Paw Withdrawal Thermal Latency Research Articles

Activation of GABAB Receptor Suppresses Diabetic Neuropathic Pain through Toll-Like Receptor 4 Signaling Pathway in the Spinal Dorsal Horn.

Diabetic neuropathic pain (DNP) is a prevalent complication in diabetes patients. Neuronal inflammation and activation of Toll-like receptor 4 (TLR4) are involved in the occurrence of DNP. However, the underlying mechanisms remain unclear. Downregulation of gamma-aminobutyric acid B (GABAB) receptor contributes to the DNP. GABAB receptor interacts with NF-κB, a downstream signaling factor of TLR4, in a neuropathic pain induced by chemotherapy. In this study, we determined the role of TLR4/Myd88/NF-κB signaling pathways coupled to GABAB receptors in the generation of DNP. Intrathecal injection of baclofen (GABAB receptor agonist), LPS-RS ultrapure (TLR4 antagonist), MIP (MyD88 antagonist), or SN50 (NF-κB inhibitor) significantly increased paw withdrawal threshold (PWT) and paw withdrawal thermal latency (PWTL) in DNP rats, while intrathecal injection of saclofen (GABAB receptor blocker) decreased PWT and PWTL in DNP rats. The expression of TLR4, Myd88, NF-κBp65, and their downstream components IL-1 and TNF-α was significantly higher in the spinal cord tissue in DNP rats compared to control rats. Following inhibition of TLR4, Myd88, and NF-κB, the expression of IL-1 and TNF-α decreased. Activation of GABAB receptors downregulated the expression of TLR4, Myd88, NF-κBp65, IL-1, and TNF-α. Blockade of GABAB receptors significantly upregulated expression of TLR4, Myd88, NF-κBp65, IL-1, and TNF-α. These data suggest that activation of the TLR4/Myd88/NF-κB signaling pathway is involved in the occurrence of DNP in rats. Activation of GABAB receptor in the spinal cord may suppress the TLR4/Myd88/NF-κB signaling pathway and alleviate the DNP.

Effect of gbpapentin on the expression of Nrf2 in dorsal root ganglion neurons of diabetic neuropathic pain rats

Objective To investigate the effect of systemic administration of gabapentin on the expression of Nrf2 in dorsal root ganglion neurons in a rat model of diabetic neuropathic pain (DNP). Methods 60 male SD rats weighing 200-220 g were randomly divided into 4 groups (n=15 each): control group (group C), DNP group, saline group (SC+ DNP group) and gabapentin group (GBP+ DNP group). Diabetes was induced with streptozocin (70 mg/kg) subcutaneously. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at 1d before and 3, 7, 10, 14, 21, and 28 d after streptozocin injection, and 21 and 28 d after administration of gabapentin (50 mg·kg-1·d-1, once) intraperitoneally. Saline and gabapentin (50 mg·kg-1·d-1) were given intraperitoneally once a day for 7 d starting from 15 d after streptozocin injection in saline and gabapentin groups respectively. The expression of Nrf2 was determined at 21 d after streptozocin injection by immune-histochemistry and Western blot. Results Compared with group C, PWMT(3.95±0.57) was significantly decreased, PWTL(6.3±0.81) was significantly shorten, Nrf2 in expression was significantly decreased in DNP, SC+ DNP and group (P 0.05). Conclusion Systemic administration of gabapentin attenuates diabetic neuropathic pain by enhancing the expression of Nrf2 in DRG neurons. (Chin J Endocrinol Metab, 2018, 34: 951-954) Key words: Gabapentin; Diabetic neuropathic pain; Dorsal root ganglion; Nrf2

Role of mammalian target of rapamycin in synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain

Objective To evaluate the role of mammalian target of rapamycin(mTOR) in the synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain. Methods Twenty-four healthy adult male Sprague-Dawley rats, weighing 180-240 g, were divided into 4 groups(n=6 each) by using a random number table method: control group(group C), inflammatory pain group(group IP), dimethyl sulfoxide(DMSO) group and mTOR inhibitor rapamycin group(group R). Inflammatory pain model was established by subcutaneous injection of 50 μl bee venom into the plantar surface of the left hindpaw. The equal volume of normal saline was subcutaneously injected into the plantar surface of the left hindpaw in group C. In group DMSO, 2% DMSO was administered by intragastric gavage for 3 days, 1 ml per day, and the inflammatory pain model was established at 1 h after administration on 3rd day. In group R, rapamycin was administered by intragastric gavage for 3 days, 1 ml per day, and the inflammatory pain model was established at 1 h after administration on 3rd day. Mechanical paw withdrawal threshold(MWT) and thermal paw withdrawal latency(TWL) were measured at 2 h after establishing the model. The rats were sacrificed after measurement of the pain threshold, and hippocampi were removed to prepare hippocampal slices. Hippocampal CA1 region and dentate gyrus(DG region) were located with an inverted microscope. Planar microelectrode array technique was used to record the number of channels and the standardized amplitude of evoked effective field excitatory postsynaptic potentials(fEPSPs)(fEPSPs amplitude>20% of the baseline value) at different stimulus intensities. Results Compared with group C, MWT was significantly decreased, TWL was shortened, the number of effective fEPSP channels at different stimulus intensities was increased, and the amplitude of standardized fEPSPs in hippocampal DG and CA1 regions was increased in group IP(P 0.05). Compared with group IP, MWT was significantly increased, TWL was prolonged, the number of effective fEPSP channels at different stimulus intensities was decreased, and the amplitude of standardized fEPSPs in hippocampal DG and CA1 regions was decreased in group R(P 0.05). Conclusion mTOR is involved in the changes in the synaptic plasticity of entorhinal area-hippocampal formation in rats with inflammatory pain. Key words: Receptor-interacting protein serine-threonine kinases; Hippocampus; Entorhinal cortex; Pain; Inflammation; Neuronal plasticity

