Targeting exosomal double-stranded RNA-TLR3 signaling pathway attenuates morphine tolerance and hyperalgesia.

Long-term morphine use leads to tolerance and hyperalgesia in patients with chronic pain, with neuroinflammation playing a key role, but its underlying mechanisms remain elusive. This study determines that repeated intrathecal morphine injections increase double-stranded RNA (dsRNA) production in spinal neurons, due to downregulated adenosine deaminase RNA specific 1 (ADAR1) expression. Lentivirus-induced ADAR1 elevation decreases the high levels of intracellular dsRNA and attenuates morphine tolerance and hyperalgesia. dsRNA is released into cerebrospinal fluid via exosomes (Exos) after repeated morphine injections and is taken up by microglia for TLR3-TRIF-IL-6 signaling activation. Blocking Exos release with GW4869 or inhibition of TLR3 signaling mitigates neuroinflammation, preventing the development of morphine tolerance and hyperalgesia. Intrathecal injection of TLR3 inhibitor alone shows analgesic effects in neuropathic pain, and co-administration with morphine amplifies the analgesic efficacy of morphine. These findings demonstrate that targeting dsRNA-TLR3 signaling to mitigate neuroinflammation could be a promising treatment for morphine tolerance.

B cells drive neuropathic pain-related behaviors in mice through IgG-Fc gamma receptor signaling.

Neuroimmune interactions are essential for the development of neuropathic pain, yet the contributions of distinct immune cell populations have not been fully unraveled. Here, we demonstrate the critical role of B cells in promoting mechanical hypersensitivity (allodynia) after peripheral nerve injury in male and female mice. Depletion of B cells with a single injection of anti-CD20 monoclonal antibody at the time of injury prevented the development of allodynia. B cell-deficient (muMT) mice were similarly spared from allodynia. Nerve injury was associated with increased immunoglobulin G (IgG) accumulation in ipsilateral lumbar dorsal root ganglia (DRGs) and dorsal spinal cords. IgG was colocalized with sensory neurons and macrophages in DRGs and microglia in spinal cords. IgG also accumulated in DRG samples from human donors with chronic pain, colocalizing with a marker for macrophages and satellite glia. RNA sequencing revealed a B cell population in naive mouse and human DRGs. A B cell transcriptional signature was enriched in DRGs from human donors with neuropathic pain. Passive transfer of IgG from injured mice induced allodynia in injured muMT recipient mice. The pronociceptive effects of IgG are likely mediated through immune complexes interacting with Fc gamma receptors (FcγRs) expressed by sensory neurons, microglia, and macrophages, given that both mechanical allodynia and hyperexcitability of dissociated DRG neurons were abolished in nerve-injured FcγR-deficient mice. Consistently, the pronociceptive effects of IgG passive transfer were lost in FcγR-deficient mice. These data reveal that a B cell-IgG-FcγR axis is required for the development of neuropathic pain in mice.

A novel animal model of symptomatic neuroma for assessing neuropathic pain.

INTRODUCTION: Following amputation, peripheral nerves lack distal targets for regeneration, often resulting in symptomatic neuromas and debilitating neuropathic pain. Animal models can establish a practical method for symptomatic neuroma formation for better understanding of neuropathic pain pathophysiology through behavioral and histological assessments. We created a clinically translatable animal model of symptomatic neuroma to mimic neuropathic pain in patients and assess sexual differences in pain behaviors. METHODS: Twenty-two male and female rats were randomly assigned to one of two experimental groups: (1) neuroma surgery, or (2) sham surgery. For the neuroma experimental group, the tibial nerve was transected in the thigh, and the proximal segment was placed under the skin for mechanical testing at the site of neuroma. For the sham surgery, rats underwent tibial nerve isolation without transection. Behavioral testing consisted of neuroma-site pain, mechanical allodynia, cold allodynia, and thermal hyperalgesia at baseline, and then weekly over 8 weeks. RESULTS: Male and female neuroma rats demonstrated significantly higher neuroma-site pain response compared to sham groups starting at weeks 3 and 4, indicating symptomatic neuroma formation. Weekly assessment of mechanical and cold allodynia among neuroma groups showed a significant difference in pain behavior compared to sham groups (p < 0.001). Overall, males and females did not display significant differences in their pain responses. Histology revealed a characteristic neuroma bulb at week 8, including disorganized axons, fibrotic tissue, Schwann cell displacement, and immune cell infiltration. CONCLUSION: This novel animal model is a useful tool to investigate underlying mechanisms of neuroma formation and neuropathic pain.

