.3. To determine the role of TRPV4-dependent calcium changes in CCD-induced TRPV4-NO pathway.Methods1. Establishemnt of CCD model and intrathecal catheter implantationMale Wistar rats weighing between 200 g-220 g (Shandong University Lab Animal Center, Jinan, China), were used in these experiments. The rats were housed in individual cages with free access to standard laboratory food and tap water for at least 7 days before surgery and kept at 23±1℃ on a 12-h light/dark cycle at 50%-60% relative humidity. Rats were randomly divided into CCD groups and sham groups. In CCD rats under pentobarbital sodium anaesthesia (50 mg/kg, i.p.) The lumbar intervertebral spaces were widened by placing a 20 ml syringe under the abdomen of the rat. A 1 cm longitudinal skin incision was made over LS-L6, lumbar vertebrae but a 1 cm lateral to the midline, a PE-10 (polyethylene) intrathecal catheter was implanted into the subarachnoid space. Then the paraspinal muscles were separated, and the transverse processes and intervertebral foramina of L4 and L5 were unilaterally exposed as previously described. Two stainless steel L-shaped rods (0.63 mm diameter and 4 mm length) were inserted into the intervertebral foramina of L4 and L5 to compress the DRG. The muscle and skin layers were then closed independently. Penicillin was injected q.d. for 3 days to prevent infection. The animals did not exhibit complete loss of sensation or the loss of any autonomy following the surgery. Finally, 10μl of sterile saline was injected, and the end of the catheter was melted to seal the catheter. Only animals with normal motor function were used.2. Chemicals and treatmentAfter 3 days recovery, the rats underwent a lidocaine infusion that determined the functionality and position of the catheter tip in the subarachnoid space, and intrathecal doses of 10μl/day LV-NC and LV-TRPV4 was continuously infused over the course of 3 days. On the 7th day after surgery, rats received an intraperitoneal (i.p.) injection of mibefradil dihydrochloride (a calcium channels inhibitor, sigma, M5441-5MG, USA), in a volume of 0.1 ml/kg. All chemicals were dissolved in saline. Mechanical and thermal paw withdrawal thresholds were assessed to determine the analgesic effects. Each rat was tested in a random order, and experimenters were blind to the treatment of each rat. The i.t. delivery caused no adverse effects in any of the animals that received LV-NC or LV-TRPV4.3. ImmunohistochemistrySix DRGs from the operated side of three rats were harvested to extract protein as one sample, which was quickly frozen in liquid nitrogen then stored at -80℃ until further examination. Protein fractions were subjected to SDS-PAGE (12% gel) and then transferred to polyvinyl fluoride membranes (Bio-Rad, Hercules, CA, USA). After incubation overnight at 4℃ with a polyclonal anti-TRPV4 preparation (1:500, Abeam, Cambridge, UK), the blots were washed and incubated with anti-rabbit IgG peroxidase conjugate secondary antibody (1:1500, Zhongshan Gold Bridge, Beijing, China) for 1 h at room temperature. Finally, immunoreactive proteins were detected with ECL plus (Millipore, Bedford, MA). The protein levels are expressed as the ratio of the density of the detected band to that of β-actin (1:5000, Abeam, Cambridge, UK).4. Western blot analysisSix DRGs from the operated side of three rats were harvested to extract protein as one sample, which was quickly frozen in liquid nitrogen then stored at -80℃ until further examination. Protein fractions were subjected to SDS-PAGE (12% gel) and then transferred to polyvinyl fluoride membranes (Bio-Rad, Hercules, CA, USA). After incubation overnight at 4℃ with a polyclonal anti-TRPV4 preparation (1:500, Abeam, Cambridge, UK), the blots were washed and incubated with anti-rabbit IgG peroxidase conjugate