Gene therapy by lentivirus-mediated RNA interference targeting extracellular-regulated kinase alleviates neuropathic pain in vivo.

Neuropathic pain is an abnormal pain, which is related to the activation of extracellular-regulated kinase (ERK) signaling. This study was to investigate the effects of ERK knockdown via lentivirus-mediated RNA interference on allodynia in rats with chronic compression of the dorsal root ganglia (DRG) and to uncover the potential mechanisms. The model of chronic compression of the dorsal root ganglia (CCD) was established in rats by surgery. Gene silence was induced by injecting rats with lentivirus expressing ERK short hairpin RNA (shRNA). Behavioral test was performed by calculating paw withdrawal mechanical threshold (PWMT) and thermal paw withdrawal latency (TPWL). We firstly generated lentivirus expressing ERK shRNA to downregulate ERK gene expression both in vitro and in vivo by using Western blot analysis and quantitative reverse transcription polymerase chain reaction. In CCD, ERK mRNA, and protein levels in DRG neurons were dramatically increased, accompanied with decreased PWMT and TPWL. Lentivirus-mediated RNA interference decreased ERK gene expression in DRG neurons and normalized the PWMT and TPWL in CCD rats, but not in rats infected with lentivirus expressing negative control shRNA. Further, calcium responses of DRG neurons to the hypotonic solution and 4α-phorbol 12,13-didecanoate were enhanced in CCD rats, which were suppressed by lentivirus-mediated ERK gene silence. Finally, the levels of transient receptor potential vanilloid 4 gene expressions in DRG neurons and L4 to L5 spinal cord isolated from CCD rats were dramatically upregulated, which were reversed by lentivirus-mediated ERK gene knockdown. Lentivirus-mediated RNA interference (RNAi) silencing targeting ERK might reverse CCD-induced neuropathic pain in rats through transient receptor potential vanilloid 4.

Vascular endothelial growth factor antibody attenuates diabetic peripheral neuropathic pain in rats

To explore the role of vascular endothelial growth factor (VEGF) in diabetic peripheral neuropathic pain in rats. Methods: Twenty-four adult male Sprague-Dawley rats aged 8 weeks were randomly divided into 3 groups (n=8 per group). The control group (C group): rats were intraperitoneally injected with sodium citrate solution at 10 mL/kg; the model group (M group): rats were intraperitoneally injected with streptozotocin at 65 mg/kg; the treatment group (T group): rats received intraperitoneal injection of anti-VEGF antibody (10 mg/kg) at the 1st, 3rd, 7th, 10th day after STZ treatment. Meanwhile, rats of C and M group were received with the same volume of sodium citrate solution. Blood glucose was measured before 1 day or at the 1st, 3rd, 7th or 14th day after receiving STZ. Body weight, paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured before 1 day or at the 1st, 3rd, 5th, 7th, 10th or 14th day after receiving STZ. All lumbar spinal cords were dissected to examine the p-protein kinase B (p-Akt) and transient receptor potential vanilloid 1 (TRPV1) expression by Western blot. Results: After injection with STZ, the body weight showed significant differences at some time point between the M, T or C group (P<0.01); body weight of rat in the C group were increased gradually. Compared with the C group, the fast blood glucose in the M or the T Group at the same time points were increased significantly (P<0.01). The PWMT and PWTL of the M, T or C group were significant difference among various time points (P<0.01). The PWMT and PWTL in the M or T group were obviously reduced compared with those in the C group (P<0.01). Compared with the M group, the PWMT and PWTL in the T group were increased at the 10th or 14th day (P<0.01 or P<0.05). Compared with the C group, the p-Akt and TRPV1 levels in the M and T group were increased (P<0.01). Compared with the M group, p-Akt and TRPV1 levels in T group were decreased (P<0.01). Conclusion: VEGF is able to regulate the expression of TRPV1 through PI3K/Akt pathway, which contributes to diabetic peripheral neuropathic pain in rats. Anti-VEGF treatment may be useful for alleviation of diabetic peripheral neuropathic pain.

