Role Of TRPV4-MAPK Pathway On Neuropathic Pain In Rats With Chronic Compression Of The Dorsal Root Ganglion

BackgroungNeuropathic pain (NP) was defined as pain caused by somatosensory system damage or disease in 2008 by NeuPSIG of IASP. The Chinese common names: neurogenic pain, neuropathy pain, neural pain, neuropathic pain, now known as neuropathic pain. Neuropathic pain is divided into two types:peripheral and central pain. Common peripheral neuropathic pain:postherpetic neuralgia, diabetes peripheral neuropathy, trigeminal neuralgia, nerve root pathological changes (cervical, thoracic, lumbar or sacral), block pressure neuropathy (carpal tunnel syndrome), posttraumatic neuropathic pain, limb pain, and so on. Common central neuropathic pain caused by:stroke, syringomyelia, ischemic spinal cord disease, compression myelopathy (spinal cervical disease, cancer), spinal cord injury and so on. Neuropathic pain has a variety of clinical manifestations and its characteristics are:1, pain appeared at local area when there was no trauma or injury stimulation-spontaneous pain; 2, pain could be induced by a slight or small changes-unnociceptive stimulation can induce pain; 3, normal painful stimulation induced inordinate pain-hyperalgesia; 4, pain properties varied, involves referred pain, shock like pain, pains like burning and pinking; 5, accompanied by paresthesia, dysesthesia, numbness, itching and other symptoms of discomfort. The pathogenesis of neuropathic pain is very complex, may be due to dysfunction and anatomical alterations in the structure, causing peripheral and/or central sensitization and activation of glial cells in spinal cord, dysfunction of the descending inhibitory system, alternation of ion channels and so on, which leads to nerve injury, neurogenic inflammation, change in the excitability of neural endings, abnormalities of the sympathetic nervous system and neural plasticity and so on, and ultimately lead to kinds of pain. IASP criteria for the diagnosis of neuropathic pain in 2008 recommend: 1, pain has clear neuroanatomical range; 2, the medical history of patients showed that there were peripheral or central sensory system disease or damage; 3, at least a supplementary examination can confirm the pain with clear neuroanatomical range; 4, at least a supplementary examination confirmed the presence of pain related illness or injury. The current treatment of neuropathic pain is poor, first-line drug are calcium ion antagonist agent, such as pregabalin, gabapentin, anti depression drugs are also first-line, give carbamazepine. Sodium channel blocking agents or lidocaine are also used commenly, but about half of the pain can not be effectively alleviated.Root neuralgia is a common clinical neuropathic pain type, when the root of the spinal nerve or ganglion were stimulated by some noxious stimulation (such as lumbar disc herniation, lumbar spinal stenosis, tumors in the spinal cord compression or inflammatory substances stimulate), nerve inflammation, excitability enhancement happened, then caused pain. CCD model is a typical experimental model of neuropathic pain. After modeling, rats will suffer spontaneous pain, hyperalgesia and allodynia, accompanied by a increasing of spontaneous discharges, and decreasing of action potential and current threshold.There are many kinds of ion channels in DRGs, of which the transient receptor potential vanilloid receptor 4(TRPV4) attracted more and more attention. Our previous research (the project of National Natural Fund 30472006) found that TRPV4 is involved in the cause of CCD-induced hyperexcitabilit of DRGs, hyperalgesia and allodynia. Giving TRPV4 antisense nucleotide interference could lead to a partial reversal of CCD-induced pain threshold decreasing, while rare changes in the basic pain threshold of rats. However, the pathways and molecular basis of TRPV4 induceing pain and abnormal pain of CCD rats still need further study. Neurons in DRGs are the first-order neurons in sensory afferent pathway, the damage of DRGs of CCD model resulted in an increase in the excitability of neuronal cell body, produced a large number of persistent ectopic discharges, not only caused the sensitization of spinal cord and upper nerve center, but also caused neuropathic pain symptoms: spontaneous pain, hyperalgesia and allodynia, became the signal source of neuropathic pain. Inhibiting the ectopic discharges of DRG neurons could decrease chronic pain signals. Nerve fibers producing discharges mainly include A6and C fibers, and different nerve