Dorsal Horn Of Spinal Cord Research Articles

Increased Expression of Fibronectin Leucine-Rich Transmembrane Protein 3 in the Dorsal Root Ganglion Induces Neuropathic Pain in Rats.

Neuropathic pain is a chronic condition that occurs frequently after nerve injury and induces hypersensitivity or allodynia characterized by aberrant neuronal excitability in the spinal cord dorsal horn. Fibronectin leucine-rich transmembrane protein 3 (FLRT3) is a modulator of neurite outgrowth, axon pathfinding, and cell adhesion, which is upregulated in the dorsal horn following peripheral nerve injury. However, the function of FLRT3 in adults remains unknown. Therefore, we aimed to investigate the involvement of spinal FLRT3 in neuropathic pain using rodent models. In the dorsal horns of male rats, FLRT3 protein levels increased at day 4 after peripheral nerve injury. In the DRG, FLRT3 was expressed in activating transcription factor 3-positive, injured sensory neurons. Peripheral nerve injury stimulated Flrt3 transcription in the DRG but not in the spinal cord. Intrathecal administration of FLRT3 protein to naive rats induced mechanical allodynia and GluN2B phosphorylation in the spinal cord. DRG-specific FLRT3 overexpression using adeno-associated virus also produced mechanical allodynia. Conversely, a function-blocking FLRT3 antibody attenuated mechanical allodynia after partial sciatic nerve ligation. Therefore, FLRT3 derived from injured DRG neurons increases dorsal horn excitability and induces mechanical allodynia.SIGNIFICANCE STATEMENT Neuropathic pain occurs frequently after nerve injury and is associated with abnormal neuronal excitability in the spinal cord. Fibronectin leucine-rich transmembrane protein 3 (FLRT3) regulates neurite outgrowth and cell adhesion. Here, nerve injury increased FLRT3 protein levels in the spinal cord dorsal root, despite the fact that Flrt3 transcripts were only induced in the DRG. FLRT3 protein injection into the rat spinal cord induced mechanical hypersensitivity, as did virus-mediated FLRT3 overexpression in DRG. Conversely, FLRT3 inhibition with antibodies attenuated mechanically induced pain after nerve damage. These findings suggest that FLRT3 is produced by injured DRG neurons and increases neuronal excitability in the dorsal horn, leading to pain sensitization. Neuropathic pain induction is a novel function of FLRT3.

Role of DLC2 and RhoA/ROCK pathway in formalin induced inflammatory pain in mice

Deficiency of deleted in liver cancer 2 (DLC2), a novel domain to inhibit RhoA activity, plays an important role in inflammatory pain. However, the underlying mechanisms remain unclear. This study investigated the role of DLC2 and its downstream cascade of RhoA/ROCK in formalin-induced inflammatory pain using DLC2-knockout (DLC2−/−) mice and compared them with DLC2 wild-type (DLC2+/+) mice. Mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filament aesthesiometer and Hargreaves test, respectively. The spinal cord dorsal horn (L3-L5) was selected for molecular and cellular identification by Western blot and immunofluorescence. DLC2−/− mice showed increased mechanical allodynia and thermal hyperalgesia. Expression of ROCK1, ROCK2 and IL-1β was significantly higher in DLC2−/− mice. Intrathecal administration of RhoA inhibitor (C3 exoenzyme) or ROCK inhibitor (Y27632) significantly attenuated formalin-induced inflammatory hyperalgesia in DLC2−/− mice. ROCK2 and IL-1β expression were reduced by C3 exoenzyme or Y27632. Spinal p38 activation was also inhibited by C3 exoenzyme or Y27632. Double-labelling immunofluorescence demonstrated co-localization of DLC2 with spinal dorsal microglia. The number of activated microglia in the spinal dorsal horn was significantly higher in DLC2−/− mice, but was reduced by Y27632. These findings indicate that DLC2 deficiency increased formalin-induced inflammatory hyperalgesia through regulating RhoA/ROCK2, and IL-1β may be a downstream effector. Our results also suggest that RhoA/ROCK enhanced p38 activation plays an important role in formalin-induced inflammatory pain. The finding that DLC2 attenuated inflammatory pain through inhibiting RhoA/ROCK2 suggests that the DLC2/RhoA/ROCK2/p38/IL-β pathway may be a potential therapeutic target to reduce inflammatory pain.

