Rat Model Of Neuropathic Pain Research Articles

Efficacy assessment of novel methanimine derivatives in chronic constriction injury-induced neuropathic model: An in-vivo, ex-vivo and In-Silico approach

The multicomponent etiology, complex clinical implications, dose-based side effect and degree of pain mitigation associated with the current pharmacological therapy is incapable in complete resolution of chronic neuropathic pain patients which necessitates the perpetual requirement of novel medication therapy. Therefore, this study explored the ameliorative aptitude of two novel methanimine imitative like (E)-N-(4-nitrobenzylidene)-4‑chloro-2-iodobenzamine (KB 09) and (E)-N-(4-methylbenzylidene)-4‑chloro-2-iodobenzamine (KB 10) in chronic constriction injury (CCI) of sciatic nerve induced neuropathic pain in rat model. Standard behavioral tests like dynamic and static allodynia, cold, thermal and mechanical hyperalgesia along with rotarod activity were performed at various experimental days like 0, 3, 7, 14 and 21. Enzyme linked immunosorbent assay (ELISA) on spinal tissue and antioxidant assays on sciatic nerve were executed accompanied by molecular docking and simulation studies. Prolonged ligation of sciatic nerve expressively induced hyperalgesia as well as allodynia in rats. KB 09 and KB 10 substantially attenuated the CCI elicited hyperalgesia and allodynia. They significantly reduced the biomarkers of pain and inflammation like Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) in ELISA and while enhanced the GSH, SOD and CAT and diminished the MDA levels during antioxidant assays. KB 09 displayed -9.62 kcal/mol with TNF-α and -7.68 kcal/mol binding energy with IL-6 whereas KB 10 exhibited binding energy of -8.20 kcal/mol with IL-6 while -11.68 kcal/mol with TNF-α and hence both trial compounds ensured stable interaction with IL-6 and TNF-α during computational analysis. The results advocated that both methanimine derivatives might be novel candidates for attenuation of CCI-induced neuropathic pain prospects via anti-nociceptive, anti-inflammatory and antioxidant mechanisms.

Antinociceptive effects of α2/α3-subtype selective GABAA receptor positive allosteric modulators KRM-II-81 and NS16085 in rats: behavioral specificity.

Recent studies suggest that amongst the GABAA receptor subtype heterogeneity, α2/α3 subunits of GABAA receptors mediate pain processing. Therefore, α2/α3-subtype selective GABAA receptor positive allosteric modulators (PAMs) may be candidate analgesics. Antinociceptive effects of α2/α3-subtype selective GABAA receptor PAMs have been reported, but the behavioral effects of these compounds have not been systematically evaluated. This study examined the behavioral effects of two α2/α3 subtype-selective GABAA receptor PAMs, KRM-II-81 and NS16085, in male rats. The antinociceptive effects of KRM-II-81 and NS16085 were examined using rat models of inflammatory (complete Freund's adjuvant) and neuropathic pain (chronic constriction injury). The effect of KRM-II-81 on affective pain was measured using the place escape/avoidance paradigm (PEAP). Rate-response of food-maintained operant responding, horizontal wire test, and the spontaneous alternation T-maze, were assessed to study the side-effect profiles of KRM-II-81 and NS16085. The benzodiazepine midazolam was used as a comparator in these studies. KRM-II-81 and NS16085 attenuated mechanical allodynia but not thermal hyperalgesia in both pain states, and their effects were attenuated by the benzodiazepine receptor antagonist flumazenil. KRM-II-81 attenuated affective pain-related behavior in the PEAP test. In the operant responding procedure and horizontal wire test, only midazolam produced significant effects at the dose that produced maximal antinociception. In the T-maze assay, only midazolam significantly decreased the percentage of alternation at an antinociceptive dose. Thus, KRM-II-81 and NS16085 but not midazolam selectively produced antinociceptive effects. Collectively, these data suggest that α2/α3-subtype selective GABAA PAMs could be a novel class of analgesics and warrant further investigation. Significance Statement This study demonstrates that α2/α3-subtype selective GABAA PAMs KRM-II-81 and NS16085 produce selective antinociceptive effects devoid of sedation, myorelaxation, cognitive impairment in two rat models of persistent pain. Unlikely classical benzodiazepines, this study supports the development of α2/α3-subtype selective GABAA PAMs as safe and novel analgesics for pain management.

