Chinese Tuina remodels the synaptic structure in neuropathic pain rats by downregulating the expression of N-methyl D-aspartate receptor subtype 2B and postsynaptic density protein-95 in the spinal cord dorsal horn.

OBJECTIVE: To investigate whether the Chinese massage system, Tuina, exerts analgesic effects in a rat model of chronic constriction injury (CCI) by remodeling the synaptic structure in the spinal cord dorsal horn (SCDH). METHODS: Sixty-nine male Sprague-Dawley rats were randomly and evenly divided into the normal group, sham group, CCI group, CCI + Tuina group, CCI + MK-801 [an -methyl D-aspartate receptor subtype 2B (NR2B) antagonist] group, and CCI + MK-801 + Tuina group. The neuropathic pain model was established using CCI with right sciatic nerve ligation. Tuina was administered 4 d after CCI surgery, using pressing manipulation for 10 min, once daily. Motor function was observed with the inclined plate test, and pain behaviors were observed by the Von Frey test and acetone spray test. At 19 d after surgery, the L3-L5 spinal cord segments were removed. Glutamate, interleukin 1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) levels were detected by enzyme-linked immunosorbent assay. The protein expression levels of NR2B and postsynaptic density protein-95 (PSD-95) were detected by Western blot, and the synaptic structure was observed by transmission electron microscopy (TEM). RESULTS: CCI reduced motor function and caused mechanical and cold allodynia in rats, increased glutamate concentration and TNF-α and IL-1ß levels, and increased expression of synapse-related proteins NR2B and PSD-95 in the SCDH. TEM revealed that the synaptic structure of SCDH neurons was altered. Most of these disease-induced changes were reversed by Tuina and intrathecal injection of MK-801 ( < 0.05 or < 0.01). For the majority of experiments, no significant differences were found between the CCI + MK-801 and CCI + MK-801 + Tuina groups. CONCLUSIONS: Chinese Tuina can alleviate pain by remodeling the synaptic structure, and NR2B and PSD-95 receptors in the SCDH may be among its targets.

Repurposing cancer drugs identifies kenpaullone which ameliorates pathologic pain in preclinical models via normalization of inhibitory neurotransmission.

Inhibitory GABA-ergic neurotransmission is fundamental for the adult vertebrate central nervous system and requires low chloride concentration in neurons, maintained by KCC2, a neuroprotective ion transporter that extrudes intracellular neuronal chloride. To identify Kcc2 gene expression­enhancing compounds, we screened 1057 cell growth-regulating compounds in cultured primary cortical neurons. We identified kenpaullone (KP), which enhanced Kcc2/KCC2 expression and function in cultured rodent and human neurons by inhibiting GSK3ß. KP effectively reduced pathologic pain-like behavior in mouse models of nerve injury and bone cancer. In a nerve-injury pain model, KP restored Kcc2 expression and GABA-evoked chloride reversal potential in the spinal cord dorsal horn. Delta-catenin, a phosphorylation-target of GSK3ß in neurons, activated the Kcc2 promoter via KAISO transcription factor. Transient spinal over-expression of delta-catenin mimicked KP analgesia. Our findings of a newly repurposed compound and a novel, genetically-encoded mechanism that each enhance Kcc2 gene expression enable us to re-normalize disrupted inhibitory neurotransmission through genetic re-programming.

Electroacupuncture may alleviate neuropathic pain via suppressing P2X7R expression.