Experimental study of the effect of the sciatic nerve elongation on pain in rats

To investigate the effect of the sciatic nerve elongation on pain in rats. Thirty-six adult male Wistar rats of SPF grade, weighing 250-300 g. Eighteen of them were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A), nerve no-elongation group (group B), and nerve ligation group (group C). The model of 10-mm sciatic nerve defect was established in all 3 groups. The sciatic nerve was extended at a speed of 1 mm/d for 14 days in group A. The group B was only installed with external fixation. The nerve stumps were ligated in the group C. At 3, 7, 10, and 14 days after operation, the foot injury was evaluated by the autotomy scoring scale. At 14 days after operation, the dorsal root ganglia (DRG) of L 4-S 1 spinal cord of rats in each group was observed by tumor necrosis factor α (TNF-α) immunohistochemical staining, and the primary antibodies were replaced by pure serum as negative control group. Another 18 rats were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A1), nerve no-elongation group (group B1), positive control group (group C1). In groups A1 and B1, the 10-mm long sciatic nerve defect model was established by the same method as groups A and B, and then fixed with external fixation. Nerve elongation was done or not done without anesthesia at 3 days after operation. In group C1, no modeling was done and 20 μL 2.5% formaldehyde was injected into the toes. After 90 minutes, the dorsal horn of spinal cord of L 4-S 1 segment of rats was cutting for c-Fos immunohistochemical staining and the number of positive cells was counted. Primary antibodies were replaced with pure serum as negative control group. The autotomy scores of rats in groups B and C gradually increased postoperatively, and group A remained stable at 0.25±0.50. The scores of group C were significantly higher than those of group A and group B at each time point postoperatively ( P<0.05). The scores of group A were significantly lower than those of group B at 10 and 14 days postoperatively ( P<0.05). TNF-α immunohistochemical staining showed that the TNF-α expression in group A was weak, slightly positive (+/-); in group B was positive (+); in group C was strongly positive (++); and the negative control group had no TNF-α expression (-). c-Fos immunohistochemical staining showed that the c-Fos expressions in groups A1 and B1 were weak positive, in group C1 was strong positive, and negative control group had no c-Fos positive expression. The number of c-Fos positive cells in groups A1, B1, C1, and negative control group were (21.5±6.6), (19.3±8.1), (95.6±7.4), and 0 cells/field, respectively, and group C1 was significantly higher than groups A1 and B1 ( P<0.05), there was no significant difference between group A1 and group B1 ( P>0.05). Nerve elongation does not cause obvious pain neither during the operation of elongation nor throughout the whole elongation.

Phα1β Spider Toxin Reverses Glial Structural Plasticity Upon Peripheral Inflammation.

The incoming signals from injured sensory neurons upon peripheral inflammation are processed in the dorsal horn of spinal cord, where glial cells accumulate and play a critical role in initiating allodynia (increased pain in response to light-touch). However, how painful stimuli in the periphery engage glial reactivity in the spinal cord remains unclear. Here, we found that a hind paw inflammation induced by CFA produces robust morphological changes in spinal astrocytes and microglia compatible with the reactive phenotype. Strikingly, we discovered that a single intrathecal injection with venom peptides that inhibit calcium channels reversed all the glial pathological features of the peripheral inflammation. These effects were more apparent in rats treated with the Phα1β spider toxin (non-specific calcium channel antagonist) than ω-MVIIA cone snail toxin (selective N-type calcium channel antagonist). These data reveal for the first time a venom peptide acting on glial structural remodeling in vivo. We, therefore, suggest that calcium-dependent plasticity is an essential trigger for glial cells to initiate reactivity, which may represent a new target for the antinociceptive effects of Phα1β and ω-MVIIA toxins in inflammatory pain conditions.