Astroglial morphological changes in periaqueductal grey in different pain and itch mice models.

BACKGROUND: The periaqueductal gray (PAG) plays a well-established pivotal role in the descending pain modulatory circuit. The objective of this study was to investigate morphological changes in the astroglia in models that are commonly used in pain and itch studies. METHODS: Five different mouse models of pain, as well as two models of chronic itch, were established using complete Freund's adjuvant (CFA), spared nerve injury (SNI), bone cancer pain (BCP), cisplatin (CIS), and paclitaxel (PTX) for pain, and diphenylcyclopropenone (DCP) and acetone and diethyl ether followed by water (AEW) for chronic itch. von Frey tests and video recordings were employed to assess pain and itching behaviors. The immunofluorescence of S100ß, pSTAT3, and glial fibrillary acidic protein (GFAP) was examined. Two- and three-dimensional studies were used to evaluate changes in astrocyte morphology. RESULTS: Significant scratching was caused by DCP and AEW, whereas the administration of CFA, SNI, BCP, CIS, and PTX produced clear mechanical allodynia. The expression of GFAP in the lPAG/vlPAG was upregulated in CFA, SNI, BCP, CIS, PTX, and DCP mice but decreased in AEW mice. According to Sholl analysis, CFA, SNI, PTX, and BCP mice showed substantially higher astrocyte intersections in the vlPAG, whereas CFA, SNI, BCP, CIS, and DCP mice presented longer peak lengths. In three-dimensional analysis, CFA, SNI, PTX, and DCP mice showed increased astrocyte surface areas, while CIS and AEW mice showed both reduced surface areas and/or volumes of astrocytes. CONCLUSION: The findings showed that different pain and itching conditions have different astrocyte morphologies, and these variations in morphological changes help to explain the pathophysiology of these conditions.

Methylene blue dose-dependently induces cutaneous inflammation and heat hyperalgesia in a novel rat model.

Methylene blue (MB) has been shown to reduce mortality and morbidity in vasoplegic patients after cardiac surgery. Though MB is considered to be safe, extravasation of MB leading to cutaneous toxicity has been reported. In this study, we sought to characterize MB-induced cutaneous toxicity and investigate the underlying mechanisms. To induce MB-induced cutaneous toxicity, we injected 64 adult male Sprague-Dawley rates with 200 µL saline (vehicle) or 1%, 0.1%, or 0.01% MB in the plantar hind paws. Paw swelling, skin histologic changes, and heat and mechanical hyperalgesia were measured. Injection of 1%, but not 0.1% or 0.01% MB, produced significant paw swelling compared to saline. Injection of 1% MB produced heat hyperalgesia but not mechanical hyperalgesia. Pain behaviors were unchanged following injections of 0.1% or 0.01% MB. Global transcriptomic analysis by RNAseq identified 117 differentially expressed genes (111 upregulated, 6 downregulated). Ingenuity Pathway Analysis showed an increased quantity of leukocytes, increased lipids, and decreased apoptosis of myeloid cells and phagocytes with activation of IL-1ß and Fos as the two major regulatory hubs. qPCR showed a 16-fold increase in IL-6 mRNA. Thus, using a novel rat model of MB-induced cutaneous toxicity, we show that infiltration of 1% MB into cutaneous tissue causes a dose-dependent pro-inflammatory response, highlighting potential roles of IL-6, IL-1ß, and Fos. Thus, anesthesiologists should administer dilute MB intravenously through peripheral venous catheters. Higher concentrations of MB (1%) should be administered through a central venous catheter to minimize the risk of cutaneous toxicity.

Reduced sympathetic activity is associated with the development of pain and muscle atrophy in a female rat model of fibromyalgia.