Neuropathic pain is a severe problem that is difficult to treat clinically. Reducing abnormal remodeling of dendritic spines/synapses and increasing the anti-inflammatory effects in the spinal cord dorsal horn are potential methods to treat this disease. Previous studies have reported that electroacupuncture (EA) could increase the pain threshold after peripheral nerve injury. However, the underlying mechanism is unclear. P2X7 receptors (P2X7R) mediate the activation of microglia and participate in the occurrence and development of neuropathic pain. We hypothesized that the effects of EA on relieving pain may be related to the downregulation of the P2X7R. Spinal nerve ligation (SNL) rats were used as a model in this experiment, and 2'(3')-O-(4-benzoyl)benzoyl ATP (BzATP) was used as a P2X7R agonist. We found that EA treatment decreased dendritic spine density, inhibited synaptic reconstruction and reduced inflammatory response, which is consistent with the decrease in P2X7R expression as well as the improved neurobehavioral performance. In contrast to the beneficial effects of EA, BzATP enhanced abnormal remodeling of dendritic spines/synapses and inflammation. Furthermore, the EA-mediated positive effects were reversed by BzATP, which is consistent with the increased P2X7R expression. These findings indicated that EA improves neuropathic pain by reducing abnormal dendritic spine/synaptic reconstruction and inflammation via suppressing P2X7R expression.

Prior perineural or neonatal treatment with capsaicin does not alter the development of spinal microgliosis induced by peripheral nerve injury.

Peripheral nerve injury is associated with spinal microgliosis which plays a pivotal role in the development of neuropathic pain behavior. Several agents of primary afferent origin causing the microglial reaction have been identified, but the type(s) of primary afferents that release these mediators are still unclear. In this study, specific labeling of C-fiber spinal afferents by lectin histochemistry and selective chemodenervation by capsaicin were applied to identify the type(s) of primary afferents involved in the microglial response. Comparative quantitative morphometric evaluation of the microglial reaction in central projection territories of intact and injured peripheral nerves in the superficial (laminae I and II) and deep (laminae III and IV) spinal dorsal horn revealed a significant, about three-fold increase in microglial density after transection of the sciatic or the saphenous nerve. Prior perineural treatment of these nerves with capsaicin, resulting in a selective defunctionalization of C-fiber afferent fibers failed to affect spinal microgliosis. Similarly, peripheral nerve injury-induced increase in microglial density was unaffected in rats treated neonatally with capsaicin known to result in a near-total loss of C-fiber dorsal root fibers. Perineural treatment with capsaicin per se did not evoke a significant increase in microglial density. These observations indicate that injury-induced spinal microgliosis may be attributed to phenotypic changes in injured myelinated primary afferent neurons, whereas the contribution of C-fiber primary sensory neurons to this neuroimmune response is negligible. Spinal myelinated primary afferents may play a hitherto unrecognized role in regulation of neuroimmune and perisynaptic microenvironments of the spinal dorsal horn.

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.

Low-Frequency Electroacupuncture Alleviates Chronic Constrictive Injury-Induced Mechanical Allodynia by Inhibiting NR2B Upregulation in Ipsilateral Spinal Dorsal Horn in Rats.

OBJECTIVE: To study the effects of electroacupuncture (EA) in chronic constrictive injury (CCI) rat model and the expression of N-methyl-D-aspartate receptor type 2B (NR2B) in ipsilateral spinal dorsal horn in rats to explore the analgesic mechanisms of EA. METHODS: According to the random number table, totally 180 rats were evenly divided into a sham group, a CCI group, and an EA group. CCI model was conducted with four 4-0 chromic gut ligatures loosely ligated around the left sciatic nerve 1 cm above the trifurcation. Rats in the EA group received 2 Hz EA therapy bilaterally at acupoints of Zusanli (ST 36) and Sanyinjiao (SP 6) once daily (30 min/d) for 30 days after surgery. Paw withdrawal thresholds (PWTs) were measured on 0 (baseline), 1, 3, 7, 15, 30 days after surgery. Rats were sacrificed on 0, 1, 3, 7, 15 and 30 days after surgery, and the L4-5 segments of spinal cord were removed to detect the expression of NR2B by immunohistochemistry and quantitative polymerase chain reaction. RESULTS: PWTs in the CCI group were significantly lower than the sham group at Day 1-30 after surgery, and reached its lowest at Day 1 (P<0.01). After EA treatment, the PWTs recovered rapidly and were significantly higher than those in the CCI group on 3, 7, 15 and 30 days after surgery (P<0.01). The numbers of NR2B-immunoreactive cells of the CCI group significantly increased after CCI surgery compared with the sham group (P<0.01). Compared with the CCI group, stimulation of EA markedly decreased the numbers of NR2B-immunoreactive cells at Day 3, 7, 15 and 30 (P<0.05). In the sham group, NR2B mRNA was expressed at a low level. It increased after CCI surgery, which increased rapidly at Day 7 (P<0.01) and reached its peak value at Day 15 (P<0.01). After EA stimulation, relative quantity of NR2B mRNA expression was less than that in the CCI group at Day 15 and 30 (P<0.05). CONCLUSIONS: Low frequency of EA had antinociceptive effect in CCI rat model. The analgesic effects of EA might be through the inhibition of NR2B.