Role of spinal glial cells in excitability of wide dynamic range neurons and the development of neuropathic pain with the L5 spinal nerve transection in the rats: Behavioral and electrophysiological study

The activation of glial cells affects the neuronal excitability in the spinal cord. Therefore, in this study, we tried to find out the modulatory role of spinal glial cells in the excitability of wide dynamic range (WDR) neurons, induction of the long-term potentiation (LTP) and development of neuropathic pain by L5 spinal nerve transection model in the rats. Forty-eight adult male Wistar rats were used to measure the paw withdrawal threshold to mechanical stimuli and also, to carry out the spinal extracellular single unit recording experiments. In these experiments, spinal nerve ligation (SNL) and a daily injection of propentofylline (1 mg/kg, ip) as a glial cell inhibitor agent, 1 h following nerve ligation during 7-day post-SNL period, were performed. Our findings showed that the mechanical allodynia, and synaptically-evoked firing were caused LTP in the Aδ-fiber, C-fiber and lesser in the Aβ-fiber after high frequency stimulation. Daily injection of propentofylline considerably decreased LTP induction in the Aδ- and C-fibers (P < .001). It was concluded that glial cell activation mediates LTP induction in the spinal cord following peripheral nerve injury. It seems that pain modulatory role of glial cells is partly parallel to changes in neural excitability of the WDR neurons in the dorsal horn of spinal cord.

Electroacupuncture Stimulation Alleviates CFA-Induced Inflammatory Pain Via Suppressing P2X3 Expression.

Chronic inflammatory pain is one of the most common complaints that seriously affects patients’ quality of life. Previous studies have demonstrated that the analgesic effect of electroacupuncture (EA) stimulation on inflammatory pain is related to its frequency. In this study, we focused on whether the analgesic effects of EA are related to the period of stimulation. Purinergic receptor P2X3 (P2X3) is involved in the pathological process underlying chronic inflammatory pain and neuropathic pain. We hypothesized that 100 Hz EA stimulation alleviated Freund’s complete adjuvant (CFA) induced inflammatory pain via regulating P2X3 expression in the dorsal root ganglion (DRG) and/or spinal cord dorsal horn (SCDH). We also assumed that the analgesic effect of EA might be related to the period of stimulation. We found that both short-term (three day) and long-term (14 day) 100 Hz EA stimulation effectively increased the paw withdrawal threshold (PWT) and reversed the elevation of P2X3 in the DRG and SCDH of CFA rats. However, the analgesic effects of 100 Hz EA were not dependent on the period of stimulation. Moreover, P2X3 inhibition or activation may contribute to or attenuate the analgesic effects of 100 Hz EA on CFA-induced inflammatory pain. This result indicated that EA reduced pain hypersensitivity through P2X3 modulation.

Ryanodine Receptor to Mitochondrial Reactive Oxygen Species Pathway Plays an Important Role in Chronic Human Immunodeficiency Virus gp120MN-Induced Neuropathic Pain in Rats.

Chronic pain is one of the most common complaints in patients with human immunodeficiency virus (HIV)-associated sensory neuropathy. Ryanodine receptor (RyR) and mitochondrial oxidative stress are involved in neuropathic pain induced by nerve injury. Here, we investigated the role of RyR and mitochondrial superoxide in neuropathic pain induced by repeated intrathecal HIV glycoprotein 120 (gp120) injection. Recombinant HIV glycoprotein gp120MN was intrathecally administered to induce neuropathic pain. Mechanical threshold was tested using von Frey filaments. Peripheral nerve fiber was assessed by the quantification of the intraepidermal nerve fiber density in the skin of the hindpaw. The expression of spinal RyR was examined using Western blots. Colocalization of RyR with neuronal nuclei (NeuN; neuron marker), glial fibrillary acidic protein (GFAP; astrocyte marker), or ionizing calcium-binding adaptor molecule 1 (Iba1; microglia marker) in the spinal cord was examined using immunohistochemistry. MitoSox-positive profiles (a mitochondrial-targeted fluorescent superoxide indicator) were examined. The antiallodynic effects of intrathecal administration of RyR antagonist, dantrolene (a clinical drug for malignant hyperthermia management), or selective mitochondrial superoxide scavenger, Mito-Tempol, were evaluated in the model. We found that repeated but not single intrathecal injection of recombinant protein gp120 induced persistent mechanical allodynia. Intraepidermal nerve fibers in repeated gp120 group was lower than that in sham at 2 weeks, and the difference in means (95% confidence interval) was 8.495 (4.79-12.20), P = .0014. Repeated gp120 increased expression of RyR, and the difference in means (95% confidence interval) was 1.50 (0.504-2.495), P = .007. Repeated gp120 also increased mitochondrial superoxide cell number in the spinal cord, and the difference in means (95% confidence interval) was 6.99 (5.99-8.00), P < .0001. Inhibition of spinal RyR or selective mitochondrial superoxide scavenger dose dependently reduced mechanical allodynia induced by repeated gp120 injection. RyR and mitochondrial superoxide were colocalized in the neuron, but not glia. Intrathecal injection of RyR inhibitor lowered mitochondrial superoxide in the spinal cord dorsal horn in the gp120 neuropathic pain model. These data suggest that repeated intrathecal HIV gp120 injection induced an acute to chronic pain translation in rats, and that neuronal RyR and mitochondrial superoxide in the spinal cord dorsal horn played an important role in the HIV neuropathic pain model. The current results provide evidence for a novel approach to understanding the molecular mechanisms of HIV chronic pain and treating chronic pain in patients with HIV.

