Decrease In Mechanical Withdrawal Threshold Research Articles

The cumulative analgesic effect of repeated electroacupuncture is modulated by Adora3 in the SCDH of mice with neuropathic pain.

Existing remedial approaches for relieving neuropathic pain (NPP) are challenging and open the way for alternative therapeutic measures such as electroacupuncture (EA). The mechanism underlying the antinociceptive effects of repeated EA sessions, particularly concerning the regulation of the Adora3 receptor and its associated enzymes, has remained elusive. This study used a mouse model of spared nerve injury (SNI) to explore the cumulative analgesic effects of repeated EA at ST36 (Zusanli) and its impact on Adora3 regulation in the spinal cord dorsal horn (SCDH). Forty-eight male mice underwent SNI surgery for induction of neuropathic pain and were randomly assigned to the SNI, SNI + 2EA, SNI + 4EA, and SNI + 7EA groups. Spinal cord (L4-L6) was sampled for immunofluorescence, adenosine (ADO) detection and for molecular investigations following repeated EA treatment. Following spared nerve injury (SNI), there was a significant decrease in mechanical withdrawal thresholds (PWTs) and thermal nociceptive withdrawal latency (TWL) in the ipsilateral hind paw on the third day post-surgery, while the contralateral hind paw PWTs showed no significant changes. On subsequent EA treatments, the SNI + EA groups led to a significant increase in pain thresholds (p < 0.05). Repeated EA sessions in SNI mice upregulated Adenosine A3 (Adora3) and cluster of differentiation-73 (CD73) expression while downregulating adenosine deaminase (ADA) and enhancing neuronal instigation in the SCDH. Colocalization analysis of Neun-treated cells revealed increased Adora3 expression, particularly in the SNI + 7EA group. In conclusion, cumulative electroacupuncture treatment reduced neuropathic pain by regulating Adora3 and CD73 expression, inhibiting ADA and most likely increasing neuronal activation in the SCDH. This study offers a promising therapeutic option for managing neuropathic pain, paving the way for further research.

Electroacupuncture improves allodynia and central sensitization via modulation of microglial activation associated P2X4R and inflammation in a rat model of migraine.

Background: Recent studies have demonstrated that activated microglia were involved in the pathogenesis of central sensitization characterized by cutaneous allodynia in migraine. Activation of microglia is accompanied by increased expression of its receptors and release of inflammatory mediators. Acupuncture and its developed electroacupuncture (EA) have been recommended as an alternative therapy for migraine and are widely used for relieving migraine-associated pain. However, it remains rare studies that show whether EA exerts anti-migraine effects via inhibiting microglial activation related to a release of microglial receptors and the inflammatory pathway. Therefore, this study aimed to investigate EA' ability to ameliorate central sensitization via modulation of microglial activation, microglial receptor, and inflammatory response using a rat model of migraine induced by repeated epidural chemical stimulation. Methods: In the present study, a rat model of migraine was established by epidural repeated inflammatory soup (IS) stimulation and treated with EA at Fengchi (GB20) and Yanglingquan (GB34) and acupuncture at sham-acupoints. Pain hypersensitivity was further determined by measuring the mechanical withdrawal threshold using the von-Frey filament. The changes in c-Fos and ionized calcium binding adaptor molecule 1 (Ibal-1) labeled microglia in the trigeminal nucleus caudalis (TNC) were examined by immunflurescence to assess the central sensitization and whether accompanied with microglia activation. In addition, the expression of Ibal-1, microglial purinoceptor P2X4, and its associated inflammatory signaling pathway mediators, including interleukin (IL)-1β, NOD-like receptor protein 3 (NLRP3), and Caspase-1 in the TNC were investigated by western blot and real-time polymerase chain reaction analysis. Results: Allodynia increased of c-Fos, and activated microglia were observed after repeated IS stimulation. EA alleviated the decrease in mechanical withdrawal thresholds, reduced the activation of c-Fos and microglia labeled with Ibal-1, downregulated the level of microglial purinoceptor P2X4, and limited the inflammatory response (NLRP3/Caspase-1/IL-1β signaling pathway) in the TNC of migraine rat model. Conclusions: Our results indicate that the anti-hyperalgesia effects of EA ameliorate central sensitization in IS-induced migraine by regulating microglial activation related to P2X4R and NLRP3/IL-1β inflammatory pathway.

Upregulation of Nav1.6 Mediated by the p38 MAPK Pathway in the Dorsal Root Ganglia Contributes to Cancer-Induced Bone Pain in Rats.

