The Effect Of Chronic Compression Of Dorsal Root Ganglion On TRPV4 And Proteomic Analysis Of Differential Proteins In Rats

Partâ… : Research of electrophysiological properties of mechanosensitive channels in cultured dorsal root ganglion neurons of neonatal ratsBackgroundAll living organisms face mechanical forces,from the fluid forces around bacterium to the high forces in a human knee during stair climbing.The process of converting physical forces into biochemical signals and integrating these signals into the cellular responses is referred to as mechanotransduction.The mechanosensitive channels(MS channels)play an important role in the mechanotransduction.The idea of MS channels arose originally from studies of specialized mechanosensory neurons. The channel open probability of MS channels changes with the membrane tension and converts mechanical force exerted on the cell membrane into electrical or biochemical signals in physiological processes.Since their discovery in embryonic chick skeletal muscle and frog muscle,MS channels have been found in many cell types.McCarter first identified the whole-cell currents activated by stretch or pressure in dorsal root ganglion(DRG)neurons of rats in 1939.After that,various MS channels activated by various stimuli were reported in DRG.The electrophysiological properties of those MS channels are diverse due to the difference in mechanical stimuli,recording methods,and the composition of pipette solution and bath solution.DRG neurons are primary afferent neurons that carry sensory signals from the skin,muscles,joints,and visceral organs to the spinal cord.Various mechanical, thermal,or noxious stimuli are known to generate action potentials at peripheral nerve endings of the DRG neurons.DRG is easily mechanical compressed in the intraforaminal or subarticular zones by degenerative changes of the lumbar vertebrae due to the special anatomical position and physiological construction features, resulted in radicular pain.Identification of the biophysics and distribution of the MS channels in the DRG can further our knowledge about the mechanism underlie the electric activity of DRG neurons.ObjectiveThe aim of this current study is to explore the electrophysiological properties and distribution of mechanosensitive channels in cultured DRG neurons of neonatal rats.MethodsAfter cultured for 4 days,the mechanosensitive channels currents of DRG neurons of neonatal rats were recorded and analyzed using cell-attached and inside-out patch-clamp technique.The electrophysiological properties such as pressure-response relationship,current-voltage relationship,channel kinetics,ion selectivity,influential factor and cell-size distribution were analyzed.Membrane stretch was achieved by the application of negative pressure(suction)to a patch pipette.Results1.Pressure-response relationshipIn cell-attached patches with·the membrane potential of-60 mV,no single-channel currents were observed before applying suction,except for a few patches,which showed spontaneous openings.The negative current was activated by pressure of-12 to -15mmHg.Those channels activated rapidly when suction was applied,kept stably during sustained application of negative pressure and quickly turned off when the suction was released.When the amplitude of the channel current was measured in cell-attached patches,the mean current amplitude was -3.40±0.04pA(n=125)at -60mV of membrane potential.Channel open possibility(NPo)increased as higher pressures were applied to the patch and reached a maximum approxiamate 50mmHg, with P1/2 was 36.66±0.455mmHg(n=25).A higher pressure would destroy the membrane.The mean NPo was 0.448±0.03(n=25)at-60mV of membrane potential and -50mmHg of pressure.2.Current-voltage relationshipIn inside-out patches with -40mmHg pressure,the MS channels exhibited a nearly linear current-voltage relationship in the symmetrical solution.The outward chord conductance was 96.15±3.73 pS(Vm is between +40mV and +60mV)and the inward slope conductance was 62.47±2.72 pS(Vm is between -60mV and 0mV). This kind of channels appeared to be an inward rectifier.The average reversal potential(Erev)was -2.33±0.255 mV.3.Channels kineticsHistograms of open or closed-time durations of MS channels activated by suction pressures were best fitted with two exponential functions.Therefore,the channels exhibited short and long openings and closings.Negative pressure of -20 mmHg activated MS channels with open time constants of 1.338±0.098ms and 13.001±0.649ms,and with closed time constants of 2.462±0.267ms and 23.386±1.206ms.When negative pressure of -40 mmHg was applied to the same channels,the duration of short openings and long openings increased significantly 1.744±0.195ms for short openings and 17.92±1.623ms for long openings(both P＜0.05).In contrast,at -40 mmHg,the duration of long closings 14.071±0.797ms (P＜0.05),but not short closings 