The mitochondrial calcium uniporter contributes to morphine tolerance through pCREB and CPEB1 in rat spinal cord dorsal horn

BackgroundThe long-term use of opioid analgesics is limited by the development of unwanted side-effects, such as tolerance. The molecular mechanisms of morphine anti-nociceptive tolerance are still unclear. The mitochondrial calcium uniporter (MCU) is involved in painful hyperalgesia, but the role of MCU in morphine tolerance has not been uncharacterised. MethodsRats received intrathecal injection of morphine for 7 days to induce morphine tolerance. The mechanical withdrawal threshold was measured using von Frey filaments, and thermal latency using the hotplate test. The effects of an MCU inhibitor, antisense oligodeoxynucleotide against cyclic adenosine monophosphate response element (CRE)-binding protein (CREB) or cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in morphine tolerance were examined. ResultsSpinal morphine tolerance was associated with an increased expression of neuronal MCU, phospho-CREB (pCREB), and CPEB1 in the spinal cord dorsal horn. MCU inhibition increased the mechanical threshold and thermal latency, and reduced the accumulation of mitochondrial calcium in morphine tolerance. Intrathecal antisense oligodeoxynucleotide against CREB or CPEB1 restored the anti-nociceptive effects of morphine compared with mismatch oligodeoxynucleotide in von Frey test and hotplate test. Chromatin immunoprecipitation with quantitative PCR assay showed that CREB knockdown reduced the interaction of pCREB with the ccdc109a gene (encoding MCU expression) promoter and decreased the MCU mRNA transcription. RNA immunoprecipitation assay suggested that CPEB1 binds to the MCU mRNA 3′ untranslated region. CPEB1 knockdown decreased the expression of MCU protein. ConclusionsThese findings suggest that spinal MCU is regulated by pCREB and CPEB1 in morphine tolerance, and that inhibition of MCU, pCREB, or CPEB1 may be useful in preventing the development of opioid tolerance.

The therapeutic potential of GABA in neuron-glia interactions of cancer-induced bone pain

The development of effective therapeutics for cancer-induced bone pain (CIBP) remains a tremendous challenge owing to its unclear mechanisms. Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). Emerging studies have shown that disinhibition in the spinal cord dorsal horn may account for the development of chronic pain. However, the role of GABA in the development of CIBP remains elusive. In addition, accumulating evidence has shown that neuroglial cells in the peripheral nervous system, especially astrocytes and microglial cells, played an important role in the maintenance of CIBP. In this study, we investigated the expression of GABA and Gamma-aminobutyric acid transporter-1 (GAT-1), a transporter of GABA. Our results demonstrate that GABA was decreased in CIBP rats as expected. However, the expression of glutamic acid decarboxylase (GAD) 65 was up-regulated on day 21 after surgery, while the expression of GAD 67 remained unchanged after surgery. We also found that the expression of GAT-1 was up-regulated mainly in the astrocytes of the spinal cord. Moreover, we evaluated the analgesic effect of exogenous GABA and the GAT-1 inhibitor. Intrathecal administration of exogenous GABA and NO-711 (a GAT-1 selective inhibitor) significantly reversed CIBP-induced mechanical allodynia in a dose-dependent manner. These results firstly show that neuron-glia interactions, especially on the GABAergic pathway, contribute to the development of CIBP. In conclusion, exogenous GABA and GAT-1 inhibitor might be alternative therapeutic strategies for the treatment of CIBP.

Colonic afferent input and dorsal horn neuron activation differs between the thoracolumbar and lumbosacral spinal cord.