Cancer-induced bone pain (CIBP) occurs frequently among advanced cancer patients. Voltage-gated sodium channels (VGSCs) have been associated with chronic pain, but how VGSCs function in CIBP is poorly understood. Here, we aimed to investigate the specific role of VGSCs in the dorsal root ganglia (DRGs) in CIBP. A CIBP rat model was generated by the intratibial inoculation of MRMT-1 breast carcinoma cells. Transcriptome sequencing was conducted to assess the gene expression profiles. The expression levels of key genes and differentiated genes related to activated pathways were measured by Western blotting and qPCR. We implanted a catheter intrathecally for the administration of lentivirus and drugs. Then, the changes in the mechanical withdrawal threshold (MWT) were measured. We identified 149 differentially expressed mRNAs (DEmRNAs) in the DRGs of CIBP model rats. The expression of Nav1.6, which was among these DEmRNAs, was significantly upregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEmRNAs showed that they were mainly enriched in the mitogen-activated protein kinase (MAPK) pathway. The decrease in MWT induced by bone cancer was attenuated by Nav1.6 knockdown. Western blot analysis revealed that a p38 inhibitor decreased the expression of Nav1.6 and attenuated pain behavior. Our study shows that the upregulation of Nav1.6 expression by p38 MAPK in the DRGs of rats contributes to CIBP.

Loss of Enrichment Induces Widespread Muscle Mechanical Hypersensitivity and Enhances Pain Related Behaviors after Ischemia with Reperfusion Injury via Macrophage Dependent Mechanisms

The development of chronic musculoskeletal pain is frequently associated with the presence of high levels of psychological stress. Multiple animal studies have shown that a variety of stress paradigms are effective at inducing deep tissue hyperalgesia and enhanced behavioral responses to subthreshold stimuli or injury. Nevertheless, the mechanisms behind stress-induced hyperalgesia are not clearly understood. Many of the current paradigms induce stress in rodents using physical stimuli that are known to induce changes in sensory function, such as repeated cold exposure or electrical shock. Non-invasive strategies to induce stress in animal models may uncover new mechanisms that drive stress related hyperalgesia. To address this issue, our lab has adapted a stress paradigm that utilizes environmental enhancement and subsequent loss of enrichment (LOE) to induce stress in mice, paired with fore limb ischemia/reperfusion injury (I/R) as a model of muscle hypersensitivity. After LOE, mice developed prolonged immobility time in a forced swimming test and presented with increased weight gain, suggesting increased stress/anxiety-like behaviors. At baseline (1 week after LOE), the mice also developed a bilateral decrease in mechanical withdrawal thresholds to plantar muscle squeezing, indicating stress related hypersensitivity. Immunohistochemical analysis of the plantar muscles showed increased macrophage infiltration one week after LOE. Mice exposed to LOE and I/R injury displayed a prolonged recovery time, compared to normal housing controls. Systemic ablation of macrophages only during the stress period facilitated the recovery after I/R. Our results suggests that LOE can significantly enhance the development of muscle pain after I/R. Together with our immunocytochemical data, results suggest that macrophages could play a role in the development of pain after LOE. These findings highlight the importance of stress in the development of long-lasting pain and may shed light in potential mechanisms behind chronic widespread muscle pain conditions like fibromyalgia. Grant Support from Anesthesia Innovation and Pilot (AIP) Program at Cincinnati Children's Hospital (LFQ) R01NS113965 and R01NS105715 (MPJ).

Tetrahydropalmatine alleviated diabetic neuropathic pain by inhibiting activation of microglia via p38 MAPK signaling pathway

Neuropathic pain is one of the common complications of diabetes. Tetrahydropalmatine(THP) is a main active component of Corydalis Rhizoma with excellent anti-inflammatory and pain-alleviating properties. This study aims to investigate the therapeutic effect of THP on diabetic neuropathic pain(DNP) and the underlying mechanism. High-fat and high-sugar diet(4 weeks) and streptozotocin(STZ, 35 mg·kg~(-1), single intraperitoneal injection) were employed to induce type-2 DNP in rats. Moreover, lipopolysaccharide(LPS) was used to induce the activation of BV2 microglia in vitro to establish an inflammatory cellular model. Fasting blood glucose(FBG) was measured by a blood glucose meter. Mechanical withdrawal threshold(MWT) was assessed with von Frey filaments, and thermal withdrawal latency(TWL) with hot plate apparatus. The protein expression levels of OX42, inducible nitric oxide synthase(iNOS), CD206, p38, and p-p38 were determined by Western blot, the fluorescence expression levels of OX42 and p-p38 in the dorsal horn of the rat spinal cord by immunofluorescence, the mRNA content of p38 and OX42 in rat spinal cord tissue by qRT-PCR, and levels of nitric oxide(NO), interleukin-1β(IL-1β), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), interleukin-10(IL-10), and serum fasting insulin(FINS) by enzyme-linked immunosorbent assay(ELISA). RESULTS:: showed that the mo-del group demonstrated significant decrease in MWT and TWL, with pain symptoms. THP significantly improved the MWT and TWL of DNP rats, inhibited the activation of microglia and p38 MAPK signaling pathway in rat spinal cord, and ameliorated its inflammatory response. Meanwhile, THP promoted the change of LPS-induced BV2 microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, suppressed the activation of the p38 MAPK signaling pathway, decreased the expression levels of inflammatory factors NO, IL-1β, IL-6, and TNF-α, and increased the expression level of anti-inflammatory factor IL-10. The findings suggested that THP can significantly ameliorate the pain symptoms of DNP rats possibly by inhibiting the inflammatory response caused by M1 polarization of microglia via the p38 MAPK pathway.