High-Frequency Spinal Stimulation Suppresses Microglial Kaiso-P2X7 Receptor Axis-Induced Inflammation to Alleviate Neuropathic Pain in Rats.

Neuropathic pain poses a persistent challenge in clinical management. Neuromodulation has emerged as a last-resort therapy. Conventional spinal cord stimulation (Con SCS) often causes abnormal sensations and provides short analgesia, whereas high-frequency spinal cord stimulation (HF SCS) is a newer therapy that effectively alleviates pain without paresthesia. However, the modes of action of 10kHz HF SCS (HF10 SCS) in pain relief remain unclear. To bridge this knowledge gap, we employed preclinical models that mimic certain features of clinical SCS to explore the underlying mechanisms of HF10 SCS. Addressing these issues would provide the scientific basis for improving and evaluating the effectiveness, reliability, and practicality of different frequency SCS in clinical settings. We established a preclinical SCS model to examine its effects in a neuropathic pain rat model. We conducted bulk and single-cell RNA sequencing in the spinal dorsal horn (SDH) to examine cellular and molecular changes under different treatments. We employed genetic manipulations through intrathecal injection of a lentiviral system to explore the SCS-mediated signaling axis in pain. Various behavioral tests were performed to evaluate pain conditions under different treatments. We found that HF10 SCS significantly reduces immune responses in the SDH by inactivating the Kaiso-P2X7R pathological axis in microglia, promoting long-lasting pain relief. Targeting Kaiso-P2X7R in microglia dramatically improved efficacy of Con SCS treatment, leading to reduced neuroinflammation and long-lasting pain relief. HF10 SCS could improve the immunopathologic state in the SDH, extending its benefits beyond symptom relief. Targeting the Kaiso-P2X7R axis may enhance Con SCS therapy and offer a new strategy for pain management. ANN NEUROL 2024;95:966-983.

Effects of tetramethylpyrazine on pharmacokinetics in plasma and brain dialysate and neuropathic pain in rat model of spared sciatic nerve injury

This study aims to explore the effects of tetramethylpyrazine(TMP) on pharmacokinetics in plasma and brain dialysate and neuropathic pain in the rat model of partial sciatic nerve injury(SNI), and to investigate the correlation between the analgesic effect of TMP and its concentrations in the plasma and brain dialysate. Male SD rats were randomized into Sham, SNI, and SNI+TMP groups. Mechanical stimulation with von frey filaments and cold spray method were employed to evaluate the mechanical sensitivity and cold sensitivity of rats. Another two groups, Sham+TMP and SNI+TMP, were used to intubate the common jugular vein and implant microdialysis probes into the anterior cingulate gyrus(ACC), respectively.After intraperitoneal injection of TMP at a dose of 80 mg·kg~(-1), automatic blood collection and intracerebral microdialysis(perfusion rate of 1 μL·min~(-1)) systems were used to collect the blood and brain dialysate for 24 h. HSS T3 C_(18) reversed-phase chromatographic column(2.1 mm×50 mm, 2.5 μm) was used for liquid chromatographic separation. Gradient elution was carried out with the mobile phase of methanol-water(containing 0.005% formic acid) at a flow rate of 0.25 mL·min~(-1). Electrospray ion source was used for mass spectrometry, and the scanning mode was multi-reaction monitoring under the positive ion mode. The ion pairs for quantitative analysis were TMP m/z 137/122 and aspirin m/z 179/137, respectively. DAS 2.11 was used to calculate the pharmacokinetic parameters. The optimal time of TMP to exert the analgesia effect and inhibit cold pain sensitivity was 60 min after treatment. The TMP in the plasma and brain dialysate of SNI rats showed the T_(max) of 15 min and 30 min, the C_(max) of(2 866.43±135.39) and(1 462.14±197.38) μg·L~(-1), the AUC_(0-t) of(241 463.30±28 070.31) and(213 115.62±32 570.07) μg·min·L~(-1), the MRT_(0-t) of(353.13±47.73) and(172.16±12.72) min, and the CL_Z of 0.73 and 0.36 L·min·kg~(-1), respectively. The analgesic effect of TMP had a significant correlation with the blood drug concentration in the ACC, which indicated that this method was suitable for the detection of TMP in rat plasma and brain dialysate. The method is accurate, reliable, and sensitive and can realize the important value of the application of correlation analysis theory of "automatic blood collection-microdialysis/PK-PD" in the research on neuropathic pain.