fibers transtucted different discharge forms. Our previous experiments (fund number:81071597) proved that, TRPV4 participates in the ectopic discharge of DRG neurons in CCD model, but the conduction mechanism is not clear yet; and TRPV4 mediated DRG abnormal discharge to what kind of fiber, and which nerve fiber palying the critical role need further study.Nociceptive pain signal from primary sensory neurons in the dorsal root ganglion, pass through the central end to the spinal dorsal horn, then terminate to the shallow and deep interneurons in the dorsal horn of the spinal cord. These neurons send nerve fibers form tractus spinothalamicus lateralis, then transent signals to the thalamus and brain. In CCD model, chronic compression of DRG, lead to ischemia and edema, promote the release of inflammatory mediators, cause the increased excitability of DRG neurons, send nociceptive signals to the dorsal horn of spinal cord, thereby resulting in the activation of the dorsal horn of spinal cord, inducing hyperalgesia and allodynia. Hyperexcitability of spinal dorsal horn may be the mechanisms of central sensitization of neuropathic pain in CCD. Our previous studies mainly focused on the peripheral sensitization mechanism after CCD, but the spinal dorsal horn is an important component of nociceptive information transmission, which needs further research. Studies have also showed that the inflammation of the peripheral tissues and nerves increases the expression of TRPV1, TRPA1 and TRPM8 in the spinal cord, but there is rare study on the change of TRPV4 in the dorsal horn of spinal cord.Peripheral mitogen activated protein kinase systems (MAPKs) widely exist in the nervous system, mainly including three subsystems:ERK (extracellular signal regulated kinases, JNK (c-Jun N-terminal kinase) and p38 (hypertonic glycerol kinase reaction), working in phosphorylated forms. Extracellular stimuli change to intracellular transcription and translation reaction through MAPK signal pathway, playing a key role. Studies confirmed that the formation and development of neuropathic pain is closely related to MAPK signaling pathway. In inflammationary pain, nerve disease, spinal cord injury and neuropathic pain model, MAPKs (ERK, JNK and p38), control the protein expression after modification, as well as the transcription regulation of some key genes, and then maintain and amplify the pain signals from nociceptive receptor or dorsal horn neurons in phosphorylated forms. Local injection of MAPK inhibitors can significantly inhibit the mechanical and thermal hyperalgesia. In CCD rats, the expression of p-ERK was increased, and the inhibition of p-ERK expression resulted in decreased excitability of DRG neurons. In CCD model, the expression of TRPV4 gene and protein were increased, and the TRPV4 channel opening caused by the low osmotic solution and 4 ?-PDD was increased, then the intracellular calcium peak was increased by TRPV4. Studies show that the increase of intracellular Ca2+ can induce the activation and phosphorylation of MAPK family, so as to mediate the biological effects of MAPK. Both in vitro and in vivo studies show that the activation and phosphorylation of p38MAPK induced by elevated intracellular concentrations of Ca2+, may lead to mechanical pain and inflammatory pain. In the autosomal recessive polycystic kidney disease, TRPV4 can also regulate the activity and expression of B-Raf/ERK by regulating the level of Ca2+ in the bile duct epithelial cells. Some studies have showed that when C fibers were stimulated, ERK and P38 were phosphorylated and activated, while A fibers were stimulated, ERK was also partially phosphorylated. In CCD model, p-ERK expression increased, the application of U0126 to inhibit the expression of p-ERK can lead to the reduction of the excitability of DRG neurons, resulting in the increase of the action potential of the current threshold and the average intensity.Thus, we hypothesized that, in CCD model, TRPV4 participates in the peripheral and central sensitization mechanism in neuropathic pain, and TRPV4 channel regulate the activity and expression of MAPK pathway, thereby regulating their functions.Based on the above progresses, in this study, we used the electrical physiological technology, real time PCR, Western blot, immunohistochemistry and immunofluorescence methods to test the function of TRPV4-MAPK pathway in neuropathic pain. At the same time, TRPV4 protein expression and distribution in the spinal dorsal horn were also been tested, in order to explore the