Blocking Pannexin 1 Channels Alleviates Peripheral Inflammatory Pain but not Paclitaxel-Induced Neuropathy.

BACKGROUND: Pannexin1 (Panx1) is a membrane channel expressed in different cells of the nervous system and is involved in several pathological conditions, including pain and inflammation. At the central nervous system, the role of Panx1 is already well-established. However, in the periphery, there is a lack of information regarding the participation of Panx1 in neuronal sensitization. The dorsal root ganglion (DRG) is a critical structure for pain processing and modulation. For this reason, understanding the molecular mechanism in the DRG associated with neuronal hypersensitivity has become highly relevant to discovering new possibilities for pain treatment. Here, we aimed to investigate the role of Panx1 in acute nociception and peripheral inflammatory and neuropathic pain by using two different approaches. METHODS: Rats were treated with a selective Panx1 blocker peptide (10Panx) into L5-DRG, followed by ipsilateral intraplantar injection of carrageenan, formalin, or capsaicin. DRG neuronal cells were pre-treated with 10Panx and stimulated by capsaicin to evaluate calcium influx. Panx1 knockout mice (Panx1-KO) received carrageenan or capsaicin into the paw and paclitaxel intraperitoneally. The von Frey test was performed to measure the mechanical threshold of rats' and mice's paws before and after each treatment. RESULTS: Pharmacological blockade of Panx1 in the DRG of rats resulted in a dose-dependent decrease of mechanical allodynia triggered by carrageenan, and nociception decreased in the second phase of formalin. Nociceptive behavior response induced by capsaicin was significantly lower in rats treated with Panx1 blockade into DRG. Neuronal cells with Panx1 blockage showed lower intracellular calcium response than untreated cells after capsaicin administration. Accordingly, Panx1-KO mice showed a robust reduction in mechanical allodynia after carrageenan and a lower nociceptive response to capsaicin. A single dose of paclitaxel promoted acute mechanical pain in wildtype (WT) but not in Panx1-KO mice. Four doses of chemotherapy promoted chronic mechanical allodynia in both genotypes, although Panx1-KO mice had significant ablation in the first eight days. CONCLUSION: Our findings suggest that Panx1 is critical for developing peripheral inflammatory pain and acute nociception involving transient receptor potential vanilloid subtype 1 (TRPV1) but is not essential for neuropathic pain chronicity.

Endophilin A2 controls touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking.

BACKGROUND: Tactile and mechanical pain are crucial to our interaction with the environment, yet the underpinning molecular mechanism is still elusive. Endophilin A2 (EndoA2) is an evolutionarily conserved protein that is documented in the endocytosis pathway. However, the role of EndoA2 in the regulation of mechanical sensitivity and its underlying mechanisms are currently unclear. METHODS: Male and female C57BL/6 mice (8-12 weeks) and male cynomolgus monkeys (7-10 years old) were used in our experiments. Nerve injury-, inflammatory-, and chemotherapy-induced pathological pain models were established for this study. Behavioral tests of touch, mechanical pain, heat pain, and cold pain were performed in mice and nonhuman primates. Western blotting, immunostaining, co-immunoprecipitation, proximity ligation and patch-clamp recordings were performed to gain insight into the mechanisms. RESULTS: The results showed that EndoA2 was primarily distributed in neurofilament-200-positive (NF200+) medium-to-large diameter dorsal root ganglion (DRG) neurons of mice and humans. Loss of EndoA2 in mouse NF200+ DRG neurons selectively impaired the tactile and mechanical allodynia. Furthermore, EndoA2 interacted with the mechanically sensitive ion channel Piezo2 and promoted the membrane trafficking of Piezo2 in DRG neurons. Moreover, as an adaptor protein, EndoA2 also bound to kinesin family member 5B (KIF5B), which was involved in the EndoA2-mediated membrane trafficking process of Piezo2. Loss of EndoA2 in mouse DRG neurons damaged Piezo2-mediated rapidly adapting mechanically activated currents, and re-expression of EndoA2 rescued the MA currents. In addition, interference with EndoA2 also suppressed touch sensitivity and mechanical hypersensitivity in nonhuman primates. CONCLUSIONS: Our data reveal that the KIF5B/EndoA2/Piezo2 complex is essential for Piezo2 trafficking and for sustaining transmission of touch and mechanical hypersensitivity signals. EndoA2 regulates touch and mechanical allodynia via kinesin-mediated Piezo2 trafficking in sensory neurons. Our findings identify a potential new target for the treatment of mechanical pain.

