Role In Development Of Neuropathic Pain Research Articles

The Role of miRNAs in Neuropathic Pain

Neuropathic pain is a debilitating condition affecting around 8% of the adult population in the UK. The pathophysiology is complex and involves a wide range of processes, including alteration of neuronal excitability and synaptic transmission, dysregulated intracellular signalling and activation of pro-inflammatory immune and glial cells. In the past 15 years, multiple miRNAs-small non-coding RNA-have emerged as regulators of neuropathic pain development. They act by binding to target mRNAs and preventing the translation into proteins. Due to their short sequence (around 22 nucleotides in length), they can have hundreds of targets and regulate several pathways. Several studies on animal models have highlighted numerous miRNAs that play a role in neuropathic pain development at various stages of the nociceptive pathways, including neuronal excitability, synaptic transmission, intracellular signalling and communication with non-neuronal cells. Studies on animal models do not always translate in the clinic; fewer studies on miRNAs have been performed involving human subjects with neuropathic pain, with differing results depending on the specific aetiology underlying neuropathic pain. Further studies using human tissue and liquid samples (serum, plasma, saliva) will help highlight miRNAs that are relevant to neuropathic pain diagnosis or treatment, as biomarkers or potential drug targets.

Simultaneous Determination of Substance P and CGRP in Rat Brainstem Tissue Using LC-ESI-MS/MS Method

Background: Substance P and CGRP are neuropeptides that belong to the thachykinin and calcitonin family, respectively. They play an important role in neuropathic pain development and regulation. Methods: In this study, simultaneous analysis of Substance P and CGRP in rat brainstem tissue was conducted by using LC-ESI-MS/MS method. A gradient mobile phase was used with a Zorbax C8 150x2.1 mm, a particle size of 3.5 µm narrow-bore column. Mobile phases consist of 0.2% formic acid in water (A) and 0.2% formic acid in acetonitrile (v/v) (B). Firstly, mass spectrometry parameters were optimized. After achieving chromatographic separation, the method has been validated and applied to rat brainstem samples. Results: SP and CGRP were eluted at 8.7 min and 10.1 min, respectively. Mass spectrum of neuropeptides was obtained in positive polarity and ion transitions of Substance P and CGRP were monitored at m/z 674.4→[600+254.4] and 952.2→1214.200, respectively. Linearity range for quantification was 5.85-438.52 ng/mL for Substance P and 0.21-155.28 ng/mL for CGRP. The limit of quantification was 5.85 ng/mL for Substance P and 0.21 ng/mL for CGRP. According to tissue samples analysis (n=6), basal endogen concentrations of Substance P and CGRP in brainstem tissue were calculated as 260.57±11.24 ng/g and 22.53±0.75 ng/g respectively. Conclusion: A novel LC-ESI-MS/MS method was successfully developed and validated according to the bioanalytical validation guideline published by the EMA including precision, accuracy, specificity and linearity. Acceptable results were achieved. Basal endogen concentrations of Substance P and CGRP in brainstem tissue were determined. Keywords: Substance P, CGRP, LC-ESI-MS/MS, rat brainstem tissue, neuropeptide, determination.

MiR-124-3p attenuates neuropathic pain induced by chronic sciatic nerve injury in rats via targeting EZH2.

Emerging evidence has suggested that microRNAs play a critical role in neuropathic pain development. However, the biological role of miRNAs in regulating neuropathic pain remains barely known. In our present study, we found that miR-124-3p was significantly downregulated in rats after chronic sciatic nerve injury (CCI). In addition, it was showed that overexpression of miR-124-3p obviously repressed mechanical allodynia and heat hyperalgesia. Meanwhile, it has been reported that neuroinflammation can contribute a lot to neuropathic pain progression. Here, we found that inflammatory cytokine (IL-6, IL-1β, and TNF-⍺) protein expression in rats after CCI greatly increased and miR-124-3p mimics depressed inflammation cytokine levels. Consistently, miR-124-3p alleviated inflammation production in lipopolysaccharide-incubated spinal microglial cells. Bioinformatics analysis revealed that EZH2 acted as a direct target of miR-124-3p, which participated in the miR-124-3p-modulated effects on neuropathic pain development and neuroinflammation. We observed that miR-124-3p was able to promote neuroinflammation and neuropathic pain through targeting EZH2. The direct correlation between them was validated in our current study using dual-luciferase reporter assays. Subsequently, it was manifested that EZH2 abrogated the inhibitory role of miR-124-3p on neuropathic pain progression in CCI rats. Taken these together, our findings highlighted a novel contribution of miR-124-3p to neuropathic pain and indicated the possibilities for developing novel therapeutic options for neuropathic pain.