new theory.Chapter IEffect of TRPV4-p38 MAPK pathway on neuropathic pain in rats with chronic compression of the dorsal root ganglionObjective1. To investigate whether TRPV4 and p38MAPK are involved in neuropathic pain in rats with chronic compression of the dorsal root ganglion.2. To investigate whether TRPV4 and p38MAPK affect each other in CCD rats.Methods1. CCD modelAfter anesthetizing the rats with 10% chloral hydrate (300 mg/100 g body weight, i.p.), the animals were shaved and sterilized. Then, their skin was cut between the bilateral spina iliaca, extending upward approximately 2 cm. Next, the deep fascia and muscle were longitudinally cut upward approximately 2 cm from the end of the right fascia triangle; the tail levator was pushed aside so that the mastoid of L4 and L5 were visible. After separating the covering muscles, the outer foramen intervertebrale of L4 and L5 were revealed; then, sterile L-shaped steel bars (diameter= 0.63 mm) were inserted into the foramen intervertebrale of L4 and L5 at a 30° oblique angle with the spinal column, keeping the other end of the steel bar out of the foramen intervertebrale. After the operation, the incisions were washed with normal saline; then, the muscle, fascia and skin were sutured in sequence, and penicillin was given to prevent infection. The rats that developed autophagy, sensory deficiency or disability were eliminated from analysis.2. Intrathecal InjectionAnimals were inhalation anesthetized with mixed isoflurane and oxygen. They were kept in a prone position and a 30 ml injector was placed under their abdomens to enlarge the intervertebral space. After sterilizing the skin on the back, the L4/L5 intervertebral space was located between the bilateral spina iliaca. The next space was the L5/L6 intervertebral space (the intrathecal injection point). The micro-injector was soaked in 75% ethanol for over 30 min and then washed three times before each injection. After the injection point was located, the L5/L6 intervertebral space was penetrated using the micro-injector (with the top of micro-injector slightly toward the head); the rats'tails flicked, indicating that the injector had penetrated into the subarachnoid space successfully. Next, the micro-injector was pumped back carefully, eliminating the possibility of blood reflux. Then,10 ?l of agentia was injected at 1 ?l per second. After injection, the injector was quickly removed, and pressure was applied to the injection site for 1 min to prevent cerebrospinal fluid leakage. When the inhalation anesthesia was stopped, the rats demonstrated the righting reflex in 1 min.3. Behavioral testingWalk gait pattern was assessed as an index of motor function. A score of 1 indicates a normal gait, without foot deformities; 2 indicates a normal gait with obvious foot deformities; 3 indicates a slight gait disturbance with foot-drop; and 4 indicates a serious gait disturbance with myasthenia. Only rats scoring 1 were used for the following experimental procedures.The behavioral testing was performed with regard to the ipsilateral hind paw of the animals prior to surgery as well as on postoperative days 2,4,6,10,14 and 28. The effects of the inhibitor or agonist on the CCD-induced allodynia were tested between 0.5-h and 8-h post injection. The paw withdrawal mechanical threshold (PWMT) was evaluated using a BME-404 Mechanical Analgesia Tester (Chinese Academy of Medical Sciences, CAMS, Beijing, China). A probe was pressed against the lateral plantar surface of the hind paws with sufficient force. A positive response was noted when the paw immediately withdrew. The procedure was repeated five times at least 5 min apart, and the average value was used as a variable.4. Western Blot AnalysisThe L4 and L5 ganglia from the operated side were harvested quickly and carefully. Protein samples of the DRGs were prepared on ice. Then, the sample of total protein was separated by 5% and 10% SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated in 5% milk for 2 h at room temperature. Then, the membranes were incubated with the primary antibody at 4? overnight followed by horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The signal was detected using the ImmobilonTM Western Chemiluminescent HRP Substrate. The primary antibodies were rabbit anti-TRPV4 polyclonal antibody (1:800, Abcam, Cambridge, UK), rabbit anti-p38 polyclonal antibody (1:200, CST, USA), and rabbit anti-P-p38 polyclonal antibody (1:1,000, CST, USA), whereas the second antibody was a goat anti-rabbit antibody (1:8,000, Zhongshan Goldenbridge, Beijing, China). The protein bands were visualized using a FluoroChem9900 imaging system (USA), and the bands' intensity was quantified with the Quantity One software and normalized to ?