The analgesic effect of early hyperbaric oxygen treatment in chronic constriction injury rats and its influence on nNOS and iNOS expression and inflammatory factor production.

Objective To observe the analgesic effect of early hyperbaric oxygen (HBO) treatment in chronic constriction injury (CCI) rats, and to analyze the influence of HBO on the expression of neuronal nitric oxide synthase and inducible nitric oxide synthase and on the levels of inflammatory factors. Methods Rats were assigned into three groups randomly: sham, CCI, and HBO groups. The CCI rat model was established, and HBO treatment at 2.5 ATA (60 min) was given one day after surgery, lasting for five consecutive days. The pain behaviors of the rats were observed at predetermined time points, and the activation of astrocytes at dorsal horns as well as the changes of the synaptic ultrastructures were observed. The expressions of inducible nitric oxide synthase and neuronal nitric oxide synthase were detected by Western blot, and the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) were detected by quantitative real-time PCR. Results Rats in the CCI group developed hyperalgesia when compared with the sham group. Mechanical withdrawal threshold decreased and thermal withdrawal latency shortened in CCI group. Also, astrocytes at the dorsal horn were activated, the synaptic structure was disordered, the expressions of inducible nitric oxide synthase and neuronal nitric oxide synthase were increased significantly, and the release of inflammatory factor (TNF-α and IL-1ß) was up-regulated. However, with early initiation of HBO treatment, rats in the HBO group showed significantly alleviated hyperalgesia, increased mechanical withdrawal threshold, and prolonged thermal withdrawal latency. HBO treatment inhibited astrocyte expression and maintained normal synaptic structure. The expressions of inducible nitric oxide synthase and neuronal nitric oxide synthase were decreased in the dorsal horn, and the release of inflammatory factor (TNF-α and IL-1ß) was reduced. Conclusions Early HBO treatment significantly improves hyperalgesia in rats with neuropathic pain. The decreased expressions of inducible nitric oxide synthase and neuronal nitric oxide synthase and reduced levels of inflammatory factors are important mechanisms by which early HBO helps to alleviate neuropathic pain.

Intraoperative electroacupuncture relieves remifentanil-induced postoperative hyperalgesia via inhibiting spinal glial activation in rats.

Background Accumulating studies have suggested that remifentanil, the widely-used opioid analgesic in clinical anesthesia, can activate the pronociceptive systems and enhance postoperative pain. Glial cells are thought to be implicated in remifentanil-induced hyperalgesia. Electroacupuncture is a complementary therapy to relieve various pain conditions with few side effects, and glial cells may be involved in its antinociceptive effect. In this study, we investigated whether intraoperative electroacupuncture could relieve remifentanil-induced postoperative hyperalgesia by inhibiting the activation of spinal glial cells, the production of spinal proinflammatory cytokines, and the activation of spinal mitogen-activated protein kinases. Methods A rat model of remifentanil-induced postoperative hyperalgesia was used in this study. Electroacupuncture during surgery was conducted at bilateral Zusanli (ST36) acupoints. Behavior tests, including mechanical allodynia and thermal hyperalgesia, were performed at different time points. Astrocytic marker glial fibrillary acidic protein, microglial marker Iba1, proinflammatory cytokines, and phosphorylated mitogen-activated protein kinases in the spinal cord were detected by Western blot and/or immunofluorescence. Results Mechanical allodynia and thermal hyperalgesia were induced by both surgical incision and remifentanil infusion, and remifentanil infusion significantly exaggerated and prolonged incision-induced pronociceptive effects. Glial fibrillary acidic protein, Iba1, proinflammatory cytokines (interleukin-1ß and tumor necrosis factor-α), and phosphorylated mitogen-activated protein kinases (p-p38, p-JNK, and p-ERK1/2) were upregulated after surgical incision, remifentanil infusion, and especially after their combination. Intraoperative electroacupuncture significantly attenuated incision- and/or remifentanil-induced pronociceptive effects, spinal glial activation, proinflammatory cytokine upregulation, and phosphorylated mitogen-activated protein kinase upregulation. Conclusions Our study suggests that remifentanil-induced postoperative hyperalgesia can be relieved by intraoperative electroacupuncture via inhibiting the activation of spinal glial cells, the upregulation of spinal proinflammatory cytokines, and the activation of spinal mitogen-activated protein kinases.