Blockade of P2X4 Receptors Inhibits Neuropathic Pain-Related Behavior by Preventing MMP-9 Activation and, Consequently, Pronociceptive Interleukin Release in a Rat Model

Neuropathic pain is still an extremely important problem in today’s medicine because opioids, which are commonly used to reduce pain, have limited efficacy in this type of pathology. Therefore, complementary therapy is needed. Our experiments were performed in rats to evaluate the contribution of the purinergic system, especially P2X4 receptor (P2X4R), in the modulation of glia activation and, consequently, the levels of nociceptive interleukins after chronic constriction injury (CCI) of the right sciatic nerve, a rat model of neuropathic pain. Moreover, we studied how intrathecal (ith.) injection of a P2X4R antagonist Tricarbonyldichlororuthenium (II) dimer (CORM-2) modulates nociceptive transmission and opioid effectiveness in the CCI model. Our results demonstrate that repeated ith. administration of CORM-2 once daily (20 μg/5 μl, 16 and 1 h before CCI and then daily) for eight consecutive days significantly reduced pain-related behavior and activation of both spinal microglia and/or astroglia induced by CCI. Moreover, even a single administration of CORM-2 on day 7 after CCI attenuated mechanical and thermal hypersensitivity as efficiently as morphine and buprenorphine. In addition, using Western blot, we have shown that repeated ith. administration of CORM-2 lowers the CCI-elevated level of MMP-9 and pronociceptive interleukins (IL-1β, IL-18, IL-6) in the dorsal L4-L6 spinal cord and/or DRG. Furthermore, in parallel, CORM-2 upregulates spinal IL-1Ra; however, it does not influence other antinociceptive factors, IL-10 and IL-18BP. Additionally, based on our biochemical results, we hypothesize that p38MAPK, ERK1/2 and PI3K/Akt but not the NLRP3/Caspase-1 pathway are partly involved in the CORM-2 analgesic effects in rat neuropathic pain. Our data provide new evidence that P2X4R may indeed play a significant role in neuropathic pain development by modulating neuroimmune interactions in the spinal cord and DRG, suggesting that its blockade may have potential therapeutic utility.

Role of dorsal root ganglion K2p1.1 in peripheral nerve injury-induced neuropathic pain.

Peripheral nerve injury-caused hyperexcitability and abnormal ectopic discharges in the primary sensory neurons of dorsal root ganglion (DRG) play a key role in neuropathic pain development and maintenance. The two-pore domain background potassium (K2P) channels have been identified as key determinants of the resting membrane potential and neuronal excitability. However, whether K2P channels contribute to neuropathic pain is still elusive. We reported here that K2P1.1, the first identified mammalian K2P channel, was highly expressed in mouse DRG and distributed in small-, medium-, and large-sized DRG neurons. Unilateral lumbar (L) 4 spinal nerve ligation led to a significant and time-dependent reduction of K2P1.1 mRNA and protein in the ipsilateral L4 DRG, but not in the contralateral L4 or ipsilateral L3 DRG. Rescuing this reduction through microinjection of adeno-associated virus-DJ expressing full-length K2P1.1 mRNA into the ipsilateral L4 DRG blocked spinal nerve ligation-induced mechanical, thermal, and cold pain hypersensitivities during the development and maintenance periods. This DRG viral microinjection did not affect acute pain and locomotor function. Our findings suggest that K2P1.1 participates in neuropathic pain development and maintenance and may be a potential target in the management of this disorder.

An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve.

Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain.