2.266±0.154 ms(P=0.545),decreased significantly compared with at -20 mmHg.Therefore,greater pressures could increase the channel activities of MS channels largely by increasing the duration of short openings and long openings and decreasing the duration of long closings.4.Ion selectivityThe MS channels were non-selective cation channels and were not permeable to anion.5.Influence factors The current activated by mechanical stimuli could be blockaded by gadolinium and colchicine.The tetrodotoxin could blockade the current in large diameter DRG neurons,with no effect on the small diameter DRG neurons.6.Cell-size distributionThe MS channels were founded mainly in small(＜20μm,48.41%)and medium (20～30μm,36.30%)diameter DRG neurons and were rarely found in relatively large diameter(＞30μm,15.29%)DRG neurons.ConclusionWe identified the electrophysiological properties and the distribution of a kind of MS channels in DRG in rats.The mechanical stimuli could activate the MS channels and transduce the mechanical signals. BackgroundChronic compression of the dorsal root ganglion (DRG) or its near nerve root is considered to be one of the most important causative factors of radicular pain. However, the evidence supporting such an argument is incomplete. The effects of pure mechanical pressure on DRG neurons have not been assessed. In vivo animal models are usually used to study the change of the morphology, function, and expression of related genes and proteins under the mechanical pressure. But the effect of the mechanical pressure on the DRG could be easily confounded by other factors and can not be exclusively evaluated in in vivo models. So it is necessary to establish a convenient, reliable, stable, and single factor in vitro cell model to explore the relationship between the pressure applied and the responses of the DRG neurons.Among the current in vitro models, the model of hydrostatic pressure is able to provide sustained compression at different pressure levels. In such a model, pure mechanical loading is generated by compressing the gas phase within a closed culture chamber that contains the culture dishes. But there is little evidence on whether this model is suitable for the study of neurons, given the specificity for the culture of the neurons.DRG neurons can transmit polymodal sensations, such as temperature and mechanical sensations. Many ion channels, including transient receptor potential vanilloid receptor 1 (TRPV1), transient receptor potential vanilloid receptor 4 (TRPV4), transient receptor potential channel of melastatin type 8 (TRPM8), and transient receptor potential subtype ankyrin 1 (TRPA1), play a potential role in mechanical nociception. Among these channels, the TRPV4 channel is a polymodal receptor, which plays an important role in nociceptive responses to hypotonic and mechanical stimuli.Recent studies suggested that the chronic compression of the dorsal root ganglion changed the expression and the electrophysiological property of the voltage-dependent ion channels, as well as the synthesis and transportation of the neurotransmitter. Furthermore, Hoyer et al. found that densities of the pressure-activated channels were significantly higher in spontaneously hypertensive and renovascular hypertensive rats compared with their respective normotensive controls. Therefore, we hypothesized the expression or function of the mechanoreceptors may be changed under direct chronic compression.Objective1. To establish an in vitro cell model to explore the relationship between the pressure applied and the responses of the DRG neurons.2. To investigate whether pure chronic mechanical compression of the dorsal root Methods1. Establishing the in vitro cell modelDRG neurons were prepared as described previously and were placed in a 37℃incubator in a 95% air-5%CO₂ atmosphere. After 2 days the culture medium was changed and the culture dishes were put into the seal chamber, which was designed for incubating the DRG cells in a range from 0 to 200 mm Hg. Compressive loading is generated by compressing the gas phase within the closed culture chamber and the pressure gas consisted of 95% air and 5%CO₂- Pressure levels are monitored continuously by manometer, with atmospheric pressure (760 mm Hg) calibrated to 0 mm Hg. Dishes were cultured under compressive loading as the test groups and other dishes were cultured without loading as the control group.2. MTT testDRGs were cultured on 96-well plates and there were 8 wells in each condition. After cultured under different pressure for different durations, cell morphology was observed under inverted microscope and the cell activation was assessed with MTT test. The original culture medium was then removed, and l00μi 1mg/ ml of MTT was added. After 150μl dimethyl-sulfoxide was added and the plates were oscillated for 10min to fully dissolve the crystal, the absorption at 490 nm was