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.

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.

Differential Effects of Regulatory T Cells in the Meninges and Spinal Cord of Male and Female Mice with Neuropathic Pain.

Immune cells play a critical role in promoting neuroinflammation and the development of neuropathic pain. However, some subsets of immune cells are essential for pain resolution. Among them are regulatory T cells (Tregs), a specialised subpopulation of T cells that limit excessive immune responses and preserve immune homeostasis. In this study, we utilised intrathecal adoptive transfer of activated Tregs in male and female mice after peripheral nerve injury to investigate Treg migration and whether Treg-mediated suppression of pain behaviours is associated with changes in peripheral immune cell populations in lymphoid and meningeal tissues and spinal microglial and astrocyte reactivity and phenotypes. Treatment with Tregs suppressed mechanical pain hypersensitivity and improved changes in exploratory behaviours after chronic constriction injury (CCI) of the sciatic nerve in both male and female mice. The injected Treg cells were detected in the choroid plexus and the pia mater and in peripheral lymphoid organs in both male and female recipient mice. Nonetheless, Treg treatment resulted in differential changes in meningeal and lymph node immune cell profiles in male and female mice. Moreover, in male mice, adoptive transfer of Tregs ameliorated the CCI-induced increase in microglia reactivity and inflammatory phenotypic shift, increasing M2-like phenotypic markers and attenuating astrocyte reactivity and neurotoxic astrocytes. Contrastingly, in CCI female mice, Treg injection increased astrocyte reactivity and neuroprotective astrocytes. These findings show that the adoptive transfer of Tregs modulates meningeal and peripheral immunity, as well as spinal glial populations, and alleviates neuropathic pain, potentially through different mechanisms in males and females.

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.

Intradermal miR-16-5p targets Akt3 and reduces RTX-induced postherpetic neuralgia-mimic pain in mice.

The molecular mechanisms of refractory pain in postherpetic neuralgia (PHN) patients are not fully understood. PHN may be related to skin abnormality after herpes zoster induced skin lesions. We previously reported 317 differentially expressed microRNAs (miRNAs) in PHN skin compared with the contralateral normal mirror skin. In this study, 19 differential miRNAs were selected and the expression was validated in other 12 PHN patients. The expression levels of miR-16-5p, miR-20a-5p, miR-505-5p, miR-3664-3p, miR-4714-3p and let-7a-5p are lower in PHN skin, which is the same as those in microarray experiment. To evaluate the effects of cutaneous miRNA on PHN, the expression of candidate miRNAs is further observed in resiniferatoxin (RTX) induced PHN-mimic mice model. In the plantar skin of RTX mice, miR-16-5p and let-7a-5p are downregulated, with the same expression trend of PHN patients. In addition, intraplantar injection of agomir-16-5p reduced mechanical hyperalgesia, and improved thermal hypoalgesia in RTX mice. Furthermore, agomir-16-5p down-regulated the expression levels of Akt3, which is the target gene of agomir-16-5p. These results suggest that intraplantar miR-16-5p may alleviate RTX induced PHN-mimic pain by inhibiting the expression of Akt3 in the skin.

Disruption of C/EBPß-Clec7a axis exacerbates neuroinflammatory injury via NLRP3 inflammasome-mediated pyroptosis in experimental neuropathic pain.