secondary antibody (1:1500, Zhongshan Gold Bridge, Beijing, China) for 1 h at room temperature. Finally, immunoreactive proteins were detected with ECL plus (Millipore, Bedford, MA). The protein levels are expressed as the ratio of the density of the detected band to that of β-actin (1:5000, Abeam, Cambridge, UK).5. Real-time quantitative RT-PCRL4 and L5 ganglions were harvested in the same manner as described above. Fragments of TRPV4 or β-actin were amplified with the following primers: TRPV4 (forward,5’-AAGTGGCGTAAGTTCGGG-3’; reverse,5’-TAAGGGTAGGGTGGCGTG-3’) and β-actin (forward,5’-AGACCTTCAACACCCCAG-3’; reverse,5’-CACGATTTCCCTCTCAGC-3’). Instrument control, automated data collection, and data analysis were all performed using the LightCycler software program, version 4.0. The 2-△△CT method was used to analyze the data.6. DRG cell cultureThe L4 and L5 ganglia were harvested from the operated side of the rats. DRGs were incubated with 1 mg/ml collagenase type I and 0.25% trypsin and cultured in Neurobasal medium supplemented with 20 ng/ml nerve growth factor (NGF,),2% B27 supplement,0.5 mM L-glutamine, and 1%(v/v) penicillin/streptomycin. DRG neurons were used for experiments after 4 days of in vitro culture.7. Measurement of [Ca2+]iNeurons were loaded with 5 umol/L fura-3 acetoxymethyl ester (Fura-3 AM) and 0.02% pluronic f-127 for 45 min at 37℃ in isotonic solution. To activate TRPV4 and cause an increase in free [Ca2+]i,4a-PDD was used. Subsequently, neurons were perfused with an isotonic solution for 10 min to permit hydrolysis of fura-3 AM before adding 4a-PDD. The volume of the confocal dish was 200 ul, and perfusion was carried out at 20-23 ℃. The dye was excited at 488 nm to indicate relative changes in [Ca2+]i. The fluorescence was acquired and analyzed with Zen 2009 imaging system software.8. Electrophoretic Mobility Shift Assay (EMSA)NF-κB activity was measured by EMSA. The sequence of the NF-κB probe was 5’-AGTTGAGGGGACTTTCCCAGGC-3’, and the sequence of the mutant probe was 5’-AGTTGAGGCTAC TTTCCCAGGC-3’. Biotins were labelled in the 5’ end oligonucleotide. The assay was carried out using a non-radioactive NF-κB EMSA kit following the manufacturer’s instructions. Briefly,10 μg of nuclear protein was incubated with lOx Gel Shift Binding buffer,1.5 μg of poly(dl-dC) and 500 fM biotin-labelled oligonucleotide bio-NF-KB probe (5’-AGTTGAGGGGACTTTCCCAGGC-3’) (total vol 15 μl) for 20 min at room temperature. Protein-DNA complexes were separated from free oligonucleotides on 6.5% non-denaturing polyacrylamide gels, electrotransfered to the banding membrane and detected with a Coollmger imaging system. The specificity of binding was also examined by a competitive assay using a 100-fold molar excess of unlabelled oligonucleotides.9. Measurement of NO content in DRGThe 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.10. Statistical analysesStatistical analyses were performed by one-way or two-way analyses of variance (ANOVAs) followed by Newman-Keuls tests or Tukey’s test where appropriate. Data are expressed as the mean ± the standard error (S.E.M). A value of P< 0.05 was considered to be significant.Results1. The transduction of neurocytes by lentivirusRats received intrathecal injection of GFP-expressing lentivirus and were sacrificed 7 days later. The lentivirus was efficiently transduced into the cells of the DRG. Strong fluorescence was observed in neurocytes of the rats from sham and CCD groups which received lentivirus treatment.2. Effects of TRPV4 knockdown in the DRGs of the ratsAfter 4 days of in vitro culture, the normal DRG neurons were transfected with TRPV4-directed siRNA (MOI=3), it showed no obvious toxic effect on cell growth and significantly decreased TRPV4 expression, compared with negative control(P< 0.05). The levels of