measured with ELISA analyzer. A mean value from 8 wells was calculated as one sample and 3 samples were taken for each condition. Cell viability of control cells was taken as 100%.3. Real-time PCR The mRNA levels of TRPV4 in DRG neurons cultured with different pressures and different durations were quantified by real-time reverse-transcriptase polymerase chain reaction (RT-PCR) using SYBR Green technology.4. Western blottingWestern blotting was performed to investigate the proteins expression of TRPV1, TRPV4, TRPM8, and TRPA1 in DRG neurons cultured with different pressures and different durations.5. Laser scanning confocal microscopeThe responses to the hypotonic solution, TRPV4 agonist, and antagonist were assessed by calcium imaging using the laser scanning confocal microscope.Results1. Effect of mechanical pressure on cell morphology and viabilityThere were no significant changes in cell morphology between the control and the compressed cells. Pressure level only at 120 mmHg significantly inhibited DRG cell growth (P<0..05 vs. 0 mmHg, Bonferroni t-test). The mechanical compression induced a significantly inhibitory effect on DRG viability only in 72h group (P<0.01, Bonferroni t-test), but not in other groups. Therefore, 80 mmHg pressure level was chosen in the current study as the test compression.2. Effect of mechanical pressure on gene expressionGene expression was monitored after sustained compression at 0 mmHg (control) and 80 mmHg for 24 hours and 48 hours. The mechanical pressure significantly increased the TRPV4 gene level (P<0.05). Furthermore, gene expression was dependent on the interaction between pressure levels and duration of compressions (P <0.001). Post hoc analysis revealed that compression at 80 mm Hg for 24h and 48h significantly increased the gene transcription of TRPV4 to 2.31-fold and 4.04-fold as compared with the control (both P<0.05, SNK), respectively. Similarly, TRPV4 mRNA level in the 48h compression-exposed cells was significant higher than that in the 24h group (P<0.05, SNK).3. Effect of mechanical pressure on the protein expression in DRG neuronsThe DRG neurons were cultured under 80 mmHg for 0 hour (control), 24 hours, and 48 hours. Mechanical pressure significantly increased the TRPV4 protein level (P<0.05). Post hoc showed compression-exposure (80 mmHg) for 24h and 48h induced a significantly up-regulation in TRPV4 protein levels, which were 1.80-fold and 4.22-fold higher than the control, respectively (both P<0.05, Tukey Test). By contrast, TRPV1, TRPM8 and TRPA1 expression showed no significant changes following sustained compression for 24 and 48h (all P>0.05).4. Effect of mechanical pressure on TRPV4 Ca²⁺ responsesHypotonic stimulation increased intracellular Ca²⁺ concentration in an osmolarity-dependent manner in DRG (P<0.001). Pre-treatment with compression caused an increase in the magnitude of Ca²⁺ response, as well as the percentage of cells responding to hypotonic stimulus. When challenged with 30% hypotonic solution, 37.0% neurons showed increase in [Ca²⁺]_i with the magnitude was 1.67±0.02. In the 24h and 48h groups, 48.4% (x²=1.86, P>0.05) and 61.2% (X²=7.16, P<0.05) neurons showed response to hypotonic solution, with the 1.74-fold and 2.52-fold increase in the magnitude of Ca²⁺ response compared to the control (both P<0.01, SNK) respectively. Similar to the hypotonic solution -induced Ca²⁺ response, the response to 4a-PDD showed the increased magnitude of Ca²⁺ response with the longer duration of mechanical compression applied, which increased to 1.48-fold and 2.09-fold compared to the control (both P<0.01, SNK), respectively. The percentage of neurons responding to 4α-PDD was also increased from 30.92% to 42.25 % (X²=2.29, P>0.05 for 24h group) and 49.18% (X⁵.30, P<0.05 for 48h group) . Furthermore, there is an interaction between pressure levels and duration of compressions on the Ca²⁺ response (P <0.001).Conclusion1. In a certain culture duration and pressure, pure mechanical pressure alone has no effect on the morphology and cell activation of DRG neurons and the model of hydrostatic pressure is suitable to investigate how the neurons respond to mechanical compression.2. Pure mechanical pressure alone can induce the upregulation of mRNA and protein expression of TRPV4 without affecting other TRPs. Meanwhile, the mechanical pressure also enhances the function of the TRPV4 channels. BackgroundMechanical compression of the nerve root and the dorsal root ganglion (DRG), by disc herniation or spinal stenosis is one of the major causes of radicular pain in humans. Chronic compression of DRG (CCD) in animals results in spontaneous pain, mechanical hyperalgesia and allodynia. In association with these painful behaviors spontaneous activity, lowered threshold currents, and action potential thresholds have been shown in the neuronal somata of the compressed ganglion. Ion channels