Changes in expression of IB4 and CGRP in neurons in dorsal root ganglia of rats with neuropathic pain

Objective To evaluate the changes in the expression of isolectin B4 (IB4) and calcium gene-related peptide (CGRP) in neurons in dorsal root ganglion (DRG) of rats with neuropathic pain (NP). Methods Forty healthy male Sprague-Dawley rats, weighing 250-280 g, were divided into 2 groups (n=20 each) using a random number table method: solvent group (group S) and group NP.NP was induced by intraperitoneally injecting resiniferatoxin 210 μg/kg, and the solvent of resiniferatoxin was intraperitoneally injected in group S. The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured in 5 rats selected before establishing the model and at 1, 3, 7 and 42 days after establishing the model.Five rats were sacrificed at 1, 3, 7 and 42 days after establishing the model, and the L4-6 segments of the DRGs were removed to determine the expression of CGRP and IB4 in neurons using immunofluorescence. Results Compared with the baseline before establishing the model, the MWT was significantly decreased at 3, 7 and 42 days after establishing the model, and the TWL was prolonged at 1, 3, 7 and 42 days after establishing the model in group NP (P<0.05). Compared with group S, the MWT was significantly decreased at 3, 7 and 42 days after establishing the model, the TWL was prolonged at 1, 3, 7 and 42 days after establishing the model, and the expression of IB4 and CGRP in neurons in DRGs was down-regulated at 1, 3, 7 and 42 days after establishing the model in group NP (P<0.05). Conclusion Down-regulated expression of IB4 expression in neurons in DRGs may be involved in the development and maintenance of mechanical hypersensitivity to pain, and down-regulated expression of CGRP may be involved in the development and maintenance of thermal analgesia in rats with NP. Key words: Neuralgia; Ganglia, spinal; Plant lectins; Calcitonin gene-related peptide

Effect of mito-TEMPO, a mitochondria-targeted antioxidant, in rats with neuropathic pain.

The therapeutic effects of mitochondria-targeted antioxidants have been demonstrated in many pathological conditions, but their effect on neuropathic pain remains unclear. The objective was to study the therapeutic effects and mechanisms of mito-TEMPO (MT), as a nitroxide conjugated with a triphenylphosphonium moiety, on neuropathic pain in rats. Rats were randomly assigned to sham control (sham), chronic constrictive injury (CCI) or MT treatment groups (sham+MT and CCI+MT). All animals received CCI of the left sciatic nerve except those in the sham group. Overall, 0.7 mg/kg of MT was intraperitoneally injected once daily for 14 consecutive days starting from day 7 after surgery. Mechanical paw withdrawal threshold and thermal paw withdrawal latency were detected to assess pain behavior. Malondialdehyde and reduced glutathione content and total superoxide dismutase activity of serum and spinal cord tissues were estimated to assess oxidative stress levels. Mitochondrial morphology and dynamin-related proteins were used to evaluate mitochondrial function, such as fusion [Mitofusin (Mfn) and optic atrophy 1 gene protein (OPA1)] and fission [dynamin-related protein (DRP1) and Fis1]. Paw withdrawal threshold and thermal paw withdrawal latency were significantly increased in the CCI+MT group compared with the CCI group. The malondialdehyde content was decreased whereas glutathione content and superoxide dismutase activity were increased in the serum of CCI+MT rats. Furthermore, MT substantially attenuated the elevated number and decreased size of mitochondria induced by CCI. Finally, MT significantly increased expressions of Mfn1 and OPA1 and significantly decreased expression of DRP1 and Fis1. The mitochondria-targeted antioxidant MT relieved neuropathic pain induced by CCI by protecting mitochondria against oxidative stress injury.

Attenuation of Remifentanil-Induced Hyperalgesia by Betulinic Acid Associates with Inhibiting Oxidative Stress and Inflammation in Spinal Dorsal Horn