Anti-hyperalgesic effect of Lippia grata leaf essential oil complexed with ß-cyclodextrin in a chronic musculoskeletal pain animal model: Complemented with a molecular docking and antioxidant screening.

BACKGROUND: Due to its unclear pathophysiology, the pharmacological treatment of fibromyalgia is a challenge for researchers. Studies using medicinal plants, such as those from the genus Lippia, complexed with cyclodextrins (CDs) have shown innovative results. OBJECTIVE: The present research intended to evaluate the effect of an inclusion complex containing ß-cyclodextrin (ßCD) inclusion complex with Lippia grata (LG) essential oil in a chronic musculoskeletal pain model, its central activity and its possible interaction with neurotransmitters involved in pain. METHODS: After acid saline-induced chronic muscle pain, male mice were evaluated for primary and secondary hyperalgesia and muscle strength. Moreover, an antagonist assay was performed to assess the possible involvement of the opioidergic, serotonergic and noradrenergic pathways. In addition, Fos protein in the spinal cord was assessed, and a docking study and antioxidant assays were performed. RESULTS: The treatment with LG-ßCD, especially in the dose of 24mg/kg, was able to significantly decrease (p<0.05) the paw withdrawal and muscle threshold. Furthermore, LG-ßCD was shown to affect the opioidergic and serotonergic pathways. There were no significant changes in muscle strength. Fos protein immunofluorescence showed a significant decrease in expression in the dorsal horn of the spinal cord. The main compounds of LG showed through the docking study interaction energies with the alpha-adrenergic and µOpioid receptors. In all antioxidant assays, LG exhibited stronger antioxidant activities than LG-ßCD. CONCLUSION: This study suggested that LG-ßCD could be considered as a valuable source for designing new drugs in the treatment of chronic pain, especially musculoskeletal pain.

Effects of Taxol on Regeneration in a Rat Sciatic Nerve Transection Model.

Recent studies describe taxol as a candidate treatment for promoting central nerve regeneration. However, taxol has serious side effects including peripheral neurotoxicity, and little information is known about the effect of taxol on peripheral nerve regeneration. We investigated the effects of taxol on regeneration in a rat sciatic nerve transection model. Rats were divided into four groups (n = 10): normal saline (i.p.) as the control, Cremophor EL vehicle, and 2 or 6 mg/kg of taxol in the Cremophor EL solution (four times in day-2, 4, 6, and 8), respectively. We evaluated neuronal electrophysiology, animal behaviour, neuronal connectivity, macrophage infiltration, location and expression levels of calcitonin gene-related peptide (CGRP), and expression levels of both nerve growth factors and immunoregulatory factors. In the high-dose taxol group (6 mg/kg), neuronal electrophysiological function was significantly impaired. Licking latencies were significantly changed while motor coordination was unaffected. Neuronal connectivity, macrophage density, and expression levels of CGRP was dramatically reduced. Expression levels of nerve growth factors and immunoregulatory factors was also reduced, while it was increased in the low-dose taxol group (2 mg/kg). These results indicate that taxol can modulate local inflammatory conditions, impair nerve regeneration, and impede recovery of a severe peripheral nerve injury.