TRPV4 mRNA and protein expression were increased significantly in the DRGs of CCD group when compared with the sham group(P< 0.05). After intrathecal injection of LV-TRPV4, TRPV4 mRNA and protein expression were markedly decreased both in sham and CCD group (P< 0.05), compared with the LV-NC group. The levels of protein expression were consistent with the levels of mRNA expression.3. Effects of intrathecal injection of LV-TRPV4 on NF-κB activity and the NO content in DRGs in CCD rats.The effects of LV-TRPV4 on NF-κB activity were confirmed by EMSA assay. We found that the activity of NF-κB in the nucleus was increased in CCD rats. Administration of LV-TRPV4, but not LV-NC, significantly downregulated NF-κB activity to nearly normal levels. Levels of the NO derivative nitrite in the DRGs of CCD-treated rats were significantly increased compared to the sham group (P< 0.05). Intrathecal injection of LV-TRPV4 significantly decreased the content of NO (P< 0.05).4. Effects of LV-TRPV4 on 4a-PDD-induced [Ca2+]i increase[Ca2+]i is reported as a pseudoratio (△F/F) according to the following formula: AF/F=(F-Fbase)/(Fbase-B). F is the measured fluorescence ratio of the Ca2+ indicator, Fbase is the fluorescence intensities in the DRG neurons before stimulation with 10 μM 4a-PDD, and B is the background signal determined from the average signal from areas adjacent to the DRG neurons. To measure the response of DRG neurons to 4a-PDD, the value of the fluorescence ratio was averaged when the stimulus-induced increase in [Ca2+]i reached its maximum plateau. Compared with the sham group, a significant increase in [Ca2+]i was observed in 31% of small and medium diameter DRG neurons of CCD rats (P< 0.05). The response is abolished with the elimination of extracellular calcium and 1μM ruthenium red (RR, an inhibitor of TRPV4). (P< 0.05). With intrathecal injection of LV-TRPV4, DRG neurons of CCD rats showed a significant decrease in [Ca2+]i, compared with LV-NC group (P< 0.05).5. Effects of mibefradil on NF-κB activity and NO content in DRGs in CCD rats.The PWLs was significantly shorter in CCD+NS group than in sham group (P< 0.05). The intraperitoneal injection of mibefradil (5,10 and 20 mg/kg) exerted a dose-dependent reversal of CCD-induced thermal hyperalgesia when compared with CCD+NS group. The highest dose used in the current study (20 mg/kg, i.p.) elicited a significant reduction of the thermal hyperalgesia at 15 min (P< 0.05), peaked at 30 min (P< 0.05), and antihyperalgesia was still evident at 60 min. Therefore, we chose the time point at 30 min after intraperitoneal injection of mibefradil for all of the following studies. Intraperitoneal injection of mibefradil dihydrochloride (20 mg/kg) significantly downregulated NF-κB activity,and meanwhile significantly decreased the content of NO metabolites nitrite, compared to CCD+NS group(P< 0.05).6. Effects of 4a-PDD on the mibefradil-induced suppression of NF-κB activity and NO content in the DRGs in CCD rats.4α-PDD (the TRPV4 selective agonist,100μmol/L) attenuated the suppressive effects of mibefradil (20mg/kg) on CCD-induced thermal hyperalgesia when compared to mibefradil (20mg/kg) alone (P< 0.05). Moreover, the downregulation of NF-κB activity and decrease in the content of NO induced by intraperitoneal injection of mibefradil (20 mg/kg) was also attenuated by pretreatment of 4a-PDD (100μmol/L) 30min post-injection (P< 0.05).ConclusionsTaken together, the results in the current study suggest that, the activation of TRPV4 in DRG may initiate an influx of Ca2+ which possibly stimulates the production of Ca2+-dependent NOS, and this in turn may result in the further activation of NF-κB to engage in the TRPV4-NO pathway in CCD-induced neuropathic hyperalgesia. |
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