Neuropeptide Y in the amygdala contributes to neuropathic pain-like behaviors in rats via the neuropeptide Y receptor type 2/mitogen-activated protein kinase axis

ABSTRACT Neuropeptide Y (NPY) is a highly conserved endogenous peptide in the central and peripheral nervous systems, which has been implicated in nociceptive signaling in neuropathic pain. However, downstream mechanistic actions remain uncharacterized. In this study, we sought to investigate the mechanism of NPY and its receptor NPY2R in the amygdala in rats with neuropathic pain-like behaviors induced by chronic constriction injury (CCI) of the sciatic nerve. The expression of NPY and NPY2R was found to be aberrantly up-regulated in neuropathic pain-related microarray dataset. Further, NPY was found to act on NPY2R in the basolateral amygdala (BLA). As reflected by the decrease in mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) as well as the increase of NPY expression in the amygdala of rats with neuropathic pain-like behaviors, NPY was closely related to the effect of amygdala nerve activity in neuropathic pain. Subsequently, mechanistic investigations indicated that NPY2R activated the MAPK signaling pathway in the amygdala. NPY2R-induced decrease of MWT and TWL were also restored in the presence of MAPK signaling pathway antagonist. Moreover, it was revealed that NPY2R overexpression promoted the viability while inhibiting the apoptosis of microglia. Taken together, NPY in the amygdala interacts with NPY2R to activate the MAPK signaling pathway, thereby promoting the occurrence of neuropathic pain.

Dexmedetomidine alleviates inflammation in neuropathic pain by suppressing NLRP3 via Nrf2 activation

The aim of the present study was to investigate the mechanism by which dexmedetomidine (DEX) alleviates neuropathic pain in a chronic constriction injury (CCI) model in rats. A CCI rat model was established through sciatic nerve ligation. CCI rats were treated with DEX, the nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor ML385, the NLR family pyrin domain containing 3 (NLRP3) antagonist MCC950 and/or the NLRP3 activator nigericin. The mechanical withdrawal threshold (MWT) was measured to assess the pain sensitivity of CCI rats. Hematoxylin and eosin staining and TUNEL staining were used to examine spinal injury and apoptosis, respectively. ELISA was used to quantify the levels of inflammatory factors. The expression levels of Nrf2 and NLRP3 were also examined. The results indicated that a decrease in MWT and increases in spinal cord injury, apoptosis and inflammatory factors were detected in CCI rats compared with control rats. Spinal inflammation was abrogated in DEX-treated CCI rats. Compared with the model group, an increase in MWT and decreases in spinal cord injury, apoptosis and inflammatory factors were detected in rats treated with MCC950, while the opposite effects were observed in rats treated with nigericin. The opposite effects on these indicators were observed in the DEX + ML385 and MCC950 + ML385 groups compared with the DEX and MCC950 groups, respectively. MWT was increased, while spinal cord injury, apoptosis and inflammation decreased in the nigericin + DEX group compared with the nigericin group. In summary, the results of the present study indicated that DEX reduced neuropathic pain in CCI rats by suppressing NLRP3 through Nrf2 activation.

Role of GABAAR in the Transition From Acute to Chronic Pain and the Analgesic Effect of Electroacupuncture on Hyperalgesic Priming Model Rats.