-tubulin (1:1,000, CST, USA).5. ImmunohistochemistryAfter the behavioral and pain tests, the rats were deeply anesthetized with 5% isoflurane and perfused transcardially with cold normal saline followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate-buffered saline (PBS, pH 6.9). Ipsilateral lumbar L4-L5 DRGs were removed rapidly after perfusion, post-fixed in the same fixative overnight at 4? and then dehydrated and paraffin infused. A series of 4-?m paraffin sections were cut using a rotary microtome. The sections were heated at 65? for at least 2 h and then deparaffinated. Antigen retrieval was accomplished with citrate buffer in a microwave oven at 92-98? for 15-20 min. The sections were washed in PBS then incubated separately in rabbit anti-TRPV4 polyclonal antibody (1:200, Abcam, Cambridge, UK), rabbit anti-p38 polyclonal antibody (1:50, CST, USA), and rabbit anti-P-p38 polyclonal antibody (1:100, CST, USA) at 4? overnight. The sections were incubated using a specific secondary antibody for 2 h at room temperature. DAB substrate solution and hematoxylin were used to develop the color. Labeled sections were examined under a Leica Quantimet 550 DMRXA automated research microscope (GER) and analyzed using IPP.6.6. Ectopic spontaneous discharge recordingFour days after CCD surgery, rats without autophagy, sensory deficiency or disability were selected. The animals were anesthetized with 10% chloral hydrate (300 mg/100 g body weight, i.p. Their skin was cut, their muscles were moved, and the vertebral plate of their L2-L6 was removed carefully to avoid spinal cord or nerve injury. The steel bars were removed from the DRGs, the peripheral nerves were cut off approximately 10 mm from the L4 and L5 DRGs to block signaling from the peripheral receptors, and the communicating branches of L4 and L5 were cut to block signals from other segments. A bath around the incision was built and filled with 35?-37? manual cerebrospinal fluid, containing 150 mmol/L NaCl,1 mmol/L MgCl2,2 mmol/L CaCl2,10 mmol/L glucose and 5 mmol/L Tris at pH 7.4. The central ends of L4 and L5 were covered with 35?-37? medicinal liquid paraffin. The nerves were divided into several 30-to 50-p.m fiber bundles under a microscope and hung on the recording electrode separately, with the reference electrode inserted into the skin nearby. Discharge signals were recorded using a BL-420E+ biological and functional experimental system (Taimeng Science and Technology Ltd, Chengdu, China).Results1. Changes in PWMT in CCD rats after the injection of agonists and inhibitorsPWMT significantly decreased from the second day after CCD surgery, lasting 14 days (P< 0.01); then, it increased to normal levels. To study the effects of TRPV4 and p38 with regard to neuropathic pain further, we sought to determine the abilities of RR,4a-PDD, SB203580 and anisomycin to enhance or block the nociception signs induced 4 days after CCD surgery. RR- and SB203580-treated rats exhibited paw withdrawals at higher mechanical forces compared with the saline group. In addition, 4?-PDD and anisomycin injections markedly decreased the paw withdrawal threshold compared with the saline group. The most prominent time point was approximately 2 h after injection; no significant dose dependence was found.2. Effects of agonists and inhibitors of TRPV4 and p38 on protein expression in CCD ratsSeparately, the concentrations of these reagents were 1 nmol/L,10 nmol/L and 100 nmol/L for RR and 4a-PDD; 10 ?mol/L,20 ?mol/L and 40 ?mol/L for SB203580; and 5 ?g/mL,25 ?g/mL and 40 ?g/mL for anisomycin. The expressions of TRPV4, p38 and P-p38 were tested at 1,2,4, and 8 h after intrathecal injection of these drugs.The control group was administered normal saline in an equal quantity.TRPV4, p38 and P-p38 were significantly inhibited by RR (2-4 h for TRPV4; 1-8 h for p38 and 1-4 h for P-p38), and both the TRPV4 and p38 changes were dose dependent. When TRPV4 expression increased by 4a-PDD (2 h), p38 and P-p38 were also up-regulated (4 h for p38; 2 h for P-p38), and all compounds were associated with the concentration of the drug administered. The administration of SB203580 significantly reduced the expression of p38 and P-p38 (4 h for p38; 2-4 h for P-p38) but significantly increased the expression of TRPV4 (1-8 h) regardless of the concentration. Finally, we administered