Remifentanil-induced hyperalgesia (RIH) is known to be associated with oxidative stress and inflammation. Betulinic acid (BA) was reported to reduce visceral pain owing to its anti-oxidative and anti-inflammatory potential. Here, we ­explored whether BA can attenuate RIH through inhibiting oxidative stress and inflammation in spinal dorsal horn. Sprague-Dawley rats were randomly divided into 4 groups: Control, Incision, RIH, and RIH pre-treated with BA. After pretreated with BA (25 mg/kg, i.g.) for 7 days, rats were subcutaneously infused with remifentanil (40 μg/kg) for 30 min during right plantar incision surgery to induce RIH. The paw withdrawal mechanical threshold (PWMT), paw withdrawal thermal latency (PWTL), spinal oxidative stress and inflammatory mediators were determined. Intraoperative remifentanil infusion induced postoperative hyperalgesia, as evidenced by the significant decrease in PWMT and PWTL (p < 0.01), and the significant increase in oxidative stress and inflammation evidenced by up-regulations of malondialdehyde, 3-nitrotyrosine, interleukin-1β and tumour necrosis factor-α (p < 0.01) in spinal dorsal horn and matrix metalloproteinase-9 (MMP-9) activity (p < 0.01) in dorsal root ganglion, as well as a decrease in manganese superoxide ­dismutase activity (p < 0.01) compared with control and ­incision groups. All these results mentioned above were markedly reversed by pre-treatment with BA (p < 0.01) compared with RIH group. These findings demonstrated that BA can effectively attenuate RIH, which associates with potentially inhibiting oxidative stress and subsequently down-regulating MMP-9-related pro-inflammatory cyokines in spinal dorsal horn.

Effect of oxycodone on function of GABAA receptors in dorsal root ganglion neurons of rats with neuropathic pain

Objective To evaluate the effect of oxycodone on function of GABAA receptors in dorsal root ganglion (DRG) neurons of rats with neuropathic pain (NP). Methods Thirty-six adult male Sprague-Dawley rats, weighing 180-220 g, aged 10 weeks, were allocated into 3 groups (n=12 each) using a random number table method: sham operation group (group S), group NP and oxycodone group (group O). The sciatic nerve was only isolated but not ligated in group S. NP was induced by chronic constriction injury.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 chromic catgut.Oxycodone 15 μg/kg was intraperitoneally injected once a day for 14 consecutive days from ligating the sciatic nerve to satisfaction in group O. The thermal paw withdrawal latency (TWL) was measured at 1 day before establishing the model (T0) and 3, 5, 7, 10 and 14 days after establishing the model (T1-5). The rats were sacrificed after measurement of pain threshold at T5, and DRG neurons were acutely isolated for recording the amplitude of GABAA receptors-activated currents using whole-cell patch-clamp technique. Results Compared with group S, the TWL was significantly shortened at T1-5, and the amplitude of GABAA receptors-activated currents in DRG neurons was decreased in NP and O groups (P<0.05). Compared with group NP, the TWL was significantly prolonged at T1-5, and the amplitude of GABAA receptors-activated currents in DRG neurons was increased in group O (P<0.05). Conclusion Oxycodone can enhance the function of GABAA receptors-activated currents in DRG neurons and thus enhance GABAA receptors-mediated presynaptic inhibition, which may be related to the mechanism of oxycodone-induced reduction of NP in rats. Key words: Oxycodone; Neuralgia; Ganglia, spinal; Receptors, GABA-A

Relationship between remifentanil-induced hyperalgesia and function of nitrated glutamate transportor-1 and glutamine synthetase in spinal cord of rats with incisional pain

Objective To investigate the relationship remifentanil-induced hyperalgesia and function of nitrated glutamate transportor-1 (GLT-1) and glutamine synthetase (GS) in the spinal cord of rats with incisional pain. Methods Thirty-two male Sprague-Dawley rats, weighing 260-280 g, aged 2-3 months, in which caudal catheters were successfully implanted, were divided into 4 groups (n=8 each) using a random number table method: control group (group C), incisional pain group (group I), remifentanil group (group R) and remifentanil plus incisional pain group (group RI). Normal saline was intravenously infused for 60 min at 0.1 ml·kg-1·min-1 in group C. The model of incisional pain was established, and normal saline was simultaneously infused for 60 min via the tail vein at 0.1 ml·kg-1·min-1 in group I. Remifentanil was infused for 60 min via the tail vein at 1.0 μg· kg-1·min-1 in group R. The model of incisional pain was established, and remifentanil was infused for 60 min via the tail vein at 1.0 μg· kg-1·min-1 in group RI.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion of remifentanil or normal saline (T0) and at 2, 6, 24 and 48 h after infusion (T1-4). The rats were sacrificed after the last measuremnet of pain threshold, and the L4-6 segment of the spinal cord was removed for determination of the expression of GLT-1 and GS (by Western blot) and expression of nitrated GLT-1 (nGLT-1) and nitrated GS (nGS) (by Western blot). Ratios of nGLT-1/GLT-1 and nGS/GS were calculated. Results Compared with group C, the MWT was significantly decreased and TWL was shortened at T1-4, the expression of GLT-1 and GS was down-regulated, the expression of nGLT-1 and nGS was up-regulated, and ratios of nGLT-1/GLT-1 and nGS/GS were increased in I, R and RI groups (P<0.05). Compared with group I and group R, the MWT was significantly decreased and TWL was shortened at T1-4, the expression of GLT-1 and GS was down-regulated, the expression of nGLT-1 and nGS was up-regulated, and ratios of nGLT-1/GLT-1 and nGS/GS were increased in group RI (P<0.05). Conclusion The mechanism of remifentanil-induced hyperalgesia may be related to impaired function of GLT-1 and GS in the spinal cord of rats with incisional pain. Key words: Piperidines; Hyperalgesia; Pain, postoperative; Glutamate plasma membrane transport proteins; Glutamate-ammonia ligase; Spinal cord