BACKGROUND: Growing evidence shows that C-Type Lectin Domain Containing 7A (Clec7a) may be involved into neuroinflammatory injury of various neurological diseases. However, its roles in neuropathic pain remain unclear. METHODS: A chronic constriction injury (CCI) rat model was constructed, and gene expression profilings in spinal cord tissues of CCI-insulted rats were detected by both microarray and RNA-seq studies. A series of bioinformatics analyses identified C/EBPß-Clec7a to be a candidate axis involved into neuropathic pain. Then, its roles in mechanical allodynia, and pathological and molecular changes during CCI progression were determined by various gain-of-function and loss-of-function experiments in vivo and in vitro. RESULTS: Significant upregulation of Clec7a at both mRNA and protein levels were verified in spinal cord tissues of CCI-insulted rats. Clec7a knockdown markedly attenuated CCI-induced mechanical allodynia, obstructed Syk, ERK and JNK phosphorylation, inhibited NLRP3 inflammasome and caspase-1 activation, GSDMD cleavage, and consequently reduced the release of pro-inflammatory cytokines (all P < 0.05). Mechanically, the rat Clec7a promoter was predicted to bind with transcription factor C/EBPß, confirmed by Luciferase assay and ChIP-qPCR. Both in vivo and in vitro assays demonstrated that C/EBPß knockdown significantly suppressed CCI- or LPS/ATP-induced Clec7a upregulation, and subsequently reduced Syk, ERK and JNK phosphorylation, NLRP3 oligomerization, caspase-1 activation, GSDMD expression and pyroptosis, which were markedly reversed by the co-transfection of Clec7a expression vector. CONCLUSIONS: This pre-clinical investigation reveals that C/EBPß-Clec7a axis may be a potential target for relieving neuropathic pain through alleviating neuroinflammation, paving its way for clinical translation as a promising approach for neuropathic pain therapy.

Masseter muscle contraction and cervical muscle sensitization by nerve growth factor cause mechanical hyperalgesia in masticatory muscle with activation of the trigemino-lateral parabrachial nucleus system in female rats.

OBJECTIVE: To establish a new rat model of craniofacial myalgia, and to clarify which central nervous system pathways are activated in the model. BACKGROUND: Craniofacial myalgia, represented by myogenous temporomandibular disorder and tension-type headache with pericranial tenderness, is more common in female patients. The pain is thought to be a type of multifactorial disorder with several coexisting causes. To our knowledge, there are no models of craniofacial muscle hyperalgesia caused by multiple types of stimuli. METHODS: We injected nerve growth factor into the trapezius muscle of female and male rats and repeatedly stimulated the masseter muscle (MM) electrically for 10 days. We determined the mechanical head-withdrawal threshold of MM and extent of phosphorylated extracellular signal-related kinase 1/2 (pERK) immunoreactivity in various regions of the lower brainstem. We conducted retrograde tract-tracing to determine the projection of mechanosensitive MM-innervating secondary neurons to the lateral parabrachial nucleus. Finally, we administered morphine in rats to determine whether increases of pERK immunoreactivity were dependent on noxious inputs. RESULTS: In female rats, but not male rats, the mechanical head-withdrawal threshold was decreased significantly from days 9 to 12. The number of pERK-immunoreactive neurons in the brainstem was increased significantly in female rats in the group with both stimuli compared to rats in other groups with a single stimulus. Mechanosensitive MM-innervating neurons in the brainstem projected to the parabrachial nucleus. Morphine administration blocked the increase in the number of pERK-immunoreactive neurons in both the brainstem and parabrachial nucleus. CONCLUSIONS: We established a model of craniofacial myalgia by combining trapezius and MM stimuli in female rats. We found mechanical hyperalgesia of the MM and activation of the pain pathway from the brainstem to parabrachial nucleus. The model reflects the characteristics of patients with craniofacial myalgia and might be helpful to clarify the pathogenic mechanisms underlying these disorders.

Lysine-specific demethylase 1 in primary sensory neurons participates in chronic compression of dorsal root ganglion-induced neuropathic pain.