The distal colon is innervated by the splanchnic and pelvic nerves, which relay into the thoracolumbar and lumbosacral spinal cord, respectively. Although the peripheral properties of the colonic afferent nerves within these pathways are well studied, their input into the spinal cord remain ill defined. The use of dual retrograde tracing from the colon wall and lumen, in conjunction with in vivo colorectal distension and spinal neuronal activation labeling with phosphorylated MAPK ERK 1/2 (pERK), allowed us to identify thoracolumbar and lumbosacral spinal cord circuits processing colonic afferent input. In the thoracolumbar dorsal horn, central projections of colonic afferents were primarily labeled from the wall of the colon and localized in laminae I and V. In contrast, lumbosacral projections were identified from both lumen and wall tracing, present within various dorsal horn laminae, collateral tracts, and the dorsal gray commissure. Nonnoxious in vivo colorectal distension evoked significant neuronal activation (pERK-immunoreactivity) within the lumbosacral dorsal horn but not in thoracolumbar regions. However, noxious in vivo colorectal distension evoked significant neuronal activation in both the thoracolumbar and lumbosacral dorsal horn, with the distribution of activated neurons correlating to the pattern of traced projections. Dorsal horn neurons activated by colorectal distension were identified as possible populations of projection neurons or excitatory and inhibitory interneurons based on their neurochemistry. Our findings demonstrate how colonic afferents in splanchnic and pelvic pathways differentially relay mechanosensory information into the spinal cord and contribute to the recruitment of spinal cord pathways processing non-noxious and noxious stimuli.NEW & NOTEWORTHY In mice, retrograde tracing from the colon wall and lumen was used to identify unique populations of afferent neurons and central projections within the spinal cord dorsal horn. We show that there are pronounced differences between the spinal cord regions in the distribution pattern of colonic afferent central projections and the pattern of dorsal horn neuron activation evoked by colorectal distension. These findings demonstrate how colonic afferent input influences spinal processing of colonic mechanosensation.

Differences Between the Prenatal Effects of Fluoxetine or Buspirone Alone or in Combination on Pain and Affective Behaviors in Prenatally Stressed Male and Female Rats.

The selective serotonin reuptake inhibitor fluoxetine and the 5-HT1A receptor agonist buspirone are used to treat depression and anxiety. Previously we demonstrated that chronic stress during pregnancy (prenatal stress) in rats, used as a model of maternal depression risk, increased inflammatory pain and depressive-like behavior in the offspring; buspirone injected to pregnant dams was protective. Clinically, the addition of buspirone to fluoxetine increases the latter’s efficacy in treating depression in patients. Here, we investigated the influence of repeated prenatal injections of fluoxetine, buspirone or their combination on pain- and depressive-like behaviors in prenatally stressed young male and female rats. Prenatal stress augmented depressive-like behavior and both thermal and inflammatory pain (formalin test), replicating our prior findings, and increased basal levels of corticosterone in the blood plasma. Both drugs and their combination reduced the effects of prenatal stress on thermal pain and depressive-like behavior independently of sex. The combination of fluoxetine and buspirone, compared with fluoxetine, was more antinociceptive in the hot plate test in both sexes, and when compared with buspirone, was more antinociceptive only in males. A detailed study of the time-course of formalin-induced pain showed a nuanced effect of these drugs that was sex-dependent. The combination of the two drugs was less effective in females than males during the initial acute phase of nociceptive behavior in flexing + shaking behaviors, whereas that combination was more effective than fluoxetine alone in the first acute phase of licking behavior in females. The antinociceptive effect of buspirone dominated that of the drug combination and of fluoxetine alone, especially during the interphase of the formalin test in both sexes for both flexing + shaking and licking, suggesting a more effective prenatal action of buspirone on the development of a descending serotonergic inhibitory system modulating pain in the spinal cord dorsal horn neurons. Our results indicate that inflammatory pain-like responses integrated at the spinal level in males were more vulnerable to prenatal stress than females. In licking, the antinociceptive effect of fluoxetine and drug combination in the interphase was more in males than females. The data underscore the importance of considering sexual dimorphism when using drug therapy.