anisomycin to the CCD rats; p38 protein expression was significantly increased (1-2 h), TRPV4 was inhibited (1-8 h) and P-p38 did not change significantly. A clear dose-dependent relationship was not found.3. Protein distribution changes after intrathecal injections of TRPV4 and p38 agonists and inhibitors among CCD ratsWe found that TRPV4 and p38 labeling were both evident in small, medium and large ganglion cell bodies (small< 30 ?m, middle 30 ?m-40 ?m, large> 40 ?m). The number of positive cells increased after CCD and was affected by the agonists and inhibitors. A quantitative analysis revealed that the number of TRPV4-positive neurons in the small and total ganglion neuron groups increased significantly (P< 0.01) compared with controls. Following the RR and SB203580 injections, the number of TRPV4-positive small neurons was reduced (P< 0.01). The total positive neuron number increased after anisomycin injection (P< 0.01), which significantly differed from the CCD group. The number of p38-positive neurons of all sizes was significantly increased after CCD compared with controls (P< 0.05, large; P< 0.01, medium, small and total). The number of p38-positive, small neurons and the total number of p38-positive neurons were significantly reduced by SB203580 (P< 0.01) as well as increased by 4a-PDD (P< 0.01) and anisomycin (P< 0.01) compared with the CCD group.4. The effects of the agonists and inhibitors on electrophysiological propertiesRare ectopic discharges occurred in normal rats. The frequencies of ectopic discharges did not markedly differ between groups. However, the amplitudes in the RR and SB203580 groups were significantly reduced (P< 0.01) but significantly increased in the 4?-PDD and anisomycin groups (P< 0.01).Conclusion1. The expression and activation of TRPV4 and p38 change after CCD significantly contributes to the alternation of PWMT and the amplitudes of ectopic discharges, acting upon each other, mostly in small neurons.2. The current studies provide evidence for the existence of a link between TRPV4 and p38, with an intermediary role for neuropathic pain. Furthermore, the link between TRPV4 and p38 is bidirectional.Chapter IIMAPK pathways are involved in neuropathic pain in rats with chronic compression of the dorsal root ganglionObjectiveTo investigate whether the MAPK pathways involved in the mechanism of neuropathic pain in rats with chronic compression of the dorsal root ganglion.Materials and methods1. Animals and surgical procedureAdult male Wistar rats weighing 180-220 g were provided by the Experimental Animal Center of Shandong University and were housed in a pathogen-free air room at a temperature of 20 ± 2? at two per cage on a 12 h light/dark cycle with water and food available ad libitum. The animals were allowed 7 days to habituate to the housing prior to manipulation and half an hour to habituate to the experimental environment before every behavioral study was performed. All experimental procedures were approved by the Animal Care and Use Committee of the Shandong University.Rats were anesthetized by 10% chloral hydrate (300mg/100g i.p.), then two stainless steel rods were implanted unilaterally into the intervertebral foramen at L4 and L5 [31,32]. Rats in sham-operation group were given the same operation but no steel bar inserting. The rats with autophagy phenomenon, feeling deficiency and disability were eliminated.2. Behavioral testingBehavioral testing was performed using the ipsilateral hind paw of the animals prior to the operation, on postoperative day 4, and 2 hours after the injection of inhibitors. The paw withdrawal mechanical thresholds (PWMTs) were evaluated with a BME-404 Mechanical Analgesia Tester (CAMS-Chinese Academy of Medical Sciences, Beijing, China) [32]. The probe was pressed against the lateral plantar surface of the hind paw with sufficient force. A positive response was noted when the paw was immediately withdrawn. The rats were tested again at least five minutes later, the tests were repeated five times, and the average was calculated and used in the statistical analyses.3. Western Blot AnalysisFour days post-surgery, CCD rats were intrathecal injected with MAPKs inhibitors for 2 h. The L4 and L5 ganglia from the operated side were quickly and carefully harvested. The samples of total protein were separated by sequential 5% and 10% SDS-PAGE, then transferred to polyvinylidene fluoride membranes. The membranes were incubated in 5% milk for 2 h at room temperature. Next, the membranes were incubated with primary antibody