Dissertation
Dissertation > Medicine, health > Clinical > Rehabilitation Medicine > Medical Rehabilitation ( Rehabilitation Therapy )

The Effects and Mechanism of Cytoskeleton on Allodynia Induced by Chronic Compression of the Dorsal Root Ganglion

Author WeiHui
Tutor YueShouWei
School Shandong University
Course Rehabilitation Medicine and Physical Therapy
Keywords CCD microtubules allodynia TRPV4CCD microfilaments TRPV4
CLC R493
Type PhD thesis
Year 2013
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Neuropathic pain is one kind of pain irritated by primary damage and malfunction of nervous systems. Chronic compression of dorsal root ganglion (DRG)(CCD) is a typical model of neuropathic pain. It presents spontaneous pain, mechanical allodynia, and thermal hyperalgesia. However, the mechanism of the allodynia is not clear.Microtubules and microfilaments are important components of cytoskeleton. Microtubules are made up of tubulin, which includes a-tubulin (TUBA),(3-tubulin (TUBB) and y-tubulin (TUBG). Taxol is a microtubule stabilizer. Colchicine binds to the subunit of tubulin heterodimers to form a tubulin-colchicine complex that subsequently inhibits the polymerization of microtubule. Microfilament cytoskeleton structure is the thinnest fiber, which is about7nm in diameter. Microfilaments are mainly composed of actin, which contains free globular actin (G-actin) and fibrous actin polymerization (F-actin); however; only the latter is functional. Cytochalasin is the depolymerizing agent, inhibits the polymerization of microfilaments. Phalloidin is the stabilizer of microfilament, inhibits the depolymerization of microfilaments. Microtubules and microfilaments are in the state of dynamic balance, which is necessary for completing the physiological function.Several findings suggest that cytoskeletal elements are involved in the mechanical transduction in sensory neurons and cytoskeletal elements may play a role in the development of chronic pain. Recent evidence shows that colchicine could relieve the hyperalgesia in rats with loose ligation of the sciatic nerve (CCI). In addition, intact actin filaments is reported to be necessary for inflammatory pain, and disruptors of the microfilaments markedly attenuate the hyperalgesia in rat paws caused by injection of epinephrine or its downstream mediators. However, the effects of microtubules and microfilaments dynamics on CCD-induced allodynia have not been clearly determined. In CCD rats, several types of ion channels are reported to contribute to the allodynia, such as voltage-gated Na+and K+, hyperpolarization-activated cation current and transient receptor potential (TRP). As one of the TRP families, transient receptor potential vanilloid4(TRPV4) is a Ca2+-permeable polymodal receptor. TRPV4can be activated by various stimuli, including osmo-stimuli, mechano-stimuli, moderate heat, endogenous substances, and4a-phorbol12,13-didecanoate (4a-PDD). TRPV4is verified to contribute to the mechanical and thermal allodynia induced by CCD surgery in our previous study.Microtubules and microfilaments interact with TRPV4and regulate the TRPV4channel activity. Disruption of the actin cytoskeleton impairs the TRPV4-mediated currents and Ca2+-influx in TRPV4expressing cells in response to hypotonic shock. Therefore, we hypothesized that TRPV4may be involved in the effects of microtubules and microfilaments on CCD-induced allodynia.In order to test the hypothesis, we investigated the effects of microtubules and microfilaments on TRPV4activity and distribution in the DRG neurons in CCD rats, so to supply a possible mechanism for the effects of microtubules and microfilaments on CCD-induced allodynia. ObjectiveTo investigate the role of microtubules in allodynia induced by CCD surgery. In addition, the effects of microtubules dynamics on TRPV4were also tested.Materials and methods1. Chronic compression of dorsal root ganglionAfter anesthetized by sodium pentobarbital (Nembutal,50mg/kg i.p.), the transverse process and intervertebral foramina of the L4and L5of CCD rats were exposed unilaterally. Two stainless steel L-shaped rods (0.63mm in diameter and4mm in length) were inserted into the L4and L5foramina respectively, to compress the DRGs. The muscle and skin layers were then sutured.2. Behavioral testingThe behavioral testing was carried out in the ipsilateral hind paw of the animals prior to surgery and on postoperative days4,6,7,14and28. The effect of colchicine on CCD-induced allodynia was tested0.5-8h post injection.Paw withdrawal mechanical threshold (PWMT) was evaluated with calibrated von Frey fibers. 