1,25-Dihydroxyvitamin D3 attenuates disease severity and induces synoviocyte apoptosis in a concentration-dependent manner in rats with adjuvant-induced arthritis by inactivating the NF-κB signaling pathway.

An aggressive proliferation of synoviocytes is the hallmark of rheumatoid arthritis (RA). Emerging evidence shows that inhibiting the NF-κB signaling pathway with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] may be a therapeutic approach for controlling inflammatory diseases. In this study, we demonstrated the protective effects of three different 1,25(OH)2D3 concentration on adjuvant-induced arthritis (AA) rats through the NF-κB signaling pathway and their pro-apoptotic roles in cultured adjuvant-induced arthritis synoviocytes (AIASs). AA rats were prepared by injecting complete Freund's adjuvant and independently given daily intraperitoneal injection of 1,25(OH)2D3 at concentrations of 50, 100, and 300ng/day/kg. Subsequently, AIASs were isolated from the inflamed joints of AA rats to test the effects of 1,25(OH)2D3 on AIASs in vitro. Intraperitoneal injection of 1,25-(OH)2D3 was found to induce a concentration- and time-dependent improvement in relieving the symptoms of AA. We found an increased paw withdrawal thermal latency (PWTL) in the affected paw of AA rats as the concentration of 1,25-(OH)2D3 increased. 1,25-(OH)2D3 treatment reduced levels of inflammatory factors in synovial tissues of AA rats. In the case of cultured AIASs, 1,25-(OH)2D3 was shown to inhibit cell proliferation and induce cell apoptosis in a concentration-dependent manner. Additionally, 1,25-(OH)2D3 inhibited the activation of the NF-κB signaling pathway. In conclusion, our study provides evidence emphasizing that 1,25(OH)2D3 has the potential to attenuate disease severity in RA potentially due to its contributory role in synoviocyte proliferation and apoptosis. The protective role of 1,25(OH)2D3 against RA depends on the NF-κB signaling pathway.

Effect of Wnt signaling pathway on pathogenesis and intervention of neuropathic pain.

Neuropathic pain (NP) is a common clinical chronic pain with very complex mechanisms. This study explored the function of activated Wnt signaling pathway in NP. A rat model of chronic constriction injury (CCI) was established. Different doses of IWP-2, a Wnt signal inhibitor, were intrathecally injected to observe the behavior indicators at different time-points, including the pain induced by mechanical stimulation and thermal stimulation. The mRNA and protein levels of Wnt-3a, Frizzled 4 and β-catenin in lumbar (L) 4–6 dorsal root ganglion (DRG) of rats in each group, as well as synaptic plasticity-related molecules in DRG region of rats were detected by RT-PCR and western blotting, respectively. Compared with Sham group and Naive group, paw withdrawal thermal latency and paw withdrawal mechanical threshold were significantly decreased after CCI, while synaptic plasticity was increased (P<0.05). Besides, activation of Wnt/β-catenin signaling pathway was observed in rats with CCI. We found that intrathecal injection of IWP-2 effectively relieved the pain behavior and reduced the synaptic plasticity in rats with neuropathic pain after CCI, suggesting that the inactivated Wnt/β-catenin signaling pathway might be the major mechanism responsible for this effect. Our data demonstrated that intrathecal injection of IWP-2 ameliorated neuropathic pain in CCI rats by inhibiting the Wnt/β-catenin pathway.