Chronic pain is a costly health problem that impairs health-related quality of life when not effectively treated. Regulating the transition from acute to chronic pain is a new therapeutic strategy for chronic pain that presents a major clinical challenge. The underlying mechanisms of pain transition are not entirely understood, and strategies for preventing this transition are lacking. Here, a hyperalgesic priming model was used to study the potential mechanism by which γ-aminobutyric acid receptor type A (GABAAR) in the dorsal root ganglion (DRG) contributes to pain transition. Furthermore, electroacupuncture (EA), a modern method of acupuncture, was administered to regulate pain transition, and the mechanism underlying EA’s regulatory effect was investigated. Hyperalgesic priming was induced by intraplanar injection of carrageenan (Car)/prostaglandin E2 (PGE2). The decrease in mechanical withdrawal threshold (MWT) induced by PGE2 returned to baseline 4 h after injection in NS + PGE2 group, and still persisted 24 h after injection in Car + PGE2 group. Lower expression of GABAAR in the lumbar DRG was observed in the model rats. Furthermore, activating or blocking GABAAR could reversed the long-lasting hyperalgesia induced by Car/PGE2 injection or produced a persistent hyperalgesia. In addition, GABAAR may be involved in Protein Kinase C epsilon (PKCε) activation in the DRG, a mark molecular of pain transition. EA considerably increased the mechanical pain thresholds of hyperalgesic priming model mammals in both the acute and chronic phases. Furthermore, EA upregulated the expression of GABAAR and inhibited the activation of PKCε in the DRG. In addition, peripheral administration of picrotoxin blocked the analgesic effect of EA on the model rats and abolished the regulatory effect of EA on PKCε activation. These findings suggested that GABAAR plays a key role in both the transition from acute to chronic pain and the analgesic effect of EA on hyperalgesic priming.

Effects of vitamin D administration on nociception and spinal cord pro-oxidant and antioxidant markers in a rat model of neuropathic pain.

Reactive oxygen species (ROS) are involved in neuropathic pain, a complicated condition after nerve tissue lesion. Vitamin D appears to improve symptoms of pain and exhibits antioxidant properties. We investigated the effects of oral administration of vitamin D3, the active form of vitamin D, on nociception, the sciatic functional index (SFI), and spinal cord pro-oxidant and antioxidant markers in rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. Vitamin D3 (500 IU/kg per day) attenuated the CCI-induced decrease in mechanical withdrawal threshold and thermal withdrawal latency (indicators of antinociception) and SFI. The vitamin prevented increased lipid hydroperoxide levels in injured sciatic nerve without change to total antioxidant capacity (TAC). Vitamin D3 prevented increased lipid hydroperoxide, superoxide anion generation (SAG), and hydrogen peroxide (H2O2) levels in the spinal cord, which were found in rats without treatment at 7 and 28 days post-CCI. A significant negative correlation was found between mechanical threshold and SAG and between mechanical threshold and H2O2 at day 7. Vitamin D3 also prevented decreased spinal cord total thiols content. There was an increase in TAC in the spinal cord of vitamin-treated CCI rats, compared to CCI rats without treatment only at 28 days. No significant changes were found in body weight and blood parameters of hepatic and renal function. These findings demonstrated, for first time, that vitamin D modulated pro-oxidant and antioxidant markers in the spinal cord. Since antinociception occurred in parallel with oxidative changes in the spinal cord, the oxidative changes may have contributed to vitamin D-induced antinociception.

Deficit in voluntary wheel running in chronic inflammatory and neuropathic pain models in mice: Impact of sex and genotype

Patients with chronic pain report decreased general activity and emotional distress. Therefore, the development of various animal models that encompass different aspects of pain are crucial for the discovery of genetic differences and the assessment of novel analgesics to improve quality of life. C57BL/6J and DBA/2J mice received unilateral intraplantar injections of 100 % CFA, paclitaxel, or CCI surgery to compare their distance traveled in a voluntary wheel running assay, paw edema diameter, and mechanical sensitivity. Mechanical withdrawal thresholds were lower in both strains of mice that received CFA when compared to their vehicle. However, a decrease in distance traveled was observed in CFA-treated C57BL/6J but not DBA/2J mice. In a separate group, chemotherapy agent paclitaxel 8 mg/kg, i.p. was administered to both strains of mice to induce CIPN which was confirmed by lower mechanical thresholds in paclitaxel-treated mice compared to vehicle-treated mice. Only female C57BL/6J mice showed attenuation of distance traveled following treatment, whereas male C57BL/6J and DBA/2J mice did not. Lastly, C57BL/6J mice underwent chronic constriction injury (CCI) or sham surgery to observe the impact of another chronic neuropathic pain model in wheel running assay. CCI mice showed a gradual decrease in mechanical withdrawal threshold and a decrease in distance traveled compared to sham 5 days following the procedure. Comparing these chronic inflammatory and neuropathic pain models in different mouse strains may help us better understand genetic differences underlying pain perception and its impact on reflexive and nonreflexive outcome measures.