Treatment with a carbon monoxide-releasing molecule (CORM-2) inhibits neuropathic pain and enhances opioid effectiveness in rats

Experiments were conducted to evaluate the contribution of P2X4 receptors to the modulation of neuropathy and their ability to amplify opioid effectiveness. The study consisted of behavioral and biochemical analysis of the effect of a carbon monoxide donor - CORM-2, on the development of neuropathic pain in a rat model of chronic constriction injury (CCI) to the sciatic nerve. Here, we exam if chronic intraperitoneal or intrathecal administration of CORM-2 influences CCI-induced allodynia and hyperalgesia. In parallel, changes of spinal microglial and/or astroglial activation were studied. CORM-2 was administered intrathecally [20μg/5μl] or intraperitoneally [10mg/kg]. Here, we report that intraperitoneal or intrathecal chronic administration of the carbon monoxide donor CORM-2 significantly reduced the allodynia/hyperalgesia induced by CCI, with a parallel reduction of spinal microglial and/or astroglial activation. Furthermore, even a single intraperitoneal administration of CORM-2 had antiallodynic potency and moreover, increased morphine/buprenorphine analgesia compared to the effects of these drugs alone, completely eliminating the neuropathic pain symptoms. When CORM-2 was administered for 7 consecutive days, the antinociceptive effect of CORM-2 after CCI was stronger on day 7 than on day 2, which indicates that this effect built up over time. We are the first to demonstrate that even a single intraperitoneal injection of CORM-2 potentiates the antihyperalgesic and antiallodynic properties of morphine/buprenorphine in a CCI rat model. Our data suggest that P2X4 receptors play a significant role in neuropathic pain development, suggesting that their blockade may have potential therapeutic utility.

Ultra-low-dose naloxone enhances the antinociceptive effect of morphine in PTX-treated rats: Regulation on global histone methylation

ObjectiveEpigenetic reprogramming may have a possible role in neuropathic pain development; the present study examined the global patterns of lysine histone modification. In this serial study we analyzed the levels of histone 3 lysine 4 monomethylation, histone 3 lysine 4 dimethylation, and histone 3 lysine 9 trimethylation in pertussis toxin (PTX)-induced thermal hyperalgesic rat spinal cords. MethodsMale Wistar rats implanted with an intrathecal catheter received a single intrathecal PTX (1μg in 5μl saline) injection. Four days later, they were randomly assigned to receive either a single injection of saline, or ultra-low-dose naloxone (15ng in 5μl saline), followed by morphine (10μg in 5μl saline) injection 30 minutes later. ResultsThe results showed that PTX injection induced thermal hyperalgesia and significant increase of global histone methylation in the spinal cords. Intrathecal morphine alone did not affect the thermal hyperalgesia and global histone methylation. In contrast, intrathecal administration of ultra-low-dose naloxone plus morphine significantly attenuated the PTX-induced thermal hyperalgesia and down-regulated the global histone methylation. ConclusionThe results suggest that ultra-low-dose naloxone might be clinical valuable for neuropathic pain management via regulating global histone modification.

Spinal SIRPα1-SHP2 interaction regulates spinal nerve ligation-induced neuropathic pain via PSD-95-dependent NR2B activation in rats

The fact that neuropathic pain mechanisms are not well understood is a major impediment in the development of effective clinical treatments. We examined whether the interaction between signal regulatory protein alpha 1 (SIRPα1) and Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2), and the downstream spinal SHP2/postsynaptic density 95 (PSD-95)/N-methyl-d-aspartate receptor NR2B subunit signaling cascade play a role in neuropathic pain. Following spinal nerve ligation (L5), we assessed tactile allodynia using the von Frey filament test and analyzed dorsal horn samples (L4-5) by Western blotting, reverse transcription polymerase chain reaction, coimmunoprecipitation, and immunofluorescence. Nerve ligation induced allodynia, SIRPα1, SHP2, phosphorylated SHP2 (pSHP2), and phosphorylated NR2B (pNR2B) expression, and SHP2-PSD-95, pSHP2-PSD-95, PSD-95-NR2B, and PSD-95-pNR2B coimmunoprecipitation in the ipsilateral dorsal horn. In allodynic rats, injury-induced SHP2 immunoreactivity was localized in the ipsilateral dorsal horn neurons and coincident with PSD-95 and NR2B immunoreactivity. SIRPα1 silencing using small interfering RNA (siRNA; 1, 3, or 5μg/rat for 7days) prevented injury-induced allodynia and the associated changes in protein expression, phosphorylation, and coimmunoprecipitation. Intrathecal administration of NSC-87877 (an SHP2 antagonist; 1, 10, or 100μM/rat) and SIRPα1-neutralizing antibodies (1, 10, or 30μg/rat) suppressed spinal nerve ligation-induced allodynia, spinal SHP2 and NR2B phosphorylation, and SHP2/phosphorylated SHP2-PSD-95 and PSD-95-NR2B/phosphorylated NR2B coprecipitation. SHP2 siRNA led to similar effects as the NSC-87877 and SIRPα1 antibody treatments, except it prevented the allodynia-associated spinal SHP2 expression. In conclusion, our results suggest that a spinal SIRPα1-SHP2 interaction exists that subsequently triggers SHP2/PSD-95/NR2B signaling, thereby playing a role in neuropathic pain development.