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 for5s. A positive response was noted if the paw was immediately withdrawn. The stimulation of the same force was repeated five times at intervals of5s. If there were no less than three withdrawal responses to any of the five applications, the next lower force fibers was repeated.Thermal allodynia was assessed using the paw withdrawal latencies (PWLs) of the animals in response to radiant heat (BME-410C) in a quiet room. Five values of the PWL were obtained for each rat at5-minute intervals. The intensity of the heat source was pre-calibrated to result in a baseline latency of approximately10s, and the cutoff time was set to20s.3. DRG neuron cultureThe L4and L5ganglia were harvested and incubated with collagenase and trypsin (Sigma-Aldrich, St. Louis, MO), followed by mechanically dissociated into single cells. Cells were plated onto coverslips coated with poly-L-lysine and cultured in Neurobasal medium (Invitrogen, Carlsbad, CA) that had been supplemented with N2, NGF and glutamine (Gibco Invitrogen, Grand Island, NY). The neurons were cultured for4days prior to use.4. HEK293cells culture and transfectionHEK293cells were transfected with TRPV4-GFP plasmids (kindly supported by Professor Yu Xiao from the physiology department of Shandong University) using Lipofectamine2000reagent and following the manufacturer’s protocol (Invitrogen, Carlsbad, CA). The fluorescent cells were used for both recording currents and measuring the levels of TRPV4gene and protein expression.5. Cell viability assayCell viability was measured via3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (the MTT test). Cells were cultured on96-well plates, and8wells were used for each condition. The mean value from the8wells for a given condition was calculated and was taken as one sample. Six samples were taken for each condition. The cell viability of the group that was incubated in the normal medium was taken to represent100%viability.6. Whole-cell current recordingCurrents were digitized at a sampling rate of5kHz and filtered at1kHz for analysis (Axon200B amplifier with pCLAMP software; Molecular Devices, Foster City, CA). The bath solution contained the following compounds (in mM):124NaCl,5KC1,1.2KH2PO4,1.3MgCl2,2.4CaCl2, and26NaHCO3(310mOsm), pH adjusted to7.35with NaOH. The pipette solution contained the following compounds (in mM):140CsCl,2NaCl,3MgCl2,10Hepes, and5EGTA (290mOsm), pH adjusted to7.25with CsOH.7. Real-time quantitative RT-PCR7days after CCD surgery, L4-L5DRGs were investigated from rats in different groups. Fragments of TRPV4or β-actin were then amplified from the cDNA via PCR The2-△△CT method was used to analyze the data according to the relative gene expression levels.8. Western blot analysis7days after CCD surgery, L4-L5DRGs were investigated from rats in different groups. The protein levels were expressed as a ratio of the density of the detected band relative to the density of the β-actin (1:4000, CST, USA).9. Immunofluorescence microscopyDRG neurons were used for immunofluorescence studies. After blocked in0.1%Triton-100and5%(w/v) heatinactivated goat serum in PBS, DRG neurons were incubated with rabbit anti-TRPV4(1:200, Abcam, Cambridge, UK) antibodies overnight at4℃. Unbound antibodies were removed by three PBS washes for15min each at room temperature. Then DRG neurons were incubated with FITC-conjugated goat anti-rabbit IgG (1:100, Zhongshan Goldenbridge, Beijing, China) for1h at room temperature. Cell nuclei were counterstained with DAPI prior to analysis under a confocal laser scanning microscope (LSM710, Zeiss, Jena, Germany).Results1. Effects of microtubules depolymerization by colchicine on CCD-induced allodyniaAfter surgery, 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 at7days post-CCD (P<0.01); we thus performed subsequent experiments at7days post-operation.When compared with intrathecal administration of saline in CCD rats, administration of colchicine (625μg/kg and1250μg/kg) produced a dose-dependent and partial reduction of allodynia in CCD rats (P<0.05); however, the similar result could not be observed in CCD rats injected with colchicine in the dose of312.5μg/kg (all P>0.05). The significant reduction in allodynia was observed at0.5h, peaked at2h, and lasted for about8h post-injection.2. Effects of colchicine on the viability of DRG neurons and HEK293-TRPV4cellsMTT results showed that, the viability of DRG neurons and HEK293-TRPV4cells were not influenced by the application of colchicine.3. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in DRG neuronsAt a holding potential of-60mV. the peak value of the inward current in DRG neurons was recorded as230.55±17.23pA. The peak current values in neurons that were incubated with colchicine at concentrations of0.01μg/ml,0.05μg/ml and0.1μg/ml were201.62±4.72pA,165.25±7.44pA, and128.41±3.75pA, respectively; these values represent colchicine-induced inhibition of12.6%,28.3%and44.3%, respectively. In general, incubation with a solution that contained colchicine suppressed the inward current, and exposure to a0.1μg/ml concentration of colchicine induced a minimal peak current value and a near maximum inhibition of the TRPV4channels (P<0.05). Thus,0.1μg/ml of colchicine was subsequently used as the preferred concentration for the inhibition of the TRPV4channels.4. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in DRG neurons in CCD ratsThe peak values of the4aPDD-induced TRPV4currents in CCD group were also suppressed by colchicine. The4aPDD-induced peak current had a value of310.19±9.17pA, and this current was depressed by26%(to230.10±12.10pA) when the neurons were exposed to colchicine. The currents of the neurons in the presence of colchicine were activated at55.7s, reached a peak at only199.1s after activation (243.6s after activation in the absence of colchicine, P<0.05), and relaxed to a pseudosteady state for the remainder of the recording.5. Effects of microtubules depolymerization by colchicine on the4aPDD-induced TRPV4channel currents in HEK293-TRPV4cellsColchicine administration also resulted in a reduction and advance of the peak current in HEK293-TRPV4cells (412.50±27.15pA and131.6s after activation for HEK293-TRPV4cells;219.53±5.24pA and42.7s after activation for HEK293-TRPV4cells in the presence of colchicine, P<0.05). HEK293-TRPV4cells incubated with colchicine expressed the typical outward rectification characteristic of TRPV4. Nevertheless, the reversal potentials shifted toward more positive voltages.6. Effects of microtubules depolymerization by colchicine on the expression of TRPV4The level of TRPV4protein expression was diminished when animals were injected with colchicine in the doses of625ug/kg and1250μg/kg, and significant decreases were observed at2h and4h post-injections (P<0.05). Pretreatment with colchicine significantly and dose-dependently depressed the expression of TRPV4mRNA with the minimum expression level occurred at2h after exposure to colchicine in the dose of1250μg/kg (P<0.05). The inhibition of colchicine on TRPV4expression was associated with colchicine-induced relief of allodynia in CCD rats.7. Effects of microtubules depolymerization by colchicine on the subcellular localization of TRPV4 Intrathecal administration of colchicine failed to alter the ratio of neurons with pronounced localization of TRPV4. TRPV4immunofluoresecnce was observed more in the plasma membrane than that in the cytoplasm, and this distribution was unaffected by colchicine. The subcellular localization of TRPV4observed in the current study was in agreement with previous findings.ConclusionDepolymerization of microtubules by colchicine could attenuate the allodynia in CCD rats. TRPV4contributes to the relief of allodynia by the depolymerization of microtubules. ObjectiveTo investigate the role of microfilaments in allodynia induced by chronic compression of the dorsal root ganglion; to investigate the effects of microfilaments on TRPV4.Materials and methods1.CCD surgeryRats were randomly divided into CCD groups and sham groups. In CCD rats, under pentobarbital sodium anesthesia (Nembutal,50mg/kg i.p.), the transverse process and intervertebral foramina of the L4and L5vertebrae were exposed unilaterally in accordance with a previously described procedure. Two hollow, stainless steel, L-shaped rods (0.66-mm diameter and4-mm length), connected to silicon tubing (0.51mm ID,0.94OD,30-40mm length) filled with heparin, were inserted into L4and L5foramen respectively, to compress the DRG. The other end of the tubing was sealed, except when injecting drugs. The muscle and skin layers were then sutured. Sham surgery involved identical surgical procedures to those described but without insertion of the rods.2. Behavioral testingAll behavioral tests were carried out in a quiet room and conducted under blind conditions. Behavioral tests were conducted on the7th day post-surgery and1,2,4,6,8, and24h after injection of chemicals or saline. Chemicals and saline were all injected by the silicon tubing.3. DRG neurons cultureThe L4and L5ganglia were removed from the operated side of each animal on7days after surgery. The neurons were cultured according to the methods in chapter I.4. Cell viability assayThe viability of DRG neurons were tested according