Fibromyalgia (FM) is characterized by chronic widespread musculoskeletal pain accompanied by fatigue and muscle atrophy. Although its etiology is not known, studies have shown that FM patients exhibit altered function of the sympathetic nervous system (SNS), which regulates nociception and muscle plasticity. Nevertheless, the precise SNS-mediated mechanisms governing hyperalgesia and skeletal muscle atrophy in FM remain unclear. Thus, we employed two distinct FM-like pain models, involving intramuscular injections of acidic saline (pH 4.0) or carrageenan in prepubertal female rats, and evaluated the catecholamine content, adrenergic signaling and overall muscle proteolysis. Subsequently, we assessed the contribution of the SNS to the development of hyperalgesia and muscle atrophy in acidic saline-injected rats treated with clenbuterol (a selective ß2-adrenergic receptor agonist) and in animals maintained under baseline conditions and subjected to epinephrine depletion through adrenodemedullation (ADM). Seven days after inducing an FM-like model with acidic saline or carrageenan, we observed widespread mechanical hyperalgesia along with loss of strength and/or muscle mass. These changes were associated with reduced catecholamine content, suggesting a common underlying mechanism. Notably, treatment with a ß2-agonist alleviated hyperalgesia and prevented muscle atrophy in acidic saline-induced FM-like pain, while epinephrine depletion induced mechanical hyperalgesia and increased muscle proteolysis in animals under baseline conditions. Together, the results suggest that reduced sympathetic activity is involved in the development of pain and muscle atrophy in the murine model of FM analyzed.

Evaluation of Anti-Nociceptive, Anti-Inflammatory, and Anti-Fibrotic effects of noscapine against a rat model of Achilles tendinopathy.

During tendinopathy, prolonged inflammation results in fibrosis and the adherence of tendons to the adjacent tissues, causing discomfort and movement disorders. As a natural compound, noscapine has several anti-inflammatory and anti-fibrotic properties. Therefore, we aimed to investigate the effects of noscapine against a rat model of tendinopathy. We created a surgical rat model of Achilles tendon damage to emulate tendinopathy. Briefly, an incision was made on the Achilles tendon, and it was then sutured using an absorbable surgical thread. Immediately, the injured area was topically treated with the vehicle, noscapine (0.2, 0.6, and 1.8 mg/kg), or dexamethasone (0.1 mg/kg) as a positive control. During the 19-day follow-up period, animals were assessed for weight, behavior, pain, and motor coordination testing. On day 20th, the rats were sacrificed, and the tendon tissue was isolated for macroscopic scoring, microscopic (H&E, Masson's trichrome, Ki67, p53) analyses, and cytokine secretion levels. The levels of macroscopic parameters, including thermal hyperalgesia, mechanical and cold allodynia, deterioration of motor coordination, tendon adhesion score, and microscopic indices, namely histological adhesion, vascular prominence and angiogenesis, and Ki67 and p53 levels, as well as fibrotic and inflammatory biomarkers (IL-6, TNF-α, TGF-ß, VEGF) were significantly increased in the vehicle group compared to the sham group (P < 0.05-0.001 for all cases). In contrast, the administration of noscapine (0.2, 0.6, and 1.8 mg/kg) attenuated the pain, fibrosis, and inflammatory indices in a dose-dependent manner compared to the vehicle group (P < 0.05-0.001). Histological research indicated that noscapine 0.6 and 1.8 mg/kg had the most remarkable healing effects. Interestingly, two higher doses of noscapine had impacts similar to those of the positive control group in both clinical and paraclinical assessments. Taken together, our findings suggested that noscapine could be a promising medicine for treating tendinopathies.

Transient immune activation without loss of intraepidermal innervation and associated Schwann cells in patients with complex regional pain syndrome.