Low back and radicular pain syndromes, usually caused by local inflammation and irritation to the nerve root and dorsal root ganglion (DRG), are common throughout medical practice, but sufficient pain relief is scarce. In this study, we employed a chronic compression of DRG (CCD)-induced radicular pain model in rats to explore whether lysine-specific demethylase 1 (LSD1), a histone demethylase and transcriptional co-repressor, is involved in the pathological process of radicular pain. We found that LSD1 was expressed in various-sized DRG neurons by immunohistochemistry. CCD induced the upregulation of LSD1 in compressed L4-L5 DRGs. Moreover, either LSD1 small interfering RNAs or LSD1 inhibitor attenuated CCD-induced pain hypersensitivities. LSD1 was also upregulated in the injured lumbar 4 (L4) DRG in a spinal nerve ligation (SNL)-induced neuropathic pain mouse model. Nevertheless, LSD1 was not altered in L3-L5 DRGs in complete Freund's adjuvant-induced inflammatory pain mouse model, paclitaxel- or streptozotocin-induced neuropathic pain models. Furthermore, knockdown of LSD1 in the injured L4 DRG reversed SNL-induced pain hypersensitivities in mice. Therefore, we speculate that nerve injury induced the upregulation of LSD1 in the injured DRGs, which contributes to neuropathic pain hypersensitivities; thus, LSD1 may serve as a potential target for the treatment of radicular pain and neuropathic pain.

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.

Sensory neurons have an axon initial segment that initiates spontaneous activity in neuropathic pain.

The axon initial segment is a specialized compartment of the proximal axon of CNS neurons where action potentials are initiated. However, it remains unknown whether this domain is assembled in sensory dorsal root ganglion neurons, in which spikes are initiated in the peripheral terminals. Here we investigate whether sensory neurons have an axon initial segment and if it contributes to spontaneous activity in neuropathic pain. Our results demonstrate that myelinated dorsal root ganglion neurons assemble an axon initial segment in the proximal region of their stem axon, enriched in the voltage-gated sodium channels Nav1.1 and Nav1.7. Using correlative immunofluorescence and calcium imaging, we demonstrate that the Nav1.7 channels at the axon initial segment are associated with spontaneous activity. Computer simulations further indicate that the axon initial segment plays a key role in the initiation of spontaneous discharges by lowering their voltage threshold. Finally, using a Cre-based mouse model for time-controlled axon initial segment disassembly, we demonstrate that this compartment is a major source of spontaneous discharges causing mechanical allodynia in neuropathic pain. Thus, an axon initial segment domain is present in sensory neurons and facilitates their spontaneous activity. This study provides a new insight in the cellular mechanisms that cause pathological pain and identifies a new potential target for chronic pain management.

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-mediated degradation of perineuronal nets promotes pain.

Activation of microglia in the spinal cord dorsal horn after peripheral nerve injury contributes to the development of pain hypersensitivity. How activated microglia selectively enhance the activity of spinal nociceptive circuits is not well understood. We discovered that after peripheral nerve injury, microglia degrade extracellular matrix structures, perineuronal nets (PNNs), in lamina I of the spinal cord dorsal horn. Lamina I PNNs selectively enwrap spinoparabrachial projection neurons, which integrate nociceptive information in the spinal cord and convey it to supraspinal brain regions to induce pain sensation. Degradation of PNNs by microglia enhances the activity of projection neurons and induces pain-related behaviors. Thus, nerve injury-induced degradation of PNNs is a mechanism by which microglia selectively augment the output of spinal nociceptive circuits and cause pain hypersensitivity.

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.

Soluble epoxide hydrolase inhibition avoid formalin-induced inflammatory hyperalgesia in the temporomandibular joint.

Epoxyeicosatrienoic acids (EETs) are endogenous molecules that exerts effective antinociceptive and resolutive actions. However, because of their rapid metabolism by the soluble epoxide hydrolase (sEH), EETs are unable to remain bioavailable. Therefore, the aim of this study was to investigate whether local sEH inhibition could prevent inflammatory hyperalgesia in the temporomandibular joint (TMJ) of rats. For that, rats were pre-treated with an intra-TMJ injection of TPPU, followed by the noxious stimulus (1.5% of formalin intra-articular) to evaluate nociceptive behavior. Histological analysis was conducted to explore the inflammatory exudate and mast cell degranulation. Periarticular tissue over the TMJ was used to measure inflammatory lipids and cytokines/chemokine by Enzyme-Linked Immunosorbent Assay (ELISA). We demonstrated that peripheral pretreatment with TPPU prevents formalin-induced inflammatory hyperalgesia in the TMJ, and this effect is strictly local. Moreover, TPPU mitigates the leukocyte exudate in the TMJ, as well as inflammatory lipids mediators. Mast cell number and degranulation were abrogated by TPPU, and the inflammatory cytokine levels were decreased by TPPU. On the other hand, TPPU up-regulated the release of interleukin 10 (IL-10), an anti-inflammatory cytokine. We provide evidence that locally sEH by intra-TMJ injection of TPPU produces an antinociceptive and anti-inflammatory effect on rats' TMJ.