Role of CXCR4 in dorsal root ganglia in incisional pain in rats

Objective To evaluate the role of C-X-C chemokine receptor type 4 (CXCR4) in the dorsal root ganglia (DRG) in incisional pain in rats. Methods Thirty-two male Sprague-Dawley rats, aged 7-10 weeks, weighing 250-300 g, in which intrathecal catheters were successfully implanted, were divided into 4 groups (n=8 each) using a random number table method: sham operation group (group S), CXCR4 antagonist AMD3100 plus sham operation group (group A+ S), incisional pain group (group I) and CXCR4 antagonist AMD3100 plus incisional pain group (group A+ I). Rats were anesthetized with sevoflurane.AMD3100 20 μg (in 10 μl of normal saline) was intrathecally injected, and no incision was made 30 min later in group A+ S.A 1-cm longitudinal incision was made through skin, fascia and muscle of the plantar aspect of the left hindpaw in group I. AMD3100 20 μg (in 10 μl of normal saline) was intrathecally injected, and 30 min later the model of incisional pain was established in group A+ I.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before surgery and 2, 4, 8, 16 and 24 h after surgery.The rats were sacrificed after the last measurement of pain threshold and the DRGs of the lumbar segment (L4-6) were removed for detecting the expression of CXCR4, phosphorylated extracellular regulated protein kinase (p-ERK) and total ERK (t-ERK) by Western blot. Results Compared with group S, MWT was significantly decreased and TWL was shortened at T1-5 in group I and group A+ I, and the expression of CXCR4 and p-ERK in DRGs was significantly up-regulated (P 0.05). Compared with group I, MWT was significantly increased and TWL was prolonged at T1-5, the expression of CXCR4 and p-ERK in DRGs was down-regulated (P 0.05). Conclusion CXCR4 in DRGs is involved in incisional pain, and the mechanism may be related to activating ERK1/2 signaling pathway in rats. Key words: Pain, postoperative; Dorsal root ganglia; Receptors, CXCR4

Effect of application of pulsed radiofrequency to dorsal root ganglia on activation of spinal astrocytes in a rat model of neuropathic pain

Objective To evaluate the effect of application of pulsed radiofrequency to dorsal root ganglia on activation of spinal astrocytes in a rat model of neuropathic pain (NP). Methods Eighty male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-250 g, were divided into 4 groups (n=20 each) using a random number table method: sham operation group (group Sham), group NP, pulsed radiofrequency group (PRF group) and sham pulsed radiofrequency group (group SPRF). NP was induced by chronic constriction injury (CCI). The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 1 day before CCI and 1, 7, 14 and 21 days after CCI.Four rats were sacrificed at 1 day before CCI and 14 and 21 days after CCI, and the L4-6 segments of the spinal cord were harvested to detect the expression of glial fibrillary acidic protein (GFAP) and interleukin-1beta (IL-1β) by Western blot. Results Compared with group Sham, the MWT was significantly decreased and the TWL was shortened at each time point after CCI, and the expression of GFAP and IL-1β was up-regulated at 14 and 21 days after CCI in NP, PRF and SPRF groups (P 0.05). Conclusion The mechanism by which pulsed radiofrequency reduces NP is probably related to inhibiting spinal astrocyte activation in rats. Key words: Catheter ablation; Ganglia, spinal; Neuralgia; Spinal cord; Astrocytes

Injectable nanocomposite analgesic delivery system for musculoskeletal pain management

Musculoskeletal pain is a major health issue which results from surgical procedures (i.e. total knee and/or hip replacements and rotator cuff repairs), as well as from non-surgical conditions (i.e. sympathetically-mediated pain syndrome and occipital neuralgia). Local anesthetics, opioids or corticosteroids are currently used for the pain management of musculoskeletal conditions. Even though local anesthetics are highly preferred, the need for multiple administration presents significant disadvantages. Development of unique delivery systems that can deliver local anesthetics at the injection site for prolonged time could significantly enhance the therapeutic efficacy and patient comfort. The goal of the present study is to evaluate the efficacy of an injectable local anesthetic nanocomposite carrier to provide sustained analgesic effect. The nanocomposite carrier was developed by encapsulating ropivacaine, a local anesthetic, in lipid nanocapsules (LNC-Rop), and incorporating the nanocapsules in enzymatically crosslinked glycol chitosan (0.3GC) hydrogels. Cryo Scanning Electron Microscopic (Cryo SEM) images showed the ability to distribute the LNCs within the hydrogel without adversely affecting their morphology. The study demonstrated the feasibility to achieve sustained release of lipophilic molecules from the nanocomposite carrier in vitro and in vivo. A rat chronic constriction injury (CCI) pain model was used to evaluate the efficacy of the nanocomposite carrier using thermal paw withdrawal latency (TWL). The nanocomposite carriers loaded with ropivacaine and dexamethasone showed significant improvement in pain response compared to the control groups for at least 7 days. The study demonstrated the clinical potential of these nanocomposite carriers for post-operative and neuropathic pain. Statement of SignificanceAcute or chronic pain associated with musculoskeletal conditions is considered a major health issue, with healthcare costs totaling several billion dollars. The opioid crisis presents a pressing clinical need to develop alternative and effective approaches to treat musculoskeletal pain. The goal of this study was to develop a long-acting injectable anesthetic formulation which can sustain a local anesthetic effect for a prolonged time. This in turn could increase the quality of life and rehabilitation outcome of patients, and decrease opioid consumption. The developed injectable nanocomposite demonstrated the feasibility to achieve prolonged pain relief in a rat chronic constriction injury (CCI) model.