at 4? overnight and subsequently with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The signals were detected with ImmobilonTM Western Chemiluminescent HRP Substrate. The primary antibodies were rabbit anti-ERK polyclonal antibody (1:1000, CST, USA), rabbit anti-p-ERK polyclonal antibody (1:2000, CST, USA), rabbit anti-JNK polyclonal antibody (1:1000, CST, USA), rabbit anti-p-JNK polyclonal antibody (1:1000, CST, USA), rabbit anti-p38 polyclonal antibody (1:200, CST, USA), and rabbit anti-P-p38 polyclonal antibody (1:1,000, CST, USA). The second antibody was goat-anti-rabbit antibody (1:8000, Zhongshan Goldenbridge, Beijing, China). The protein bands were developed with a FluoroChem9900 imaging system (USA), and the quantifications of the intensities of the bands were performed with the Quantity one software and normalized to p-tubulin (1:1,000, CST, USA).4. Immunolocalization of p38, ERK, JNK in dorsal root gangliaRats were deeply anesthetized with 5% isoflurane and perfused transcardially with cold normal saline followed by fixative containing 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 6.9). The ipsilateral lumbar L4-L5 DRGs were removed rapidly after perfusion, post-fixed in the same fixative overnight at 4? and then dehydrated and paraffin infused. A series of paraffin sections (4 um) were cut using a rotary microtome. The sections were incubated separately in mixtures of the following primary antibodies at 4? overnight:rabbit anti-ERK polyclonal antibody (1:200, CST, USA), rabbit anti-JNK polyclonal antibody (1:200, CST, USA), and rabbit anti-p38 polyclonal antibody (1:50, CST, USA). The primary antibodies were combined with mouse-anti-NF200 polyclonal antibody (1:1,000, Abcam, Cambridge, UK). Then the sections were incubated in Alexa Fluor 488-conjugated Affinipure Donkey Anti-Rabbit IgG (H+L) and Alexa Fluor 594-conjugated Donkey Anti-Mouse IgG (H+L) for 2 h at room temperature. DAPI was used to stain the cell nuclei. Labeled sections were examined under an Olympus-u-rfl-t/dp 72 automatic fluorescence microscope using the image analysis system of the microscope (JA) and analyzed using the IPP.6 by software. For the quantitative analyses of the numbers of positive neurons, three immunofluorescent stained non-consecutive sections were imaged per ganglion. The data were collected from three animals for each inhibitor (SB203580, UO126 and SP600125).5. Real-time quantitative RT-PCRL4 and L5 ganglions were harvested in the same manner as described above. Fragments of p38, JNK, ERK or ?-actin were amplified with the following primers:p38 (forward,5'-CCTGCGAGGGCTGAAGTA-3'; reverse,5'-ACGGACCAAATATCCACTGTC-3'), JNK(forward, 5'-AGCCTTGTCCTTCGTGTC-3'; reverse,5'-AAAGTGGTCAACAGAGCC-3'), ERK1(forward,5'-CCAGAGTGGCTATCAAGA AG-31; reverse, 5'-TCCATGAGGTCCTGAACAA-3'), ERK2(forward, 5'-TGCCGTGGAACAGGTTGT-3'; reverse, 5'-TGGGCTCATCACTTGGGT-3') and p-actin (forward, 5'-AGACCTTCAACACCCCAG-3';reverse,5'-CACGATTTCCCTCTCAGC-3'). Instrument control, automated data collection, and data analysis were all performed using the Light Cycler software program, version 4.0. The 2-AACT method was used to analyze the data.6. Chemicals and reagentsThe following chemicals were used in this study:SB203580 (p38 inhibitor, CST, USA, recommended concentration= 40?mol/L), SP600125 (JNK inhibitor, CST, USA, recommended concentration= 50 ?mol/L) and UO126 (ERK inhibitor, CST, USA, recommended concentration= 40?mol/L). All of the chemicals were dissolved in DMSO, and the final experimental dilutions were made in normal saline on the day of the experiment.Results1. PWMT changes after the CCD operation.The CCD group developed evident mechanical allodynia hyperalgesia in the ipsilateral hind paw compared with the control group. The PWMT significantly decreased at 4 days after the CCD operation (n=8 in each group,**P<0.01). Furthermore, CCD-induced allodynia were attenuated by SB203580, SPOO125 and UO126 (n=8 in each group,#P<0.05), while there was no significant difference between sham group and control group (n=8 in each group).2. Changes in protein expressions of p38, ERK and JNK in the DRGsThe levels of p38, JNK, ERK and P-p38, P-JNK, P-ERK protein expression in the CCD rats significantly increased (n=5,*p<0.05,**p<0.01 compared with control group;#p<0.05,##p<0.01 compared with sham group). These CCD-induced increases in protein expression level were diminished significantly by inhibitors (SB203580, SP600125 and UO126) administration (&p<0.05,&&p<0.01 compared with CCD groups).3. Changes in gene expressions