Pulsed Ultrasound Remedies Post-thoracotomy Hypersensitivity and Increases Spinal Anti-inflammatory Cytokine in Rats

The purpose of the experiment was to study the effect of pulsed ultrasound (PUS) on post-thoracotomy pain and local tissue temperature and to correlate the findings with the alteration in spinal anti-inflammatory and pro-inflammatory cytokines. Mechanical sensitivity, subcutaneous temperature and spinal interleukin-10 (IL-10), IL-6 or tumor necrosis factor-alpha (TNF-α) expression were examined in a rat model of experimental post-thoracotomy pain. Group 1 received a sham surgery where thoracotomy was performed except for rib retraction. Group 2 underwent thoracotomy with rib retraction (TRR). Group 3 received the TRR procedure followed by PUS. Group 4 underwent the TRR procedure followed by only the massage with the ultrasound turned off. Compared with group 1 (sham), groups 2–4 showed a decrease in mechanical withdrawal thresholds on postoperative days (PODs) 10 and 11. On PODs 16, 23 and 30, group 3 (TRR+PUS-1) displayed an increase in mechanical withdrawal thresholds compared with groups 2 and 4. Subcutaneous and body temperatures in group 3 were not prominently different from group 1, group 2 (TRR only) or group 4 (TRR+PUS-0). Compared with group 2, group 3 had an increase in spinal IL-10 level on POD 30 and a decrease in spinal IL-6 or TNF-α expression on PODs 16 and 30. We concluded that mechanical hypersensitivity after TRR is postponed by PUS, and its effect continues for 3 wk. A PUS dose not increase local tissue temperature. The beneficial effect of PUS appears related to upregulation of spinal anti-inflammatory cytokine and downregulation of spinal pro-inflammatory cytokines.

Persistent pain induces mood problems and memory loss by the involvement of cytokines, growth factors, and supraspinal glial cells

Lesions of peripheral nerves lead to pain, hyperalgesia, and psychological comorbidities. However, the relationship between mood disorders and neuropathic pain is unclear, as well as the underlying mechanisms related to these disorders. Therefore, we investigated if nerve injury induces depression, anxiety, and cognitive impairment and if there were changes in cytokines, growth factors, and glial cell activation in cortical sites involved in processing pain and mood in animals with nerve injury. Nerve injury was induced by partial sciatic nerve ligation (PSNL) in male Swiss mice and compared to sham-operated animals. Nociceptive behavioral tests to mechanical and thermal (heat and cold) stimuli confirmed the development of hyperalgesia. We further examined mood disorders and memory behaviors. We show nerve injury induces a decrease in mechanical withdrawal thresholds and thermal latency to heat and cold. We also show that nerve injury causes depressive-like and anxiety-like behaviors as well as impairment in short-term memory in mice. There were increases in proinflammatory cytokines as well as Brain-Derived Neurotrophic Factor (BDNF) in the injured nerve. In the spinal cord, there were increases in both pro and anti-inflammatory cytokines, as well as of BDNF and Nerve Growth Factor (NGF). Further, in our data was a decrease in the density of microglia and astrocytes in the hippocampus and increased microglial density in the prefrontal cortex, areas associated with neuropathic pain conditions.

Electroacupuncture alleviates morphine‑induced hyperalgesia by regulating spinal CB1�receptors and ERK1/2 activity

Electroacupuncture (EA), a traditional Chinese therapeutic technique, is considered an effective method for treating certain painful neuropathies induced by various neuropathological damage. The current study investigated the effect of EA on intrathecal (IT) morphine-induced hyperalgesia (MIH) and examined the hypothesis that activation of cannabinoid receptor 1 (CB1) could enhance the antinociceptive effect of EA on MIH via regulation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Using a rat model of IT MIH, mechanical and thermal hyperalgesia were evaluated by an electronic von Frey filament and hotplate at baseline (1 day before IT administration) and at days 1, 3, 5 and 7 after IT administration. Rats received IT normal saline, IT morphine or IT morphine + EA at ST36-GB34. The protein levels of ERK1/2, phosphorylated (p)-ERK1/2 and CB1 in the spinal cord were assayed by western blotting. Furthermore, the effect of IT injection of the CB1 agonist WIN 55,212-2 and the CB1 antagonist SR141716 on the antinociceptive effect of EA in rats with MIH was investigated. Nociceptive behavior and ERK1/2, phosphorylated (p)-ERK1/2 and CB1 protein levels were evaluated as mentioned above. The results revealed that chronic IT injections of morphine induced a significant decrease in mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) accompanied with remarkable upregulation of p-ERK1/2 in the spinal cord, which could be attenuated by EA at the ST36-GB34 acupoints. In the rat model of MIH, IT injection of WIN 55,212-2 combined with EA induced a significant increase in MWT and TWL accompanied with a significant decrease in p-ERK1/2 and a significant increase in CB1 protein level compared with EA alone, while SR141716 induced the opposite results. The present study suggests that EA alleviates hyperalgesia induced by IT injection of morphine partially through the inhibition of ERK1/2 activation. Activation of the CB1 receptor enhances the antinociceptive effect of EA in rats with MIH partly through the regulation of the spinal CB1-ERK1/2 signaling pathway.