Activation of 5-HT5A receptor in the ventrolateral orbital cortex produces antinociceptive effects in rat models of neuropathic and inflammatory pain

The ventrolateral orbital cortex (VLO) is identified as an integral component of the endogenous analgesic system comprising a spinal cord - thalamic nucleus submedius - VLO - periaqueductal gray (PAG) - spinal cord loop. The present study investigates the effects of 5-HT5A receptor activation in the VLO on allodynia induced by spared nerve injury and formalin-evoked flinching behavior and spinal c-Fos expression in male SD rats, and further examines whether GABAergic modulation is involved in the effects evoked by VLO 5-HT5A receptor activation. We found an upregulation of 5-HT5A receptor expression in the VLO during neuropathic and inflammatory pain states. Microinjection of the non-selective 5-HT5A receptor agonist 5-CT into the VLO dose dependently alleviated allodynia, and flinching behavior and spinal c-Fos expression, which were blocked by the selective 5-HT5A receptor antagonist SB-699551. Moreover, application of the GABAA receptor antagonist bicuculline in the VLO augmented the analgesic effects induced by 5-CT in neuropathic and inflammatory pain states, whereas the GABAA receptor agonist muscimol attenuated these analgesic effects. Additionally, the 5-HT5A receptors were found to be colocalized with GABAergic neurons in the VLO. These results provide new evidence for the involvement of central 5-HT5A receptors in the VLO in modulation of neuropathic and inflammatory pain and support the hypothesis that activation of 5-HT5A receptors may inhibit the inhibitory effect of GABAergic interneurons on output neurons projecting to the PAG (GABAergic disinhibitory mechanisms), consequently activating the brainstem descending inhibitory system that depresses nociceptive transmission at the spinal cord level.

5-HT3a receptor contributes to neuropathic pain by regulating central sensitization in a rat with brachial plexus avulsion

PurposeAs a frequently occurring complication resulting from brachial plexus avulsion (BPA), neuropathic pain significantly impacts the quality of life of patients and places a substantial burden on their families. Recent reports have suggested that the 5-HT3a receptor may play a role in the development and regulation of neuropathic pain. The current study aimed to explore the involvement of the 5-HT3a receptor in neuropathic pain resulting from BPA in rats. MethodsA rat model of neuropathic pain was induced through brachial plexus avulsion (BPA). The pain thresholds of the rats were measured after BPA. The spinal dorsal horn (SDH) of rats was collected at day 14 after surgery, and the expression and distribution of the 5-HT3a receptor were analyzed using immunohistochemistry and western blotting. The expression levels of various factors related to central sensitization were measured by western blot, including c-Fos, GFAP, IBA-1, IL-1β and TNF-α. The effects of 5-HT3a receptor antagonists on hyperalgesia were assessed through behavioral tests after intrathecal administration of ondansetron. Additionally, at 120 min postinjection, the SDH of rats was acquired, and the change of expression levels of protiens related to central sensitization were measured by western blot. ResultsBPA induced mechanical and cold hypersensitivity in rats. The 5-HT3a receptor was increased and mainly distributed on neurons and microglia in the SDH after BPA, and the level of central sensitization and expression of inflammatory factors, such as c-Fos, GFAP, IBA-1, IL-1β and TNF-α, were also increased markedly. Ondansetron, which is a selective 5-HT3a receptor antagonist, reversed the behavioral changes caused by BPA. The antagonist also decreased the expression of central sensitization markers and inflammatory factors. ConclusionThe results suggested that the 5-HT3a receptor is involved in neuropathic pain by regulating central nervous system sensitization in a rat brachial plexus avulsion model. Targeting the 5-HT3a receptor may be a promising approach for treating neuropathic pain after brachial plexus avulsion.