of p38, ERK and JNK in the DRGsPharmacological inhibitors of MAPKs were not only diminished the protein expression of p38, ERK and JNK in DRGs of CCD rats, but also affected the level of gene expressions. The levels of p38, JNK and ERK gene expression in the CCD rats significantly increased (n=6,*p<0.05,**p<0.01 compared with control group). These CCD-induced increases in gene expression level were diminished significantly by inhibitors (SB203580, SP600125 and UO126) administration (n=6,##p<0.01 compared with CCD groups).4. Changes in p38, ERK and JNK distributions in DRG neuronsp38, JNK and ERK were expressed both in the nuclei and cytoplasm, the proportions of NF200 positive large size neurons among all of the neurons in the DRG tissues increased significantly (n=6,*p<0.05,**p<0.01) after CCD surgey compared with control groups. After SB203580, SP600125 or U0126 administration, the proportions of NF200 positive neurons significantly decreased (n=6,#p<0.05, ##p<0.01 compared with CCD group). As to NF200 negative small size neurons, though there were some chenges in proportion, we could not found any obvious regulation of these changes.Conclusion1. pecific inhibitors of MAPKs contributed to the attenuation of mechanical allodynia in CCD rats and the large size MAPKs positive neurons in dorsal root ganglia were crucial.2. MAPK pathways are involved in the mechanism of neuropathic pain in CCD rats.Chapter IIIAlternation of TRPV4 protein expression and distribution in spinal dorsal horn in chronic compression of the dorsal root ganglion model of ratsObjectiveTo investigate the alternation of transient receptor potential vanilloid 4 (TRPV4) protein expression and distribution in spinal dorsal horn in chronic compression of the dorsal root ganglion model of rats.Methods1. GroupsA total of 30 Wistar rats were used, they were randomly divided into a control group and operated group (4,7,14 and 28 days after operation,5 rats in each group;5 rats in immunofluorescence group).2. CCD operationChronic compression of dorsal root ganglion (DRG) model was established according to the method in Chapter I.3. Test of the paw withdrawal mechanical thresholdAt 4,7,14 and 28 days after CCD operation, the paw withdrawal mechanical threshold was tested according to the method in Chapter I.4. Western blottingThe expression of TRPV4 in ipsilateral and contralateral spinal dorsal horn was tested after behavioral testing compared to control group.5. Immunolocalization of TRPV4 in spinal dorsal hornThe distribution of TRPV4 positive neurons in ipsilateral and contralateral spinal dorsal horn were detected.Results:1. Alternation of the paw withdrawal mechanical threshold after CCD surgeryThe paw withdrawal mechanical threshold of CCD group was tested before and 4,7,14 and 28 days after CCD surger, that of control group was tested at the same time. The results showed that, at 4,7,14 days after CCD surgery, the paw withdrawal mechanical threshold decreased significantly compared with control group (n=5, P<0.001); there was no significant difference in control group at different time.2. Changes of TRPV4 protein expression in spinal dorsal hornThe expression of TRPV4 in ipsilateral and contralateral spinal dorsal horn was tested after behavioral testing compared to control group. Compared with control group, at 4 and 7 days after CCD surgery, TRPV4 protein expression increased significantly (n=5, p<0.01); there was no significant differences at 14 and 28 days. TPRV4 protein expression in contralateral spinal dorsal horn was similar with control group.3. Changes of TRPV4 positive neurons in spinal dorsal hornThe distribution of TRPV4 positive neurons in ipsilateral and contralateral spinal dorsal horn were detected at 4 days after CCD surgery. TRPV4 positive neurons were appeared both in ipsilateral and contralateral spinal dorsal horn. Fluorescence signal could be found in cytoplasm and cell membrane, and gathered at axon hillock.5 spine cords were harvested, and 5 discontinued sections were chosen, the TRPV4 positive neurons in ipsilateral and contralateral spinal dorsal horn were counted. We found that, compared with contralateral spinal dorsal horn, the number of TRPV4 positive neurons was significantly increased (n=5, p=0.0008).ConclusionAfter CCD operation, the paw withdrawal mechanical threshold decreased, the expression of TRPV4 in ipsilateral spinal dorsal horn was up-regulated and the number of TRPV4 increased. TRPV4 may take part in the central sensitization mechanism of neuropathic pain of CCD rats.