Effects of 1,8-cineole on neuropathic pain mediated by P2X2 receptor in the spinal cord dorsal horn

As an intractable health threat, neuropathic pain is now a key problem in clinical therapy, which can be caused by lesions affecting the peripheral nervous systems. 1,8-cineole is a natural monoterpene cyclic ether present in eucalyptus and has been reported to exhibit anti-inflammatory and antioxidant effects. Research has shown that 1,8-cineole inhibits P2X3 receptor-mediated neuropathic pains in dorsal root ganglion. The P2X2 and P2X3 receptors participate in the transmission of algesia and nociception information by primary sensory neurons. In the present study, We thus investigated in the spinal cord dorsal horn whether 1,8-cineole inhibits the expression of P2X2 receptor-mediated neuropathic pain. This study used rats in five random groups: group of chronic constriction injury(CCI) with dimethysulfoxide control (CCI + DMSO); group of CCI; sham group(Sham); group of CCI treated with a low dose 1,8-cineole (CCI + 50 mg/kg); group of CCI with a high dose (CCI + 100 mg/kg). We observed the effects of 1,8-cineole on thermal withdrawal latency (TWL) and mechanical withdrawal threshold (MWT). We examined P2X2 receptors mRNA change in rat spinal cord dorsal horn by In situ nucleic acid hybridization(ISH) and Quantitative realtime polymerase chain reaction (qRT-PCR) methods. Western Blotting and Immunohistochemical staining methods were used to observe P2X2 receptor protein expressions in the rat spinal cord dorsal horn. It demonstrated that oral administration of 1,8-cineole inhibits over-expression of P2X2 receptor protein and mRNA in the spinal cord and dorsal horn in the CCI rats. And the study explored new methods for the prevention and treatment of neuropathic pain.

AAV-encoded CaV2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain.

Transmission of pain signals from primary sensory neurons to secondary neurons of the central nervous system is critically dependent on presynaptic voltage-gated calcium channels. Calcium channel-binding domain 3 (CBD3), derived from the collapsin response mediator protein 2 (CRMP2), is a peptide aptamer that is effective in blocking N-type voltage-gated calcium channel (CaV2.2) activity. We previously reported that recombinant adeno-associated virus (AAV)-mediated restricted expression of CBD3 affixed to enhanced green fluorescent protein (EGFP) in primary sensory neurons prevents the development of cutaneous mechanical hypersensitivity in a rat neuropathic pain model. In this study, we tested whether this strategy is effective in treating established pain. We constructed AAV6-EGFP-CBD3A6K (AAV6-CBD3A6K) expressing a fluorescent CBD3A6K (replacing A to K at position 6 of CBD3 peptide), which is an optimized variant of the parental CBD3 peptide that is a more potent blocker of CaV2.2. Delivery of AAV6-CBD3A6K into lumbar (L) 4 and 5 dorsal root ganglia (DRG) of rats 2 weeks following tibial nerve injury (TNI) induced transgene expression in neurons of these DRG and their axonal projections, accompanied by attenuation of pain behavior. We additionally observed that the increased CaV2.2α1b immunoreactivity in the ipsilateral spinal cord dorsal horn and DRG following TNI was significantly normalized by AAV6-CBD3A6K treatment. Finally, the increased neuronal activity in the ipsilateral dorsal horn that developed after TNI was reduced by AAV6-CBD3A6K treatment. Collectively, these results indicate that DRG-restricted AAV6 delivery of CBD3A6K is an effective analgesic molecular strategy for the treatment of established neuropathic pain.

P2X3 receptors contribute to muscle pain induced by static contraction by a mechanism dependent on neutrophil migration.

P2X3 receptors are involved with several pain conditions. Muscle pain induced by static contraction has an important socioeconomic impact. Here, we evaluated the involvement of P2X3 receptors on mechanical muscle hyperalgesia and neutrophil migration induced by static contraction in rats. Also, we evaluated whether static contraction would be able to increase muscle levels of TNF-α and IL-1β. Male Wistar rats were pretreated with the selective P2X3 receptor antagonist, A-317491, by intramuscular or intrathecal injection and the static contraction-induced mechanical muscle hyperalgesia was evaluated using the Randall-Selitto test. Neutrophil migration was evaluated by measurement of myeloperoxidase (MPO) kinetic-colorimetric assay and the cytokines TNF-α and IL-1β by enzyme-linked immunosorbent assay. Intramuscular or intrathecal pretreatment with A-317491 prevented static contraction-induced mechanical muscle hyperalgesia. In addition, A-317491 reduced static contraction-induced mechanical muscle hyperalgesia when administered 30 and 60min of the beginning of static contraction, but not after 30 and 60min of the end of static contraction. Intramuscular A-317491 also prevented static contraction-induced neutrophil migration. In a period of 24h, static contraction did not increase muscle levels of TNF-α and IL-1β. These findings demonstrated that mechanical muscle hyperalgesia and neutrophil migration induced by static contraction are modulated by P2X3 receptors expressed on the gastrocnemius muscle and spinal cord dorsal horn. Also, we suggest that P2X3 receptors are important to the development but not to maintenance of muscle hyperalgesia. Therefore, P2X3 receptors can be pointed out as a target to musculoskeletal pain conditions induced by daily or work-related activities.