BACKGROUND: Complex regional pain syndrome (CRPS) develops after injury and is characterized by disproportionate pain, oedema, and functional loss. CRPS has clinical signs of neuropathy as well as neurogenic inflammation. Here, we asked whether skin biopsies could be used to differentiate the contribution of these two systems to ultimately guide therapy. To this end, the cutaneous sensory system including nerve fibres and the recently described nociceptive Schwann cells as well as the cutaneous immune system were analysed. METHODS: We systematically deep-phenotyped CRPS patients and immunolabelled glabrous skin biopsies from the affected ipsilateral and non-affected contralateral finger of 19 acute (< 12 months) and 6 chronic (> 12 months after trauma) CRPS patients as well as 25 sex- and age-matched healthy controls (HC). Murine foot pads harvested one week after sham or chronic constriction injury were immunolabelled to assess intraepidermal Schwann cells. RESULTS: Intraepidermal Schwann cells were detected in human skin of the finger-but their density was much lower compared to mice. Acute and chronic CRPS patients suffered from moderate to severe CRPS symptoms and corresponding pain. Most patients had CRPS type I in the warm category. Their cutaneous neuroglial complex was completely unaffected despite sensory plus signs, e.g. allodynia and hyperalgesia. Cutaneous innate sentinel immune cells, e.g. mast cells and Langerhans cells, infiltrated or proliferated ipsilaterally independently of each other-but only in acute CRPS. No additional adaptive immune cells, e.g. T cells and plasma cells, infiltrated the skin. CONCLUSIONS: Diagnostic skin punch biopsies could be used to diagnose individual pathophysiology in a very heterogenous disease like acute CRPS to guide tailored treatment in the future. Since numbers of inflammatory cells and pain did not necessarily correlate, more in-depth analysis of individual patients is necessary.

Endothelial Glycocalyx in the Peripheral Capillaries is Injured Under Oxaliplatin-Induced Neuropathy.

Oxaliplatin, a platinum-based anticancer drug, is associated with peripheral neuropathy (oxaliplatin-induced peripheral neuropathy, OIPN), which can lead to worsening of quality of life and treatment interruption. The endothelial glycocalyx, a fragile carbohydrate-rich layer covering the luminal surface of endothelial cells, acts as an endothelial gatekeeper and has been suggested to protect nerves, astrocytes, and other cells from toxins and substances released from the capillary vessels. Mechanisms underlying OIPN and the role of the glycocalyx remain unclear. This study aimed to define changes in the three-dimensional ultrastructure of capillary endothelial glycocalyx near nerve fibers in the hind paws of mice with OIPN. The mouse model of OPIN revealed disruption of the endothelial glycocalyx in the peripheral nerve compartment, accompanied by vascular permeability, edema, and damage to the peripheral nerves. To investigate the potential treatment interventions, nafamostat mesilate, a glycocalyx protective agent was used in tumor-bearing male mice. Nafamostat mesilate suppressed mechanical allodynia associated with neuropathy. It also prevented intra-epidermal nerve fiber loss and improved vascular permeability in the peripheral paws. The disruption of endothelial glycocalyx in the capillaries that lie within peripheral nerve bundles is a novel finding in OPIN. Furthermore, these findings point toward the potential of a new treatment strategy targeting endothelial glycocalyx to prevent vascular injury as an effective treatment of neuropathy as well as of many other diseases. PERSPECTIVE: OIPN damages the endothelial glycocalyx in the peripheral capillaries, increasing vascular permeability. In order to prevent OIPN, this work offers a novel therapy approach that targets endothelial glycocalyx.

Interleukin-17: A Putative Novel Pharmacological Target for Pathological Pain.

Pathological pain imposes a huge burden on the economy and the lives of patients. At present, drugs used for the treatment of pathological pain have only modest efficacy and are also plagued by adverse effects and risk for misuse and abuse. Therefore, understanding the mechanisms of pathological pain is essential for the development of novel analgesics. Several lines of evidence indicate that interleukin-17 (IL-17) is upregulated in rodent models of pathological pain in the periphery and central nervous system. Besides, the administration of IL-17 antibody alleviated pathological pain. Moreover, IL-17 administration led to mechanical allodynia which was alleviated by the IL-17 antibody. In this review, we summarized and discussed the therapeutic potential of targeting IL-17 for pathological pain. The upregulation of IL-17 promoted the development of pathological pain by promoting neuroinflammation, enhancing the excitability of dorsal root ganglion neurons, and promoting the communication of glial cells and neurons in the spinal cord. In general, the existing research shows that IL-17 is an attractive therapeutic target for pathologic pain, but the underlying mechanisms still need to be investigated.

Potential anxiolytic and antidepressant-like effects of luteolin in a chronic constriction injury rat model of neuropathic pain: Role of oxidative stress, neurotrophins, and inflammatory factors.