Amitriptyline influences the mechanical withdrawal threshold in bone cancer pain rats by regulating glutamate transporter GLAST.

Patients with cancer, especially breast, prostate, and lung cancer, commonly experience bone metastases that are difficult to manage and are associated with bone cancer pain. Amitriptyline is often used to treat chronic pain, such as neuropathic pain. In this study, the effects of amitriptyline on the mechanical withdrawal threshold and its underlying mechanisms were evaluated in rat models of bone cancer pain. Walker 256 rat mammary gland carcinoma cells were injected into the bone marrow cavity of the right tibia of rats to provoke bone cancer pain. Then, amitriptyline was intraperitoneally administered twice daily from fifth day after the operation. Rats with bone cancer showed an apparent decline in the mechanical withdrawal threshold at day 11 after Walker 256 cells inoculation. The levels of the glutamate-aspartate transporter in the spinal cord dorsal horn decreased remarkably, and the concentration of the excitatory amino acid glutamate in the cerebrospinal fluid increased substantially. Amitriptyline injection could prevent the decline of mechanical withdrawal threshold in bone cancer pain rats. In addition, glutamate-aspartate transporter was upregulated on the glial cell surface, and glutamate levels were reduced in the cerebrospinal fluid. However, amitriptyline injection could not prevent the bone cancer pain-induced reduction in glutamate-aspartate transporter in the glial cell cytosol, it further downregulated cytosolic glutamate-aspartate transporter. Amitriptyline had no significant effect on GLAST messenger RNA expression, and bone cancer pain-invoked protein kinase A/protein kinase C upregulation was prevented. Taken together, these results suggest that the intraperitoneal injection of amitriptyline can prevent the decrease of mechanical withdrawal threshold in bone cancer pain rats, the underlying mechanisms may be associated with the inhibition of protein kinase A/protein kinase C expression, thus promoting glutamate-aspartate transporter trafficking onto the glial cell surface and reducing excitatory amino acid concentrations in the cerebrospinal fluid.

Stimulation-induced expression of immediate early gene proteins in the dorsal horn is increased in neuropathy

Background and aimsPeripheral neuropathic pain is described as a pain state caused by an injury or dysfunction of the nervous system, and could have clinical manifestations such as hyperalgesia, allodynia and spontaneous pain. The development of neuropathic pain may depend on long-term forms of neuronal plasticity in the spinal cord (SC). Expression of the immediate early gene proteins (IEGPs) Arc, Zif268, and c-Fos are implicated in establishment of long-term potentiation (LTP) induced by conditioning stimulation (CS) of primary afferent fibres. However, the impact of the neuropathic state (Bennett's model) on CS-induced expression of IEGPs has not been studied. The aim of this study was to compare the levels of Arc, c-Fos and Zif268 immunoreactivity prior to and after conditioning stimulation in animals with developed neuropathic pain, with sham operated, non-ligated controls. MethodsTwenty-four animals were divided equally into the neuropathic and non-neuropathic groups. Neuropathic pain was induced in all animals by conducting a loose ligation of the sciatic nerve with Chromic Catgut 4.0 sutures 7 days prior to conditioning stimulation or sham operation. The loose ligation was performed by placing sutures around the sciatic nerve compressing the nerve slightly just enough to reduce but not completely diminish the perineural circulation. A state of neuropathy was confirmed by a significant decrease in mechanical withdrawal threshold measured by von Frey's fibres. Immunohistochemical analysis was performed on transverse sections obtained from the L3–L5 segments of the SC at 2 and 6h post-CS and IEGP positive cells were counted in lamina I and II of the dorsal horn. During statistical analyses, the groups were compared by means of analysis of variance (univariate general linear model). If significant differences were found, each set of animals was compared with the sham group with post hoc Tukey's multiple comparison test. ResultsStrikingly, all IEGPs exhibited a significant increase in immunoreactivity at both time points compared to time-matched, sham operated controls. Maximal IEGP expression was found 2h after CS in neuropathic rats, and there was a smaller but still significant increase 6h after CS. The unstimulated side of the dorsal horn in stimulated animals did not show any significant change of the number of IEGP positive cells and was approximately at the same level as sham operated animals. The number of IEGP positive cells in sham operated controls (non-neuropathic and non-stimulated animals) showed same immunoreactivity in 2 and 6h post sham operation. Conclusions and implicationsThe neurophysiological process of neuropathic pain development is complex and needs to be studied further in order to clarify its nature and components. This present study is meant to reveal a step towards further understanding the role of Arc, c-Fos and Zif268 in neuropathic pain. Moreover, this study might contribute to the knowledge base for further research on better therapeutic possibilities for neuropathic pain.