Suppression of WNK1-SPAK/OSR1 Attenuates Bone Cancer Pain by Regulating NKCC1 and KCC2

Our preliminary experiment indicated the activation of with-nolysine kinases 1 (WNK1) in bone cancer pain (BCP) rats. This study aimed to investigate the underlying mechanisms via which WNK1 contributed to BCP. A rat model of BCP was induced by Walker-256 tumor cell implantation. WNK1 expression and distribution in the lumbar spinal cord dorsal horn and dorsal root ganglion were examined. SPS1-related proline/alanine-rich kinase (SPAK), oxidative stress-responsive kinase 1 (OSR1), sodium-potassium-chloride cotransporter 1 (NKCC1), and potassium-chloride cotransporter 2 (KCC2) expression were assessed. Pain behaviors including mechanical allodynia and movement-evoked pain were measured. BCP rats exhibited significant mechanical allodynia, with increased WNK1 expression in the dorsal horn and dorsal root ganglion neurons, elevated SPAK/OSR1 and NKCC1 expression in the dorsal root ganglion, and decreased KCC2 expression in the dorsal horn. WNK1 knock-down by small interfering alleviated mechanical allodynia and movement-evoked pain, inhibited WNK1-SPAK/OSR1-NKCC1 activities, and restored KCC2 expression. In addition, closantel (a WNK1-SPAK/OSR1 inhibitor) improved pain behaviors, downregulated SPAK/OSR1 and NKCC1 expression, and upregulated KCC2 expression in BCP rats. Activation of WNK1-SPAK/OSR1 signaling contributed to BCP in rats by modulating NKCC1 and KCC2 expression. Therefore, suppression of WNK1-SPAK/OSR1 may serve as a potential target for BCP therapy. PerspectiveOur findings demonstrated that the WNK1-SPAK/OSR1 signaling contributed to BCP in rats via regulating NKCC1 and KCC2. Suppressing this pathway reduced pain behaviors. Based on these findings, the WNK1-SPAK/OSR1 signaling may be a potential target for BCP therapy.

Ultrasound therapy reduces persistent post-thoracotomy tactile allodynia and spinal substance P expression in rats

BackgroundTherapeutic ultrasound (TU) alleviates nerve injury-associated pain, while the molecular mechanisms are less clear. This is an investigator-initiated experimental study to evaluate the mechanisms and effects of ultrasound on prolonged...

Dynamic expression of glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 in the mouse spinal cord dorsal horn under pathological pain states

ABSTRACTObjective: Animal models of chronic pain have demonstrated that glial cells are promising target for development of analgesic drugs. However, preclinical studies on glial response under chronic pain conditions vary depending on the cellular markers, the species used, the experimental design and model. Therefore, we investigate the expression profile of GFAP and Iba-1 during the behavioral manifestation of sensory disorder in inflammatory and neuropathic pain models.Methods: the expression profile of fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule-1 (Iba-1) were quantitated in the spinal dorsal horn of Balb/C mice submitted to six models of chronic pain. Protein analysis was performed by western blot and the results colligated with pain-related behavior.Results: Using the same method to quantitate proteins we observed that while GFAP is upregulated after axotomy, partial nerve injury and cutaneous inflammation, its expression is not changed during muscle inflammation, non-inflammatory muscle pain, and in a viral-associated pain. Differently, Iba-1 is downregulated after axotomy but upregulated after partial lesion of peripheral nerve as well as after virus inoculation and during non-inflammatory muscle pain. Cutaneous and muscle inflammation induced no change in Iba-1 expression in the dorsal horn.In spite of a marked time-dependent variation in protein expression, mechanical allodynia was present at any time of all the models investigated.Discussion: Under distinct pain conditions, GFAP and Iba-1 expression is dependent on the origin of the stimulus, disease progression and tissue affected. Moreover, their expression and is not necessarily associated to the behavior manifestation of pain.