This study aimed to examine the effects of luteolin (LUT) on chronic neuropathic pain (NP)-induced mood disorders (i.e., anxiety and depression) by regulating oxidative stress, neurotrophic factors (NFs), and neuroinflammation. Chronic constrictive injury (CCI) was used to induce NP in the animals. Animals in the treatment groups received LUT in three doses of 10, 25, and 50 mg/kg for 21 days. The severity of pain and mood disorders were examined. Finally, animals were sacrificed, and their brain tissue was used for molecular and histopathological studies. CCI led to cold allodynia and thermal hyperalgesia. Mood alterations were proven in the CCI group, according to the behavioral tests. Levels of glial cell-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), B-cell lymphoma-2 (Bcl2), superoxide dismutase (SOD), catalase (CAT), and nuclear factor erythroid-2-related factor 2 (Nrf2) were reduced in the hippocampus (HPC) and prefrontal cortex (PFC). Furthermore, the levels of MDA, Bcl-2-associated X protein (Bax), and inflammatory markers, including nuclear factor kappa B (NF-κB), NLR family pyrin domain containing 3 (NLRP3), interleukin-1ß (IL-1ß), IL-18, IL-6, and tumor necrosis factor-α (TNF-α) significantly increased in the HPC and PFC following CCI induction. LUT treatment reversed the behavioral alterations via regulation of oxidative stress, neurotrophines, and inflammatory mediators in the HPC and PFC. Findings confirmed the potency of LUT in the improvement of chronic pain-induced anxiety- and depressive-like symptoms, probably through antioxidant, anti-inflammatory, and neuroprotective properties in the HPC and PFC.

Cilostazol Alleviates NLRP3 Inflammasome-Induced Allodynia/Hyperalgesia in Murine Cerebral Cortex Following Transient Ischemia: Focus on TRPA1/Glutamate and Akt/Dopamine/BDNF/Nrf2 Trajectories.

Global cerebral ischemia/reperfusion (I/R) provokes inflammation that augments neuropathic pain. Cilostazol (CLZ) has pleiotropic effects including neuroprotection in several ravaging central disorders; nonetheless, its potential role in transient central ischemic-induced allodynia and hyperalgesia has not been asserted before. Rats were allocated into 4 groups; sham, sham + CLZ, and 45 min-bilateral carotid occlusion followed by a 48 h-reperfusion period either with or without CLZ (50 mg/kg; p.o) post-treatment. CLZ prolonged latency of hindlimb withdrawal following von Frey filaments, 4 °C cold, and noxious mechanical stimulations. Histopathological alterations and the immunoexpression of glial fibrillary acidic protein induced by I/R were reduced by CLZ in the anterior cingulate cortex (ACC) area, while, CLZ enhanced intact neuronal count. Meanwhile, CLZ modulated cerebral cortical glutamate, dopamine neurotransmission, and transient receptor potential ankyrin 1 (TRPA1). CLZ anti-inflammatory potential was mediated by the downregulated p65 NF-κB and sirtuin-1 enhancement to reduce nucleotide-binding domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), active caspase-1, and interleukin-1ß, indicative of inflammasome deactivation. It also revealed an antioxidant capacity via boosting nuclear factor E2-related factor (Nrf2) enhancing glutathione through forkhead box protein O3a (FOXO3a) reduction. Additionally, CLZ triggered neuronal survival by promoting the p-content of Akt, TrkB, and CREB as well as BDNF content. A novel approach of CLZ in hindering global cerebral I/R-mediated neuropathy is firstly documented herein to forward its adjunct action via deactivating the NLRP3 inflammasome, besides enhancing Nrf2 axis, neuronal survival, and dopamine neurotransmission as well as inhibiting TRPA1 and excitotoxicity.

Attenuation of Some Inflammatory Markers by Endurance Training in the Spinal Cord of Rats with Diabetic Neuropathic Pain.