Gabapentin decreases microglial cells and reverses bilateral hyperalgesia and allodynia in rats with chronic myositis.

In the present work, we investigated the antinociceptive effect of gabapentin in a chronic myositis model and its interference in spinal glial cells. Chronic myositis was induced by injection of Complete Freund Adjuvant (CFA) into the right gastrocnemius (GS) muscle of rats and tests for evaluating mechanical hyperalgesia, thermal hyperalgesia and tactile allodynia were performed. Pharmacological treatment with gabapentin was administrated intrathecally and 100µg and 200µg doses were tested. For analyzing astrocytes and microglia in the spinal cord, immunochemistry assay was performed. It was found that gabapentin 200µg reverted CFA-induced chronic muscle pain bilaterally, in all applied tests and it was able to attenuate microglial but not astrocytes activation in the dorsal horn of spinal cord. In conclusion, gabapentin was able to inhibit hyperalgesia and allodynia in chronic myositis and also to attenuate spinal microglial activation. Therefore, gabapentin could be used as treatment for targeting chronic muscle pain.

Pioglitazone rapidly reduces neuropathic pain through astrocyte and nongenomic PPARγ mechanisms.

Repeated administration of peroxisome proliferator-activated receptor gamma (PPARγ) agonists reduces neuropathic pain-like behavior and associated changes in glial activation in the spinal cord dorsal horn. As PPARγ is a nuclear receptor, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. However, we recently reported that a single intrathecal (i.t.) injection of pioglitazone, a PPARγ agonist, reduced hyperalgesia within 30 minutes, a time frame that is typically less than that required for genomic mechanisms. To determine the very rapid antihyperalgesic actions of PPARγ activation, we administered pioglitazone to rats with spared nerve injury and evaluated hyperalgesia. Pioglitazone inhibited hyperalgesia within 5 minutes of injection, consistent with a nongenomic mechanism. Systemic or i.t. administration of GW9662, a PPARγ antagonist, inhibited the antihyperalgesic actions of intraperitoneal or i.t. pioglitazone, suggesting a spinal PPARγ-dependent mechanism. To further address the contribution of nongenomic mechanisms, we blocked new protein synthesis in the spinal cord with anisomycin. When coadministered intrathecally, anisomycin did not change pioglitazone antihyperalgesia at an early 7.5-minute time point, further supporting a rapid nongenomic mechanism. At later time points, anisomycin reduced pioglitazone antihyperalgesia, suggesting delayed recruitment of genomic mechanisms. Pioglitazone reduction of spared nerve injury-induced increases in GFAP expression occurred more rapidly than expected, within 60 minutes. We are the first to show that activation of spinal PPARγ rapidly reduces neuropathic pain independent of canonical genomic activity. We conclude that acute pioglitazone inhibits neuropathic pain in part by reducing astrocyte activation and through both genomic and nongenomic PPARγ mechanisms.

pERK1/2 immunofluorescence in rat dorsal horn and paraventricular nucleus neurons as a marker for sensitization and inhibition in the pain pathway.

The aim of the present study was to investigate whether the phosphorylation of ERK1/2 in the rat lumbar dorsal horn and in the parvocellularis part of the paraventricular nucleus can be used to visualize neuronal activity. pERK1/2 fluorescence-immunohistochemistry is specifically suited to mirror neuronal activity in the pain pathway following an acute noxious stimulation. The rat hind paw was either stimulated by noxious heat or by a sequence of mustard oil and noxious heat. Two and 10 min after the thermal stimulation a 3-4-fold increase in cells with pERK1/2 immunoreactivity was observed in lamina I/II of the L3-L5 dorsal horn. The combination of mustard oil with heat led to a 5-6-fold increase in the pERK1/2 signal. The pERK1/2 immunoreactivity in the parvocellularis part of the paraventricular nucleus increased by 2-fold following the heat stimulus, with no further increase following the sequential mustard oil and heat stimulus. A pretreatment with the opioid analgesic morphine or the NMDA antagonist MK-801 markedly attenuated ERK1/2 phosphorylation in both areas of the pain pathway. The present findings support the concept that the pERK1/2 immunofluorescence signal can be used as a quantitative marker for sensitization or inhibition in the pain pathway at spinal and hypothalamic level.