Modulation of spinal cord synaptic activity by tumor necrosis factor α in a model of peripheral neuropathy

BackgroundThe cytokine tumor necrosis factor α (TNFα) is an established pain modulator in both the peripheral and central nervous systems. Modulation of nociceptive synaptic transmission in the spinal cord dorsal horn (DH) is thought to be involved in the development and maintenance of several pathological pain states. Increased levels of TNFα and its receptors (TNFR) in dorsal root ganglion (DRG) cells and in the spinal cord DH have been shown to play an essential role in neuropathic pain processing. In the present experiments the effect of TNFα incubation on modulation of primary afferent synaptic activity was investigated in a model of peripheral neuropathy.MethodsSpontaneous and miniature excitatory postsynaptic currents (sEPSC and mEPSCs) were recorded in superficial DH neurons in acute spinal cord slices prepared from animals 5 days after sciatic nerve transection and in controls.ResultsIn slices after axotomy the sEPSC frequency was 2.8 ± 0.8 Hz, while neurons recorded from slices after TNFα incubation had significantly higher sEPSC frequency (7.9 ± 2.2 Hz). The effect of TNFα treatment was smaller in the slices from the control animals, where sEPSC frequency was 1.2 ± 0.2 Hz in slices without and 2.0 ± 0.5 Hz with TNFα incubation. Tetrodotoxin (TTX) application in slices from axotomized animals and after TNFα incubation decreased the mEPSC frequency to only 37.4 ± 6.9% of the sEPSC frequency. This decrease was significantly higher than in the slices without the TNFα treatment (64.4 ± 6.4%). TTX application in the control slices reduced the sEPSC frequency to about 80% in both TNFα untreated and treated slices. Application of low concentration TRPV1 receptors endogenous agonist N-oleoyldopamine (OLDA, 0.2 μM) in slices after axotomy induced a significant increase in mEPSC frequency (175.9 ± 17.3%), similar to the group with TNFα pretreatment (158.1 ± 19.5%).ConclusionsOur results indicate that TNFα may enhance spontaneous transmitter release from primary afferent fibres in the spinal cord DH by modulation of TTX-sensitive sodium channels following sciatic nerve transection. This nerve injury also leads to enhanced sensitivity of presynaptic TRPV1 receptors to endogenous agonist. Modulation of presynaptic receptor activity on primary sensory terminals by TNFα may play an important role in neuropathic pain development.

Identification, characterization and quantification of specific neuropeptides in rat spinal cord by liquid chromatography electrospray quadrupole ion trap mass spectrometry