Role of interleukin-17 in peripheral nerve in blood-nerve barrier injury in a rat model of neuropathic pain

Objective To evaluate the role of interleukin-17 (IL-17) in the peripheral nerve in blood-nerve barrier injury in a rat model of neuropathic pain(NP). Methods Forty healthy adult male Sprague-Dawley rats, weighing 250-280 g, were divided into 4 groups (n=10 each) using a random number table method: sham operation group(Sham group), NP group, blank vector group (BV group) and IL-17 siRNA recombinant lentivirus group (siRNA group). The rats were anesthetized with chloral hydrate, and intrathecal catheters were implanted.NP was produced by chronic constriction injury (CCI) to the sciatic nerve.Normal saline 10 μl was intrathecally injected at day 3 after CCI in Sham and NP groups.Blank vector and IL-17 siRNA-GFP recombinant lentivirus 1×107 TU 10 μl were intrathecally injected, and then the tube was washed using normal saline 10 μl with the total volume of 20 μl once a day for 4 consecutive days.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured on day 1 before CCI and days 1, 7, 10, 11, 12, 13 and 14 after CCI.Animals were sacrificed after the last behavior testing, and a segment of injured sciatic nerve 7 mm in length and containing 3 ligatures were harvested for examination of the ultrastructure and for detection of the expression of IL-17, occludin and claudin-5 by Western blot. Results Compared with Sham group, the MWT was significantly decreased, TWL was shortened, the expression of IL-17 was up-regulated, and the expression of occludin and claudin-5 was down-regulated at each time point after CCI in the other groups (P<0.05 or 0.01). Compared with NP group or with BV group, the MWT was significantly increased, TWL was prolonged, the expression of IL-17 was down-regulated, the expression of occludin and claudin-5 was up-regulated (P<0.05), and the pathological changes of sciatic nerve were significantly attenuated in siRNA group. Conclusion The mechanism of blood-nerve barrier injury may be related to increased level of IL-17 in the peripheral nerve in a rat model of NP. Key words: Interleukin-17; Neuralgia; Blood-nerve barrier

Role of spinal CCR5 in remifentanil-induced hyperalgesia in rats with incisional pain

Objective To evaluate the role of spinal C-C motif chemokine receptor 5 (CCR5) in remifentanil-induced hyperalgesia in rats with incisional pain. Methods Thirty-two male Sprague-Dawley rats, weighing 240-260 g, aged 2-3 months, in which intrathecal and caudal catheters were successfully implanted, were divided into 4 groups (n=8 each) using a random number table: control group (group C), CCR5 antagonist maraviroc group (group M), remifentanil plus incisional pain group (group R+ I) and maraviroc plus remifentanil plus incisional pain group (group M+ R+ I). Phosphate buffer solution (PBS) 10 μl was intrathecally injected and normal saline was infused for 60 min at 1 μg·kg-1·min-1 via the caudal vein in group C. Maraviroc 100 pmol (in 10 μl of PBS) was intrathecally injected and normal saline was infused for 60 min at 1 μg·kg-1·min-1 via the caudal vein in group M. PBS 10 μl was intrathecally injected, then the model of incisional pain was established, and remifentanil 1 μg·kg-1·min-1 was infused for 60 min via the caudal vein in group R+ I.Maraviroc 100 pmol (in 10 μl of PBS) was intrathecally injected, then the model of incisional pain was established, and remifentanil 1 μg·kg-1·min-1 was infused for 60 min via the caudal vein in group M+ R+ I.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 24 h before infusion of remifentanil or normal saline (T0) and 2, 6, 24 and 48 h after stopping infusion (T1-4). The rats were sacrificed after the last measurement of pain threshold, and L4-6 segments of the spinal cord were removed for determination of the expression of glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule-1 (Iba-1) by Western blot. Results Compared with group C, the MWT was significantly decreased and TWL was shortened at T1-4, and the expression of GFAP and Iba-1 in the spinal cord was up-regulated in R+ I and M+ R+ I groups (P 0.05). Compared with group R+ I, the MWT was significantly increased and TWL was prolonged at T1-4, and the expression of GFAP and Iba-1 in the spinal cord was down-regulated in group M+ R+ I (P<0.05). Conclusion Spinal CCR5 is involved in remifentanil-induced hyperalgesia in the rats with incisional pain, and the mechanism may be related to activating astrocytes and microglias. Key words: Piperidines; Hyperalgesia; Pain, postoperative; Receptors, CCR5; Spinal cord

Electro-acupuncture stimulation prevents remifentanil-induced postoperative hyperalgesia by suppressing spinal microglia in rats.