A synthetic peptide disturbing GluN2A/SHP1 interaction in dorsal root ganglion attenuated pathological pain

Src Homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) interacts specifically with GluN2A subunit of N-methyl-D-aspartate (NMDA) subtype of glutamate receptors in spinal cord dorsal horn. This molecular interaction is involved in the development of GluN2A-dependent spinal sensitization of nociceptive behaviors. Intrathecal application of a GluN2A-derived polypeptide (short for pep-GluN2A) has been shown to disturb spinal GluN2A/SHP1 interaction and inhibit inflammatory pain. Here we found that SHP1 was also located at dorsal root ganglion (DRG) neurons and formed complexes with GluN2A subunit. Peripheral inflammation activated SHP1 in DRG neurons, which promoted GluN2A tyrosine phosphorylation. The SHP1 binding to GluN2A facilitated the glutamate release from primary afferent fibers and exaggerated nociceptive synaptic transmission onto postsynaptic spinal cord neurons. Our data showed that intradermal application of pep-GluN2A disrupted GluN2A/SHP1 interaction in DRG neurons, attenuated the ability of GluN2A subunit-containing NMDA receptors to regulate the presynaptic glutamate release and more importantly, alleviated the pain hypersensitivity caused by carrageenan, complete Freund's adjuvant and formalin. The neuropathic pain induced by spared nerve injury was also ameliorated by intradermal pep-GluN2A application. These data suggested that disruption of GluN2A/SHP1 interaction in DRG neurons generated an effective analgesic action against pathological pain.

Leaky expression of channelrhodopsin-2 (ChR2) in Ai32 mouse lines.

Optogenetics enables the selective activation of genetically-targeted neuronal populations using light-sensitive ion channels. Genetic strategies using Cre-dependent mouse strains, especially the Ai32 line expressing Channelrhodopsin (ChR2)-EYFP fusion protein, have been a popular means to drive opsin expression in a cell-type specific manner. Here we report a low level of leaky ‘off-target’ (Cre-independent) ChR2-EYFP expression in Ai32/Ai32 homozygous mice throughout the nervous system. This leaky off-target expression was characterized in multiple prevalent nervous system regions using anti-EYFP immunostaining. Expression of full-length ChR2-EYFP protein was confirmed using immunoprecipitation followed by Western blotting. Notably, light stimulation of these ChR2-EYFP expressing neurons in the spinal cord dorsal horn did not induce detectable photocurrents in juvenile 4-week old mice. Given the wide use of the Ai32 line by many labs, our results suggest researchers should be vigilant of possible off-target ChR2-EYFP expression in their region of interest, especially when generating Ai32/Ai32 homozygotes to drive high levels of ChR2-EYFP expression in adult mice.

NNOS-PSD95 interactions activate the PKC-ε isoform leading to increased GluN1 phosphorylation and the development of neuropathic mechanical allodynia in mice

It has been suggested that interactions of neuronal nitric oxide synthase (nNOS) with postsynaptic density 95 (PSD95) play important roles in the development of chronic neuropathic pain. Here we examine the possible role of nNOS-PSD95 interactions in central sensitization as represented by phosphorylation of the NMDA receptor GluN1 subunit (pGluN1) in mice with chronic constriction injury (CCI) of the sciatic nerve. Intrathecal administration of the nNOS-PSD95 interactions inhibitor, IC87201 on post-operative days 0–3 significantly reduced the CCI-induced increase in total NO levels in the lumbar spinal cord dorsal horn. IC87201 administration on post-operative days 0–3 also attenuated the CCI-induced development of mechanical allodynia (MA) and PKC-dependent (Ser896) pGluN1. Sciatic nerve injury elicited a significant translocation of the PKC-ε isoform from the cytosol to the membrane fraction in the lumbar spinal cord dorsal horn on day 3 post-CCI surgery. Administration of IC87201 significantly inhibited this translocation of PKC-ε, while the expression of PKC-α and -ξ in the cytosol and membrane fractions was unaffected by sciatic nerve injury or injection of IC87201. Furthermore, administration of the PKC-ε inhibitor, εV1-2 on post-operative days 0–3 attenuated the CCI-induced development of MA and pGluN1. Collectively these results demonstrate that spinal nNOS-PSD95 interactions play an important role in PKC-dependent GluN1 phosphorylation via activation of the PKC-ε isoform, and ultimately contributes to the development of MA in peripheral neuropathy.