Nervous inflammation is an important component of the pathogenesis of neurodegenerative diseases including chronic diabetic neuropathic pain. In order to obtain a decrease in the progression of diabetic neuronal damage, it may be necessary to examine therapeutic options that involve antioxidants and anti-inflammatory agents. The aim of this study was to investigate the attenuation of inflammatory factors with endurance training in the spinal cord of rats with neuropathic pain. Thirty-two 8-week-old male Wistar rats (with a weight range of 204 ± 11.3 g) were randomly divided into 4 groups (n = 8), including (1) diabetic neuropathy (50 mg/kg streptozotocin intraperitoneal injection), (2) diabetic neuropathy training (30 minutes of endurance training at 15 meters per minute, 5 days a week for 6 weeks), (3) healthy training, and (4) healthy control. After confirmation of diabetic neuropathy by behavioral tests, training protocol and supplementation were performed. The NLRP3, P38 MAPK, TNF-α, and IL-1ß gene expressions were measured by a real-time technique in the spinal cord tissue. One-way analysis of variance and Tukey's post hoc test were used for statistical analysis. Endurance training reduced the sensitivity of the nervous system to thermal hyperalgesia and mechanical allodynia; also, compared to the diabetic neuropathy group, the gene expressions of NLTP3, P38 MAPK, TNF-α, and IL-1ß were significantly reduced by endurance training (P < 0.05). Endurance training modulates NLRP3, P38 MAPK, and TNF-α, IL-1ß gene expressions and improves the sensitivity of nociceptors to pain factors. Accordingly, it is recommended to use endurance training to reduce neuropathic pain for diabetics.

Microglia-independent peripheral neuropathic pain in male and female mice.

ABSTRACT: The dominant view in the field of pain is that peripheral neuropathic pain is driven by microglia in the somatosensory processing region of the spinal dorsal horn. Here, to the contrary, we discovered a form of neuropathic pain that is independent of microglia. Mice in which the nucleus pulposus (NP) of the intervertebral disc was apposed to the sciatic nerve developed a constellation of neuropathic pain behaviours: hypersensitivity to mechanical, cold, and heat stimuli. However, NP application caused no activation of spinal microglia nor was pain hypersensitivity reversed by microglial inhibition. Rather, NP-induced pain hypersensitivity was dependent on cells within the NP which recruited macrophages to the adjacent nerve. Eliminating macrophages systemically or locally prevented NP-induced pain hypersensitivity. Pain hypersensitivity was also prevented by genetically disrupting the neurotrophin brain-derived neurotrophic factor selectively in macrophages. Moreover, the behavioural phenotypes as well as the molecular mechanisms of NP-induced pain hypersensitivity were not different between males and females. Our findings reveal a previously unappreciated mechanism for by which a discrete peripheral nerve lesion may produce pain hypersensitivity, which may help to explain the limited success of microglial inhibitors on neuropathic pain in human clinical trials.

Dorsal horn neuronal sparing predicts the development of at-level mechanical allodynia following cervical spinal cord injury in mice.

Spinal cord injury (SCI) frequently results in immediate and sustained neurological dysfunction, including intractable neuropathic pain in approximately 60-80% of individuals. SCI induces immediate mechanical damage to spinal cord tissue followed by a period of secondary injury in which tissue damage is further propagated, contributing to the development of anatomically unique lesions. Variability in lesion size and location influences the degree of motor and sensory dysfunction incurred by an individual. We predicted that variability in lesion parameters may also explain why some, but not all, experimental animals develop mechanical sensitivity after SCI. To characterize the relationship of lesion anatomy to mechanical allodynia, we utilized a mouse cervical hemicontusion model of SCI that has been shown to lead to the development and persistence of mechanical allodynia in the ipsilateral forelimb after injury. At four weeks post-SCI, the numbers and locations of surviving neurons were quantified along with total lesion volume and nociceptive fiber sprouting. We found that the subset of animals exhibiting mechanical allodynia had significantly increased neuronal sparing in the ipsilateral dorsal horn around the lesion epicenter compared to animals that did not exhibit mechanical allodynia. Additionally, we failed to observe significant differences between groups in nociceptive fiber density in the dorsal horn around the lesion epicenter. Notably, we found that impactor probe displacement upon administration of the SCI surgery was significantly lower in sensitive animals compared with not-sensitive animals. Together, our data indicate that lesion severity negatively correlates with the manifestation of at-level mechanical hypersensitivity and suggests that sparing of dorsal horn neurons may be required for the development of neuropathic pain.

Microglial deactivation by adeno-associated virus expressing small-hairpin GCH1 has protective effects against neuropathic pain development in a spinothalamic tract-lesion model.