Glutaminase 1 is a potential biomarker for chronic post-surgical pain in the rat dorsal spinal cord using differential proteomics.

Chronic post-surgical pain (CPSP) is a normal and significant symptom in clinical surgery, such as breast operation, biliary tract operation, cesarean operation, uterectomy and thoracic operation. Severe chronic post-surgical pain could increase post-surgical complications, including myocardial ischemia, respiratory insufficiency, pneumonia and thromboembolism. However, the underlying mechanism is still unknown. Herein, a rat CPSP model was produced via thoracotomy. After surgery, in an initial study, 5 out of 12 rats after surgery showed a significant decrease in mechanical withdrawal threshold and/or increase in the number of acetone-evoked responses, and therefore classified as the CPSP group. The remaining seven animals were classified as non-CPSP. Subsequently, open-chest operation was performed on another 30 rats and divided into CPSP and non-CPSP groups after 21-day observation. Protein expression levels in the dorsal spinal cord tissue were determined by 12.5% SDS-PAGE. Finally, differently expressed proteins were identified by LC MS/MS and analyzed by MASCOT software, followed by Gene Ontology cluster analysis using PANTHER software. Compared with the non-CPSP group, 24 proteins were only expressed in the CPSP group and another 23 proteins expressed differentially between CPSP and non-CPSP group. Western blot further confirmed that the expression of glutaminase 1 (GLS1) was significantly higher in the CPSP than in the non-CPSP group. This study provided a new strategy to identify the spinal proteins, which may contribute to the development of chronic pain using differential proteomics, and suggested that GLS1 may serve as a potential biomarker for CPSP.

Influence of Dexmedetomidine on Toxicity of Intrathecal Ketamine on Neonatal Rat Spinal Function

Purpose: To investigate the influence of dexmedetomidine on the effect of ketamine on developing spinal cord.Methods: Postnatal day 3 (P3) and postnatal day 7 (P7) rat pups received intrathecal ketamine (10 mg/kg b.wt) and/or dexmedetomidine (10 μg/kg b.wt). Spinal reflex function was assessed by evaluating the sensory stimuli based on mechanical withdrawal threshold. Spinal tissue was analysed for activated caspase-3 using monoclonal anti-activated caspase-3. Apoptosis count of the spinal tissue was also measured by Fluoro-Jade C staining while glial reactivity was assessed by ionized calcium binding adapter molecule 1 on Day 7 day following injection. Long-term spinal function in rat pups on postnatal day 35 (P35) was evaluated by measuring the hindlimb withdrawal threshold and gait analysis.Results: Exposure to intrathecal ketamine at 10 mg/kg in P3 and P7 pups decreased mechanical withdrawal thresholds and increase apoptosis and microglial activation in the spinal cord. Altered spinal function, as presented by a decrease in mechanical withdrawal threshold and altered static gait, was observed in P35 rats exposed to intrathecal ketamine on P3. Dexmedetomidine administration did not alter the spinal function and histology of spinal tissue. Co-administration of dexmedetomidine and ketamine suggests that the former reduced apoptosis counts, altered glial responses, increased mechanical withdrawal threshold and improved gait.Conclusion: Dexmedetomidine protects against intrathecal ketamine-induced spinal toxicity in neonatal rats.Keywords: Dexmedetomidine, Intrathecal ketamine, Spinal toxicity, Mechanical withdrawal threshold,Glial reactivity, Gait analysis, Activated caspase-3, Apoptosis count

Assessment of diabetic peripheral neuropathy in streptozotocin-induced diabetic rats with magnetic resonance imaging