to the method in chapter I.5. Whole-cell current recordingThe whole-cell current of TRPV4were tested according to the method in chapter I.6. Immunofluorescence microscopyThe localization of TRPV4is tested according to the method in chapter I.Results1. Effects of microfilaments dynamics on the allodynia in CCD ratsAll rats walked normally after the CCD surgery, indicating that CCD surgery did not injury the motor behavior.Both of two disrupting agents (CB and CD) resulted in attenuation of CCD-induced mechanical and thermal allodynia (n=9in each group, both P<0.01). Compared with the administration of saline in CCD rats, administration of cytochalasin B (200μg and300μg) produced a dose-dependent and partial reduction of allodynia in CCD rats. However, similar result could not be observed in CCD rats injected with cytochalasin B in the dose of100ug (P>0.05, n=9in each group). The significant reduction in allodynia was observed at1h, peaked at2h, and lasted less than24h post-injection.Phalloidin didn’t affect the mechanical and thermal allodynia induced by CCD surgery (all P>0.05, n=9in each group). Phalloidin was injected to investigate whether it could prevent cytochalasin B-induced loss of allodynia in CCD rats, confirming that the inhibition of CCD-induced allodynia by anti-microfilaments agents was due to microfilaments disruption. Injection of phalloidin1h preceding administration of cytochalasin B significantly inhibited the cytochalasin B-induced attenuation of allodynia in CCD rats (n=9).2. Effects of microfilaments dynamics on TRPV4currents in DRG neurons in CCD ratsSimilar to CCD-induced allodynia in vivo, whether the potentiation of TRPV4in CCD rats was dependent on intact actin filaments was tested. The peak inward current was significantly higher in CCD group than in sham group (n=12in CCD group, n=10in sham group, P<0.05). The effects of dynamic changes of microfilaments on TRPV4-mediated channel activity were tested next.Exposure of cultured compressed DRG neurons to disruptors (cytochalasin B and cytochalasin D, both10-4M) for2h prior to the patch clamp significantly inhibited the potentiation of TRPV4(the peak value is310.19±9.17pA in CCD group,245.48±12.12pA in DRG neurons exposure to cytochalasin B, and237.81±10.17pA in DRG neurons exposure to cytochalasin D, n=12in each group, P<0.05), which are consistent with the attenuation of the effects of disruptors on CCD-induced allodynia. DRG neurons incubated with disruptors expressed the typical outward rectification characteristic of TRPV4. Nevertheless, the reversal potentials shifted toward more positive voltages, which are in accordance with the inhibition of TRPV4currents by disruption of microfilaments. The time course was analyzed to further confirm the effect of disruption of microfilaments on TRPV4channel opening. A delayed peak time was observed in DRG neurons from CCD rats (243.60±20.00s for CCD group and200.37±15.40s for sham group, P<0.05). Disruption of microfilaments induced an advance of the peak current in DRG neurons of CCD rats (123.44±10.10s for DRG neurons exposure to cytochalasin B, and193.76±12.40s in DRG neurons exposure to cytochalasin D, P<0.05).Phalloidin (10-5M) resulted in a reduction and delay of TRPV4current (80.87±5.42pA and311.72±14.56s in DRG neuron exposure to phalloidin, n=12, P<0.01), which is not consistent with the effect of phalloidin on CCD-induced allodynia. In association with the inhibition of TRPV4currents, the reversal potentials shifted toward more positive voltages. MTT tests were carried out to exclude the effects of chemicals (cytochalasin B, cytochalasin D and phalloidin) on the cell viabilities of DRG neurons. The results showed that both disruptors and stabilizer of microfilaments had no significant influence on the cell viabilities of DRG neurons (P>0.05).3. Effects of microfilaments dynamics on the subcellular localization of TRPV4in DRG neurons in CCD ratsResults demonstrated that more TRPV4immunofluoresecnce was observed in the plasma membrane than the cytoplasm in all groups. Cytochalasin B (10-4M) resulted in an apparent decrease of the plasma membrane association of TRPV4(83.62±2.14%in CCD group and69.63±3.41%in cytochalasin B group, P<0.05). Meanwhile, phalloidin (10-5M) also leaded to a decrease of plasma membrane association of TRPV4(54.24±2.44%, P<0.05).ConclusionsThis study demonstrates that microfilaments play an important role in allodynia induced by chronic compression of the dorsal root ganglion and TRPV4contributes to the attenuation of allodynia by depolymerization of microfilaments.

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