AIMS: Neuropathic pain after spinal cord injury is one of the most difficult clinical problems after the loss of mobility, and pharmacological or neuromodulation therapy showed limited efficacy. In this study, we examine the possibility of pain modulation by a recombinant adeno-associated virus (rAAV) encoding small-hairpin RNA against GCH1 (rAAV-shGCH1) in a spinal cord injury model in which neuropathic pain was induced by a spinothalamic tract (STT) lesion. METHODS: Micro-electric lesioning was used to damage the left STT in rats (n = 32), and either rAAV-shGCH1 (n = 19) or rAAV control (n = 6) was injected into the dorsal horn of the rats at the same time. On postoperative days 3, 7, and 14, we evaluated neuropathic pain using a behavioral test and microglial activation by immunohistochemical staining. RESULTS: A pain modulation effect of shGCH1 was observed from postoperative days 3 to 14. The mechanical withdrawal threshold was 13.0 ± 0.95 in the shGCH1 group, 4.3 ± 1.37 in the control group, and 3.49 ± 0.85 in sham on postoperative day 3 (p < 0.0001) and continued to postoperative day 14 (shGCH1 vs. control: 11.4 ± 1.1 vs. 2.05 ± 0.60, p < 0.001 and shGCH1 vs. sham: 11.4 ± 1.1 vs. 1.43 ± 0.54, p < 0.001). Immunohistochemical staining of the spinal cord dorsal horn showed deactivation of microglia in the shGCH1 group without any change of delayed pattern of astrocyte activation as in STT model. CONCLUSIONS: Neuropathic pain after spinal cord injury can be modulated bilaterally by deactivating microglial activation after a unilateral injection of rAAV-shGCH1 into the dorsal horn of a STT lesion spinal cord pain model. This new attempt would be another therapeutic approach for NP after SCI, which once happens; there is no clear curative options still now.

Central and peripheral effects of environmental enrichment in a mouse model of arthritis.

This study analyzed whether environmental enrichment (EE) modulates the nociceptive and inflammatory responses in the mouse model of arthritis induced by Complete Freund's Adjuvant (CFA). Ninety male mice (C57BL/6-JUnib, 4-weeks-old; 20-25 g) were distributed into EE and standard (SE) groups. For EE, mice were kept in bigger cages using an alternation of materials to chew (wood and paper), for nesting (cotton), to use as hiding places (plastic tunnels), and for voluntary exercise (wheel running). Arthritis was induced by an injection of CFA (50 µL) into the right hind paw or saline solution in the control group. Separate groups received the anti-inflammatory drug dexamethasone (0.5 mg/kg; every 48 h). Inflammatory and pain measurements were performed from 1 to 35 days after CFA administration. EE per se reduced the acute paw edema formation and arthritis scores. The serum levels of tumor necrosis factor (TNF) were undetectable in any experimental groups. EE diminished the immunopositivity for the microglia marker IBA1 in the pre-frontal cortex, with slight changes for hippocampal GFAP-positive activated astrocytes. Finally, EE induced a marked increment of brain-derived nerve factor (BDNF) expression in the hippocampus, an effect that was fully prevented by dexamethasone. These data bring novel evidence on the peripheral and central effects of EE in a mouse arthritis model.

Neuroprotective effect of l-theanine in a rat model of chronic constriction injury of sciatic nerve-induced neuropathic pain.

BACKGROUND/PURPOSE: We investigated the protective efficacy of l-theanine (LT), the major amino acid components of green tea, on chronic constriction injury (CCI) of sciatic nerve-induced neuropathic pain (NP) development and neuronal functional changes in rats. METHODS: Rats with NP induced by CCI of the left sciatic nerve and sham-operated rats received LT or saline solution, with pain sensitive tests of thermal hyperalgesia and mechanical allodynia. Motor and sensory nerve conduction velocities were measured after surgery. Subsequently, the rats were sacrificed; the sciatic nerve was excised, homogenized, prepared and subjected for estimation of nitric oxide (NO), malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), myeloperoxidase (MPO), and caspase-3. RESULTS: CCI produced a significant increase in hyperalgesia and allodynia, an increase in SFI, a decrease in nerve conduction velocity, increases in NO, MDA, TNF-α, IL-1ß, IL-6, MPO, and caspase-3 levels, as well as reduction of GSH, SOD, and CAT in the rat sciatic nerve. LT treatment significantly and dose-dependently alleviated CCI-induced nociceptive pain thresholds and ameliorated abnormal nerve conduction and functional loss in rats with CCI. Moreover, LT treatment reduced NO and MDA levels, increased antioxidative strength, and markedly suppressed the levels of neuroinflammatory and apoptotic markers in injured sciatic nerves. CONCLUSION: This is the first report on the ameliorative effect of LT in CCI-induced NP in rats. This effect might be attributed to its anti-oxidative, anti-inflammatory, anti-apoptotic, and neuroprotective, thus making it potentially useful as an adjuvant to conventional treatment.