The aim of the present study was to assess the effect of electro-acupuncture (EA) stimulation on remifentanil-induced postoperative hyperalgesia (RIPH) and the possible involvement of spinal microglia suppression. A model of RIPH was established using adult male Sprague-Dawley rats by administration of remifentanil at 0.08 mg/kg intravenously for 60 min. The Huantiao and Yanglingquan acupoints were stimulated continuously by EA (2 Hz, ~1 mA) for 90 min from before paw incision to the end of remifentanil administration. Sham acupoints were stimulated by EA in the sham group. Paw withdrawal threshold (PWT) and paw thermal withdrawal latency (PWL) were determined. Cluster of differentiation (CD)11b, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 levels in spinal cord samples were measured using immunohistochemistry and ELISA. PWT and PWL values were decreased following the administration of remifentanill; however, following EA, PWT and PWL values increased compared with the sham group (P<0.05), indicating that EA alleviates remifentanil-induced RIPH. CD11b, TNF-α, IL-1β and IL-6 levels were increased following remifentanil administration and these effects were counteracted by EA (all P<0.05). In the sham group, no significant differences were observed in PWT and PWL values or CD11b, TNF-α, IL-1β and IL-6 levels compared with the control group, suggesting that EA was responsible for the reduction in CD11b and pro-inflammatory cytokine expression following remifentanil administration. The results of the present study demonstrated that EA at the Huantiao and Yanglingquan acupoints may reduce remifentanil-induced postoperative hyperalgesia, likely by inhibiting spinal microglia via reduction of CD11b and pro-inflammatory cytokine expression. However, these results are preliminary and require further validation.

β-Elemene alleviates bone cancer-related pain in rats by modulating N-methyl-D-aspartate receptor 2B subunit

Purpose : To investigate the effect of β-elemene on bone cancer-related pain (BCRP) and its underlying mechanism in a rat model. Methods : A rat model of BCRP was established by injection of Walker 256 cells into the medullary cavity in adult male Sprague-Dawley rats. The effects of β-elemene on pain-related behaviours were assessed by paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL). Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to analyse the transcription and translation levels of N-methyl-D-aspartate receptor 2B subunit (NR2B), respectively. Results : This study found that PWMT and PWTL were significantly decreased in rats as evidence of successful establishment of a rat model of BCRP. Behavioral test showed that β-elemene treatment could attenuate pain-related behaviours. This analgesic effect began 1 h after drug injection, reached the highest value at 2 h, declined at 4 h, and disappeared by 24 h. qRT-PCR and western blotting indicated that intrathecal administration of β-elemene down-regulated mRNA and protein levels of NR2B. Conclusion : The results show that β-elemene alleviates behaviours related to pain in a rat model of BCRP probably by modulating NR2B, thus indicating a new potential treatment method for cancerrelated pain. Keywords : β-Elemene, Bone cancer, Pain relief, N-methyl-D-aspartate receptor 2B subunit (NR2B), Sensitisation

Effects of Cannabinoid Type 2 Receptor Agonist AM1241 on Morphine-Induced Antinociception, Acute and Chronic Tolerance, and Dependence in Mice

Morphine is a potent opioid analgesic used to alleviate moderate or severe pain, but the development of drug tolerance and dependence limits its use in pain management. Previous studies showed that cannabinoid type 2 (CB2) receptor ligands may modulate opioid effects. However, there is no report of the effect of CB2 receptor agonist on acute morphine tolerance and physical dependence. We therefore investigated the effect of a CB2 receptor agonist (AM1241) on morphine-induced morphine tolerance and physical dependence in mice. Repeated coadministration of AM1241 (1 or 3mg/kg intraperitoneally) and morphine (10mg/kg subcutaneously) for 7days increased the mechanical paw withdrawal threshold in mice as measured by the von Frey filament test, and 3mg/kg AM1241 in combination with morphine increased the thermal paw withdrawal latency as measured by the hot-plate test. Combination with 3mg/kg AM1241 and morphine increased acute morphine antinociception. Coadministration of 1 or 3mg/kg AM1241 and morphine reduced acute morphine tolerance, and 3mg/kg AM1241 reduced chronic morphine tolerance. Coadministration of 1 or 3mg/kg AM1241 and morphine reduced naloxone-precipitated withdrawal jumping, but not diarrhea. Coadministration of AM1241 and morphine did not inhibit spontaneous locomotor activity. Pretreatment with 3mg/kg AM1241 decreased the chronic morphine-induced Iba1 expression in spinal cord. Coadministration of AM1241 (3 mg/kg) reduced the production of interleukin-1β, tumor necrosis factor-α, and interleukin-6 induced by long-term and acute morphine treatment. Our findings suggest that the coadministration of the CB2 receptor agonist and morphine could increase morphine antinociception and reduce morphine tolerance and physical dependence in mice.Perspective:The combination of a CB2 agonist and morphine may provide a new strategy for better treatment of acute and chronic pain and prevention of opioid tolerance and dependence. This finding may also provide a clue for the treatment of opioid tolerance and dependence in clinics.