Characteristics of sensory innervation in synovium of rats within different knee osteoarthritis models and the correlation between synovial fibrosis and hyperalgesia.

Introduction: Knee osteoarthritis (KOA) showed synovial fibrosis and hyperalgesia, although the correlation between the two is unclear. Besides, the specific changes of sensory innervation in animal models are still controversial, which makes it difficult to choose the modeling methods for KOA pain research. Objectives: Study the characteristics of sensory innervation within three commonly used KOA rat models and the correlation between synovial fibrosis and hyperalgesia. Methods: KOA models were induced by destabilization of medial meniscus (DMM), anterior cruciate ligament transection (ACLT), and monoiodoacetate (MIA), respectively. Mechanical, cold and thermal withdrawal threshold (MWT, CWT and TWT) were measured. The harvested tissues were used for pathological sections, immunofluorescence and quantitative analysis. Results: KOA synovium showed more type I collagen deposition, increased expression of CD31, VEGF and TGF-ß. These changes were most pronounced in surgical models, with DMM presenting the most prominent at Day 14 and ACLT at Day 28. Day 14, changes in mechanical hyperalgesia and cold hyperalgesia were most typical in DMM model and statistically different from MIA. There was a negative correlation between the percentage of type I collagen and MWT value (r = -0.88), as well as CWT value (r = -0.95). DMM synovium showed more axonal staining, upregulated CGRP, TRPV1, NGF and Netrin1 compared with MIA. Above changes were also observed at Day 28, but ACLT replaced DMM as the most typical. In DRG, only the levels of CGRP and NGF were different among KOA models at Day 14, and the highest in DMM, which was statistically different compared with MIA. Conclusions: This study described the details of sensory innervation in different KOA model of rats, and the degree of synovial fibrosis was positively correlated with the pain sensitivity of KOA model rats. Additionally, surgical modeling especially ACLT method is more recommended for KOA pain research.

Effects of buprenorphine on model development in an adjuvant-induced monoarthritis rat model.

Complete Freund's adjuvant (CFA)-induced arthritis in rats is a common animal model for studying chronic inflammatory pain. However, modelling of the disease is associated with unnecessary pain and impaired animal wellbeing, particularly in the immediate post-induction phase. Few attempts have been made to counteract these adverse effects with analgesics. The present study investigated the effect of buprenorphine on animal welfare, pain-related behaviour and model-specific parameters during the disease progression in a rat model of CFA-induced monoarthritis. The aim was to reduce or eliminate unnecessary pain in this model, in order to improve animal welfare and to avoid suffering, without compromising the quality of the model. Twenty-four male Sprague Dawley rats were injected with 20 µl of CFA into the left tibio-tarsal joint to induce monoarthritis. Rats were treated with either buprenorphine or carprofen for 15 days during the disease development, and were compared to a saline-treated CFA-injected group or a negative control group. Measurements of welfare, pain-related behaviour and clinical model-specific parameters were collected. The study was terminated after 3 weeks, ending with a histopathologic analysis. Regardless of treatment, CFA-injected rats displayed mechanical hyperalgesia and developed severe histopathological changes associated with arthritis. However, no severe effects on general welfare were found at any time. Buprenorphine treatment reduced facial pain expression scores, improved mobility, stance and lameness scores and it did not supress the CFA-induced ankle swelling, contrary to carprofen. Although buprenorphine failed to demonstrate a robust analgesic effect on the mechanical hyperalgesia in this study, it did not interfere with the development of the intended pathology.