Comparative study of cold hyperalgesia and mechanical allodynia in two animal models of neuropathic pain: different etiologies and distinct pathophysiological mechanisms

Abstract Neuropathic pain (NP) affects more than 8% of the global population. The proposed action of the transient receptor potential ankyrin 1 (TRPA1) as a mechanosensor and the characterization of the transient receptor potential melastatin 8 (TRPM8) as a cold thermosensor raises the question of whether these receptors are implicated in NP. Our study aimed to evaluate the involvement of TRPA1 and TRPM8 in cold and mechanical signal transduction to obtain a comparative view in rat models of streptozotocin-induced diabetes (STZ) and chronic constriction injury of the sciatic nerve (CCI). The electronic von Frey test showed that STZ rats presented mechanical allodynia that was first evidenced on the 14th day after diabetes confirmation, and four days after CCI. This phenomenon was reduced by the intraplantar (ipl) administration of a TRPA1 receptor antagonist (HC-030031; 40 µL/300 µg/paw) in both NP models. Only CCI rats displayed cold hyperalgesia based on the cold plate test. The pharmacological blocking of TRPA1 through the injection of the antagonist attenuated cold hyperalgesia in this NP model. STZ animals showed a reduction in the number of flinches induced by the intraplantar injection of mustard oil (MO; TRPA1 agonist; 0.1%/50 µL/paw), or intraplantar injection of menthol (MT; TRPM8 agonist; 0.5% and 1%/50 µL/paw). The response induced by the ipl administration of MT (1%/50 µL/paw) was significantly different between the CCI and SHAM groups. Together, these data suggest a different pattern in nociceptive behavior associated with different models of NP, suggesting a variant involvement of TRPA1 and TRPM8 in both conditions

Intrathecal IGF2 siRNA injection provides long-lasting anti-allodynic effect in a spared nerve injury rat model of neuropathic pain.

Previous studies have shown an increase of insulin-like growth factor-2 (IGF2) in animal models of neuropathic pain. We aimed to examine the hypothesis that reducing the expression of IGF2 using intrathecal IGF2 small-interfering RNA (siRNA) would attenuate the development of neuropathic pain in rats after spared nerve injury (SNI). Male Wistar rats were divided into three groups: sham-operated group, in which surgery was performed to cut the muscles without injuring the nerves; SNI group, in which SNI surgery was performed to sever the nerves; and SNI + siRNA IGF2 group, in which SNI surgery was performed, and IGF2-siRNA was administered intrathecally 1 day after SNI. The rats were assessed for mechanical allodynia and cold allodynia 1 day before surgery (baseline), and at 2, 4, 6, 8, and 10 days after siRNA treatment. The rat spinal cord was collected for quantitative polymerase chain reaction and western blot analysis. Compared with the SNI group, rats that received IGF2 siRNA showed a significantly increased SNI-induced paw-withdrawal threshold to metal filament stimulation from Day 4 to Day 10 after SNI surgery. IGF2 siRNA significantly decreased the response duration from the acetone test from Day 2 to Day 10 following SNI surgery. SNI increased IGF2 mRNA expression on Day 2 and increased IGF2 protein expression on Day 8 and Day 10 in the spinal cord of the SNI rats. However, the above-mentioned effects of IGF2 mRNA and protein expression were significantly inhibited in the SNI + IGF2 siRNA group. We demonstrated that intrathecal administration of IGF2 siRNA provided significant inhibition of SNI-induced neuropathic pain via inhibition of IGF2 expression in the spinal cord. The analgesic effect lasted for 10 days. Further exploration of intrathecal IGF2 siRNA administration as a potential therapeutic strategy for neuropathic pain is warranted.