1,25-Dihydroxyvitamin D3 prevents bone loss of the secondary spongiosa in arthritic rats by an increase of bone formation and mineralization and inhibition of bone resorption.

BACKGROUND: Active vitamin D metabolites have been shown to have protective effects in experimental arthritis especially when used as preventive treatment. However, because the direct effects of 1,25-dihydroxyvitamin D3 (1,25(OH) 2D3) on bone formation and resorption are very complex, the net effect of 1,25(OH)2D3 on histomorphometric parameters of bone turnover and mineralisation should be investigated. Therefore, we examined the influence of 1,25(OH)2D3 therapy on arthritis-induced alterations of periarticular and axial bone as well as disease activity, inflammation and joint destruction in antigen-induced arthritis (AIA) of the rat. METHODS: AIA was induced in 20 eight-week-old female Wistar rats. 10 rats without arthritis were used as healthy controls. AIA rats received 1,25(OH)2D3 (0.2 µg/kg/day, i.p., n = 10) or vehicle (n = 10) at regular intervals for 28 consecutive days beginning 3 days before arthritis induction. Bone structure of the secondary spongiosa of the periarticular and axial bone was analyzed using histomorphometry. Parameters of mineralization were investigated using tetracycline labelling. Clinical disease activity, inflammation and joint destruction were measured by joint swelling and histological investigation, respectively. RESULTS: AIA led to significant periarticular bone loss. 1,25(OH)2D3 treatment resulted in a highly significant increase in trabecular bone volume and bone formation rate in comparison to both vehicle-treated AIA and healthy controls at periarticular (p < 0.01 and p < 0.001, respectively) and axial bone (p < 0.001 and p < 0.001, respectively). In addition, bone resorption was reduced by 1,25(OH)2D3 at the axial bone (p < 0.05 vs. vehicle-treated AIA). Joint swelling as well as histological signs of inflammation and joint destruction were not influenced by 1,25(OH)2D3. CONCLUSIONS: The results of the study indicate a marked osteoanabolic effect of 1,25(OH)2D3 presumably due to a substantial increase in mineralization. Thus, 1,25(OH)2D3 may be an effective osteoanabolic treatment principle to antagonize the inflammation-associated suppression of bone formation in rheumatoid arthritis.

Current-modulated electrical stimulation as a treatment for peripheral nerve regeneration in diabetic rats.

PURPOSE: To study if electrical stimulation (ES) can be a useful tool to improve functional recovery after neuronal injury in the peripheral nervous system. METHODS: We studied the effects of 2 Hz of percutaneous ES at different intensities of 1, 10 and 20 mA on peripheral nerve regeneration in rats with diabetes induced by streptozotocin. Non-stimulated diabetic rats were used as the sham-controls. A10-mm gap was made in the rat sciatic nerve by suturing the stumps into silicone rubber tubes and stimulation was carried out every other day for 3 weeks starting 1 week after surgery. RESULTS: After 4 weeks of recovery, the diabetic rats showed that ES of 1 mA or above could increase the cutaneous blood flow in their ipsilateral hindpaw to the injury. ES of 10 mA could improve the amplitude and the area of evoked muscle action potentials with faster target muscle reinnervation. ES of 10 mA could also ameliorate the calcitonin gene-related peptide expression in lamina I-II regions in the dorsal horn ipsilateral to the injury and the number of macrophages in the diabetic distal sciatic nerve. The impaired growth and maturation of regenerating axons in diabetic rat could be improved by ES of 10 mA or above. CONCLUSIONS: All these results lead to the conclusion that ES of 10 mA or above might be necessary to improve regeneration after a dissect lesion of the sciatic nerve in the diabetic rat.