such as voltage-gated Na⁺ and K⁺, and hyperpolarization-activated cation current may contribute to the hyperexcitability of DRG neurons after CCD. However, to date the ionic mechanisms underlying mechanical hyperalgesia and allodynia are not fully understood.Ion channels in DRG neurons, including the transient receptor potential vanilloidreceptor 2 (TRPV2), transient receptor potential vanilloid receptor 4 (TRPV4), and transient receptor potential subtype ankyrin 1 (TRPA1), may also play a role in mechanical nociception. TRPV4 is a polymodal receptor and it has been shown to mediate nociceptive behaviors to hypotonic and mechanical stimuli under pathological conditions. Chemotherapy (Taxol) has been shown to increase the percentage of the DRG neurons respective to TRPV4 agonists. However, no significant changes in the expression or function of TRPV4 were found in traumatic or diabetic neuropathy pain models. Thus, the role of TRPV4 channel in neuropathic pain is controversial.We have shown in the second part of the thesis that the pure sustained mechanical pressure can increase the expression levels and sensitized the ion function of TRPV4. In CCD model, the mechanical compression associated with the secondary inflammatory process, such as ischemic, edema, and inflammatory cells infiltrate makes the pathophysiological conditions much more complex. Therefore, the aim of this study was to investigate the effects of CCD on the levels of TRPV4 mRNA and protein expression to determine the role of TRPV4 in CCD-induced mechanical allodynia.Objective1. To investigate the effects of CCD on the expression levels of mRNA and protein and the function of TRPV4.2. To determine the role of TRPV4 in CCD-induced mechanical allodynia.Methods1. Establish CCD modelNinty-nine rats were randomly divided into three groups, corresponding to 7, 14,and 28 days post-CCD. Each group was sub-divided into a sham and a CCD group (n=13 for sham and n=20 for CCD). In CCD rats, under pentobarbital sodium anesthesia, the paraspinal muscles were separated to expose the transverse process and intervertebral foramina of L₄ and L₅ unilaterally as previously described. A stainless steel U-shaped rod (0.63 mm diam and 4 mm length) was inserted into each foramen to compress the DRG, one at L₄ and the other at the L₅ ganglion. The muscle and skin layers were then sutured. Penicillin was injected to prevent infection. Sham group underwent the same surgical procedure as described, but without the insertion of the rods. Animals were sacrificed at 7,14, and 28 days post-CCD, respectively.2. Antisense oligodeoxynucleotide treatmentTo determine the effects of antisense oligodeoxynucleotide (ODN) treatment on CCD-induced mechanical allodynia and calcium response, 18 normal rats and 27 CCD rats were treated with 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) (n=6 in normal rats and n=9 in CCD rats for each sub-group). 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.3. Neuroethology measurementThe motor function and MWT were measured pre-CCD and right before the animals were sacrificed at 7,14, and 28 days post-CCD, respectively.1) Walk 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 myashtnia.2) Mechanical withdrawal threshold (MWT) was measured with a von Frey hair monofilament with logarithmically incremental stiffness (0.09-17.30 g). 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. A positive response was noted if the paw was immediately withdrawn. The upper limit for testing was 17.30 g hair.4. Real-time PCRThe levels of TRPV4 gene expression in different groups were assessed using real-time RT-PCR.5. Western blottingThe levels of TRPV4 protein expression in different groups were assessed using Western blotting.6. Laser scanning confocal microscope measurementThe calcium responses to hypotonic solution and 4a-phorbol 12, 13-didecanoate (4a-PDD) were assessed following sham surgery, CCD, spinal application of TRPV4 antisense ODN, and mismatch ODN with laser scanning confocal microscope.Results1. Neuroethology measurement1) 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. 2) The MWT in CCD group was significantly lower than the sham group (F=39.97, P<0.001). There was also a significant difference in MWT at 7, 14, and 28 days post-CCD (F=8.03, P<0.001). Furthermore, the MWT was dependent on the interaction between the groups and the days post-CCD (F=3.16, P<0.05). The MWT was lower in the CCD group than the sham group at 7, 14, and 28 days post-surgery (SNK, all P<0.05), with the lowest level at 7 days post-CCD.2. The effect of CCD on the levels of TRPV4 mRNA expressionThe levels of TRPV4 mRNA expression were increased significantly in CCD group when compared with the sham group (F=240.02, P<0.001). There were also significant differences in the levels of TRPV4 mRNA expression at different days post-CCD (F=14.77, P<0.001). The levels of