To determine the role of magnetic resonance (MR) imaging and quantitative T2 value measurements in the assessment of diabetic peripheral neuropathy (DPN). Sequential MR imaging, T2 measurement, and quantitative sensory testing of sciatic nerves were performed in streptozotocin-induced diabetic rats (n = 6) and normal control rats (n = 6) over a 7-week follow-up period. Histological assessment was obtained from 48 diabetic rats and 48 control rats once weekly for 7weeks (n = 6 for each group at each time point). Nerve signal abnormalities were observed, and the T2 values, mechanical withdrawal threshold (MWT), and histological changes were measured and compared between diabetic and control animals. Sciatic nerves in the diabetic rats showed a gradual increase in T2 values beginning at 2weeks after the induction (P = 0.014), while a decrease in MWT started at 3weeks after the induction (P = 0.001). Nerve T2 values had a similar time course to sensory functional deficit in diabetic rats. Histologically, sciatic nerves of diabetic rats demonstrated obvious endoneural oedema from 2 to 3weeks after the induction, followed by progressive axonal degeneration, Schwann cell proliferation, and coexistent disarranged nerve regeneration. Nerve T2 measurement is potentially useful in detecting and monitoring diabetic neuropathy. • Sciatic nerves in diabetic rats showed a gradual increase in T2 values • Nerve T2 values were negatively correlated with sensory function impairment • Longitudinal T2 values can be used to monitor the disease progress • Nerve degeneration contributed mainly to progressive prolongation of nerve T2 values.

Peripheral Neuropathy Induces Cutaneous Hypersensitivity in Chronically Spinalized Rats

The present study was aimed at the issue of whether peripheral nerve injury-induced chronic pain is maintained by supraspinal structures governing descending facilitation to the spinal dorsal horn, or whether altered peripheral nociceptive mechanisms sustain central hyperexcitability and, in turn, neuropathic pain. We examined this question by determining the contribution of peripheral/spinal mechanisms, isolated from supraspinal influence(s), in cutaneous hypersensitivity in an animal model of peripheral neuropathy. Adult rats were spinalized at T8-T9; 8 days later, peripheral neuropathy was induced by implanting a 2-mm polyethylene cuff around the left sciatic nerve. Hind paw withdrawal responses to mechanical or thermal plantar stimulation were evaluated using von Frey filaments or a heat lamp, respectively. Spinalized rats without cuff implantation exhibited a moderate decrease in mechanical withdrawal threshold on ~day 10 (P < 0.05) and in thermal withdrawal threshold on ~day 18 (P < 0.05). However, cuff-implanted spinalized rats developed a more rapid and significant decrease in mechanical (~day 4; P < 0.001) and thermal (~day 10; P < 0.05) withdrawal thresholds that remained significantly decreased through the duration of the study. Our findings demonstrate an aberrant peripheral/spinal mechanism that induces and maintains thermal and to a greater degree tactile cutaneous hypersensitivity in the cuff model of neuropathic pain, and raise the prospect that altered peripheral/spinal nociceptive mechanisms in humans with peripheral neuropathy may have a pathologically relevant role in both inducing and sustaining neuropathic pain.

Salvinorin A Reduces Mechanical Allodynia and Spinal Neuronal Hyperexcitability Induced by Peripheral Formalin Injection

BackgroundSalvinorin A (SA), the main active component of Salvia Divinorum, is a non-nitrogenous kappa opioid receptor (KOR) agonist. It has been shown to reduce acute pain and to exert potent antinflammatory effects. This study assesses the effects and the mode of action of SA on formalin-induced persistent pain in mice. Specifically, the SA effects on long-term behavioural dysfuctions and changes in neuronal activity occurring at spinal level, after single peripheral formalin injection, have been investigated. Moreover, the involvement of microglial and glial cells in formalin-induced chronic pain condition and in SA-mediated effects has been evaluated.ResultsFormalin induced a significant decrease of mechanical withdrawal threshold at the injected and contralateral paw as well as an increase in the duration and frequency, and a rapid decrease in the onset of evoked activity of the nociceptive neurons 7 days after formalin injection. SA daily treatment significantly reduced mechanical allodynia in KOR and cannabinoid receptor 1 (CB1R) sensitive manner. SA treatment also normalized the spinal evoked activity. SA significantly reduced the formalin-mediated microglia and astrocytes activation and modulated pro and anti-inflammatory mediators in the spinal cord.ConclusionSA is effective in reducing formalin-induced mechanical allodynia and spinal neuronal hyperactivity. Our findings suggest that SA reduces glial activation and contributes in the establishment of dysfunctions associated with chronic pain with mechanisms involving KOR and CB1R. SA may provide a new lead compound for developing anti-allodynic agents via KOR and CB1R activation.