Substance P and CGRP play a central role in neuropathic pain development and maintenance. Additionally, dynorphin A is an endogenous ligand of opioid receptors implicated in the modulation of neurotransmitters including neuropeptides, such as substance P and CGRP. This manuscript proposes a method to characterize, identify and quantify substance P, CGRP and dynorphin A in rat spinal cord by HPLC-ESI/MS/MS. Rat spinal cords were collected and homogenized into a TFA solution. Samples were chromatographed using a microbore C(8) 100 x 1 mm column and a 19 min linear gradient (0:100 --> 40:60; ACN:0.2% formic acid in water) at a flow rate of 75 microL/min for a total run time of 32 min. The peptides were identified in rat spinal cord based on full-scan MS/MS spectra. Substance P, CGRP and dynorphin A were predominantly identified by the presence of specific b CID fragments. Extracted ion chromatogram (XIC) suggested selected mass transitions of 674 --> [600 + 254], 952 --> [1215 + 963] and 717 --> [944 + 630] for substance P, CGRP and dynorphin A can be used for isolation and quantitative analysis. A linear regression (weighted 1/x) was used and coefficients of correlations (r) ranging from 0.990 to 0.999 were observed. The precision (%CV) and accuracy (%NOM) observed were 10.9-14.4% and 8.9-14.2%, 8.8-13.0% and 91.0-110.2% and 97.2-107.3% and 91.8-97.3% for substance P, CGRP and dynorphin A respectively. Following the analysis of rat spinal cords, the mean endogenous concentrations were 110.7, 2541 and 779.4 pmol/g for substance P, CGRP and dynorphin A respectively. The results obtained show that the method provides adequate figures of merit to support targeted peptidomic studies aimed to determine neuropeptide regulation in animal neuropathic and chronic pain models.

BDNF contributes to animal model neuropathic pain after peripheral nerve transection

The outcome of peripheral nerve injury is often impaired by neuropathic pain, which is resistant to most analgesics and presents a serious clinical problem. The mechanisms underlying post-traumatic neuropathic pain remain unclear, but they are likely associated with the regeneration processes. Brain-derived neurotrophic factor (BDNF) is known to enhance peripheral nerve regeneration and is also considered to be an endogenous modulator of nociceptive responses following spinal cord lesion. The aim of this work was to examine the local effect of BDNF in a neuropathic pain model. Sciatic nerves of adult male rats were transected and supplied with connective tissue chambers filled with (1) fibrin only, (2) fibrin with BDNF, or (3) fibrin with antibodies against BDNF. In control animals the nerve was transected and no chamber was applied. During follow-up, autotomy behavior was assessed. Seven weeks after the operation, the number of surviving and regenerating neurons in dorsal root ganglia was counted and the neuroma incidence was examined. We found that local inactivation of BDNF decreased the incidence as well as severity of autotomy and neuroma formation, but did not influence neuron regeneration into the chambers. These results indicate that BDNF plays a locally crucial role in neuropathic pain development after peripheral nerve injury.

Spinal dorsal horn calcium channel alpha2delta-1 subunit upregulation contributes to peripheral nerve injury-induced tactile allodynia.

Peripheral nerve injury induces upregulation of the calcium channel alpha2delta-1 structural subunit in dorsal root ganglia (DRG) and dorsal spinal cord of spinal nerve-ligated rats with neuropathic pain, suggesting a role of the calcium channel alpha2delta-1 subunit in central sensitization. To investigate whether spinal dorsal horn alpha2delta-1 subunit upregulation derives from increased DRG alpha2delta-1 subunit and plays a causal role in neuropathic pain development, we examined spinal dorsal hornalpha2delta-1 subunit expression with or without dorsal rhizotomy in spinal nerve-ligated rats and its correlation with tactile allodynia, a neuropathic pain state defined as reduced thresholds to non-noxious tactile stimulation. We also examined the effects of intrathecal alpha2delta-1 antisense oligonucleotides on alpha2delta-1 subunit expression and neuropathic allodynia in the nerve-ligated rats. Our data indicated that spinal nerve injury resulted in time-dependentalpha2delta-1 subunit upregulation in the spinal dorsal horn that correlated temporally with neuropathic allodynia development and maintenance. Dorsal rhizotomy diminished basal level expression and blocked injury-induced expression of the spinal dorsal hornalpha2delta-1 subunit and reversed injury-induced tactile allodynia. In addition, intrathecal alpha2delta-1 antisense oligonucleotides blocked injury-induced dorsal horn alpha2delta-1 subunit upregulation and diminished tactile allodynia. These findings indicate that alpha2delta-1 subunit basal expression occurs presynaptically and postsynaptically in spinal dorsal horn. Nerve injury induces mainly presynaptic alpha2delta-1 subunit expression that derives from increased alpha2delta-1 subunit in injured DRG neurons. Thus, changes in presynaptic alpha2delta-1 subunit expression contribute to injury-induced spinal neuroplasticity and central sensitization that underlies neuropathic pain development and maintenance.