TRPV4 mRNA expression were also dependent on the interaction between the groups and the days post-CCD (F=14.22, P<0.001). The levels of gene expression were significantly increased at 7, 14, and 28 days in CCD group when compared with the sham group (SNK, all P<0.05). There was no significant changes in the level of mRNA expression in sham group (SNK, all P>0.05), but the mRNA level was significantly higher at 7 days than that at 14 and 28 days post-CCD (SNK, both P<0.05).3. The effect of CCD on the levels of TRPV4 protein expressionThe levels of TRPV4 protein expression were increased significantly in CCD group when compared with the sham group (F=451.52, P<0.001). There were also significant differences in the levels of TRPV4 protein expression at different days post-CCD (F=25.99, P<0.001). The levels of TRPV4 protein expression were also dependent on the interaction between the groups and the days post-CCD (F=23.99, P<0.001). There were significantly increased at 7, 14, and 28 days in CCD group, compared with the sham group (SNK, all P<0.05). The protein level was significantly higher at 7 days than at 14 and 28 days post-CCD (SNK, both P<0.05).Furthermore, the sustained increase in the levels of TRPV4 mRNA and protein expression was associated with the decreases in MWT in CCD group.4. The effect of antisense oligodeoxynucleotide treatment on CCD-induced mechanical allodyniaThe antisense ODN, but not the mismatch ODN, significantly inhibited the TRPV4 expression in normal rats (13.75±1.05%, 2.65±0.071%, 13.8±0.7% for control, AS, and MM group, respectively; F=77.79, P<0.005) and CCD rats (42.2±7.19%, 8.75±0.25%, 44.6±6% for control, AS, and MM group, respectively; F=13.93, P<0.05). In CCD rats, the MWTs were similar at baseline (Bonferroni t-test, all P>0.05), but after intrathecal ODN treatment, the MWT was decreased significantly in control group and MM group when compared with the baseline (Bonferronit-test, both P<0.05). In contrast, mechanical allodynia was partly reversed in AS group (Bonferroni t-test, P>0.05 compared with baseline). The MWT of normal rats were not changed following intrathecal AS or MM (F=0.0751, P= 0.93).5. Laser scanning confocal microscope measurementThe fluorescence ratio of calcium responsive to 30% hypotonic solution and 3μM 4a-PDD were significantly differed between different groups (F=148.46, P<0.01; F=173.26, .P<0.01, respectively). The percentage of the DRG neurons responsive to hypotonicity was upregulated in CCD group (52.6%, X²=5.14, P<0.05) and in MM group (50.8%, X²=4.32, P<0.01) when compared with sham group. The hypotonicity-induced increase in fluorescence ratio of calcium was also enhanced in CCD group and MM group when compared with sham group (Tukey Test, both P<0.01). Treatment with TRPV4 antisense significantly decreased the fluorescence ratio when compared with the CCD group and MM group (Tukey Test, both P<0.05). The percentage was also significantly decreased in AS group (29.1%) when compared with the CCD group ( X²=8.65, P<0.01) and MM group X²=7.59, P<0.01). Similar to the hypotonic solution-induced Ca²⁺ response, the percentage of DRG neurons responsive to the 4α-PDD was significantly upregulated in CCD group (46.1%, X²=4.25, P<0.05) and MM group (47.6%, X²=4.95, P<0.05) when compared with the sham group. The fluorescence ratio of calcium was enhanced in CCD group and MM group compared with the sham group (Tukey Test, both P<0.05). The percentage was significantly decreased in AS group (26.3%, X²=7.31, P<0.01 vs. CCD group; X²=8.22, P<0.01 vs. MM group) and the fluorescence ratio reduced significantly compared with CCD group and MM group (Tukey Test, both P<0.05).Conclusions1. It was shown that CCD in rats increases the levels of TRPV4 mRNA and protein expression, in addition to an enhancement in the calcium response to hypotonic stimuli and 4α-PDD.2. TRPV4 plays a crucial role in CCD-induced mechanical allodynia. 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 hemiation 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. The chronic compression of the DRG 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.Global gene expression and proteomics studies have discovered a large number of genes and proteins that are regulated in vivo after peripheral nerve injury in animal models of neuropathic pain. Many genes and proteins have been identified to be related to neuronal cell death and regeneration, including cytoskeletal proteins, neurotransmitter metabolizing enzymes, neuropeptides, growth factors, and signal transduction molecules, inflammation factor, protein synthesis/maturation, and myelination in the nerve. These studies have extended our knowledge about cellular and molecular changes in the degenerating nerve and its microenvironment after peripheral nerve injury. Since there are apparent behavioral and morphological differences among the most utilized animal neuropathy models, comparison of protein expression patterns from different types of injuries might help us understand elaborate molecular and cellular mechanisms underlying diverse peripheral neuropathy.Among the neuropathic pain models, the chronic compression of the dorsal root ganglion (CCD) model is unique in that the injury and inflammation are located in the ganglion. It is reported that in the CCD model, the acute compression to the nerve root induced endoneurial edema and the associated DRG edema. The ischemia along with any mechanical trauma caused by the compression could initiate an inflammatory reaction and the release of a variety of inflammatory mediators derived from the blood circulation, neurons as well as nonneuronal cells in the ganglion. Thus, the molecular and cellular changes that underlie peripheral nerve injury are complex and identification of proteins involved will contribute significantly to our understanding of the mechanisms of neuropathic pain and neuroprotection.ObjectiveThe aim of the study was to identify the differential protein expressions related to neuropathic pain and neuroprotection in DRG following CCD in rats.Methods1. CCD surgery Seventy-eight adult male Wistar rats weighing 150-180 g (Shandong University) were randomly divided into CCD group and normal control group, 39 in each group. In CCD rats, under pentobarbital sodium anesthesia, the stainless steel U-shaped rods were inserted into the intervertebral foramina of L₄ and L₅ as described in part 3. Animals were sacrificed at 28 days after surgery by decapitation2. Measurement of mechanical withdrawal threshold and thermal withdrawal latencyMechanical withdrawal threshold (MWT) was measured with von Frey monofilaments as described in part 3. Thermal allodynia was assessed with paw withdrawal latencies to radiant heat. The radiant heat source beneath the glass floor was focused on the plantar surface of the ipsilateral hind paw when in contact with the floor. The paw withdrawal latencies per animal were obtained for five times with intervals of 5 min.3. 2-DE and image analysisThe proteins from L₄ and L₅ DRGs of 20 rats in each group were extracted and quantificated with the Bradford method. The differential proteins expression between CCD group and control group were detected using two-dimensional gel electrophoresis (2-DE) followed by silver staining visualization and comparative analysis with the software of ImageMaster 2D Platinum 5.0. Three independent repeats of each sample were performed. The threshold as the significant change in 2-DE spots was defined as 3-folds of change in spot volume upon comparison of average gels between the CCD and control groups. 4. Mass spectrometry for protein identificationSelected spots were located and digested with trypsin. Then the peptide mass fingerprints (PMFs) of differential proteins expressed between the CCD and control groups were generated by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and interpreted utilizing Mascot produced by Matrix Science against the NCBInr database.5. Western blottingSome of the identified proteins were further examined by Western blotting as described in part 2.6. Real-time RT-PCRThe gene expression of annexin A2 and PKCεwere measured with real-time RT-PCR as described in part 2.Results1. Neuroethology measurementThe chronic compression produced marked mechanical and thermal allodynia, indicated by significantly decreased mechanical thresholds and thermal withdrawal latency compared with the normal control group (F=95.40, P<0.05; F=86.19, P<0.01, respectively). All rats walked normally after surgery which indicated that the surgery did not injury the motor behavior.2. Identification of the differential proteinsA total of 98 protein spots were detected with significant changes in their expression levels after CCD and 15 protein spots were identified by MALDI-TOF MS analysis. Of these proteins, annexin A2, protein kinase C epsilon (PKCe), glyceraldehyde-3-phosphate dehydrogenases (GAPDH), and heat shock protein 70 (HSP70) were up-regulated significantly compared with the normal control.3. Confirmation by Western blottingThe results of Western blotting showed consistent data with the results of the proteomic analysis. There was no significant difference in protein levels ofβ-actin between control and CCD groups (t = -0.139, P= 0.896).4. Effect of mechanical pressure on gene expressionQuantitative real-time RT-PCR experiments indicated that CCD-induced increase the gene levels of annexin A2 and PKCe.ConclusionsThese proteomic results suggest that chronic DRG compression is associated with the upregulation of annexin A2 and PKCεand their related genes, with may be related to the neuropathic pain. The upregulation of GAPDH and HSP70 suggests that there exist concurrent processes of nervous injury and neuroprotection in the course of neuropathic pain.