The Serotonin-Mediated Anti-Allodynic Effect of Yokukansan on Paclitaxel-Induced Neuropathic Pain.

Refractory peripheral neuropathy can occur as a side effect in 60-70% of patients receiving Paclitaxel (PTX). Yokukansan (YKS) is a Japanese herbal medicine reported to have analgesic properties for entrapment nerve injuries. Therefore, we investigated the anti-allodynic effect of Yokukansan on Paclitaxel-induced neuropathic pain. All experiments used 6-week-old male Sprague Dawley rats. Mechanical allodynia was evaluated using a dynamic plantar aesthesiometer. A mobile touch-stimulator unit applied progressively increasing force to the mid-plantar region of the hind paw in a vertical direction until the animal withdrew its paw. This was carried out before the Paclitaxel administration and during the first, second, third, and fourth weeks. Using a rat model of PTX-induced neuropathic pain (PTX rat), we injected PTX (intraperitoneally, 2 mg/kg) five times every 2 days. Using the dynamic plantar test, we evaluated the anti-allodynic effect of YKS (orally administered, 1 g/kg). YKS administration on a daily basis significantly enhanced the withdrawal threshold in PTX rats and reduced the expression level of activated microglia immunostaining with Iba1, a specific marker for microglia. The intrathecal administration of WAY-100635 (5-hydroxytryptamine [5-HT]1A receptor antagonist) and Ketanserin (5-HT2A/2C receptor antagonist) inhibited the protective effects of YKS. YKS exhibited an anti-allodynic effect in a rodent model of PTX-induced neuropathic pain by reducing the sensitivity to pain stimuli. These results suggest that Yokukansan may activate 5-HT receptors in the spinal cord, mediating Paclitaxel-induced neuropathic pain.

A Cross-Sectional and Longitudinal Integrated Study on Brain Functional Changes in a Neuropathic Pain Rat Model.

Human and animal imaging studies demonstrated that chronic pain profoundly alters the structure and the functionality of several brain regions and even causes mental dysfunctions such as depression and anxiety disorders. In this article, we conducted a multimodal study cross-sectionally and longitudinally, to evaluate how neuropathic pain affects the brain. Using the spared nerve injury (SNI) model which promotes long-lasting mechanical allodynia, results showed that neuropathic pain deeply modified the intrinsic organization of the brain functional network 2 weeks after injury. There are significant changes in the activity of the left thalamus (Th_L) and left olfactory bulb (OB_L) brain regions after SNI, as evidenced by both the blood oxygen level-dependent (BOLD) signal and c-Fos expression. Importantly, these changes were closely related to mechanical pain behavior of rats. However, it is worth noting that after morphine administration for analgesia, only the increased activity in the TH region is reversed, while the decreased activity in the OB region becomes more prominent. Functional connectivity (FC) and c-Fos correlation analysis further showed these two regions of interest (ROIs) exhibit different FC patterns with other brain regions. Our study comprehensively revealed the adaptive changes of brain neural networks induced by nerve injury in both cross-sectional and longitudinal dimensions and emphasized the abnormal activity and FC of Th_L and OB_L in the pathological condition. It provides reliable assistance in exploring the intricate mechanisms of diseases.

Analgesic effect of neuromodulation using the AT-04 portable magnetic field-generating device in a patient with neuropathic pain: a case report

BackgroundNeuromodulation by magnetic field through the AT-04 (ait® (AT-04); Peace of Mind Co., Ltd., Kumamoto, Japan) has improved allodynia in neuropathic pain model rats. This report focuses on neuromodulation through magnetic field exposure using the AT-04 that provided an analgesic effect in a patient with neuropathic pain.Case presentationA 47-year-old man presented with flaccid paralysis and extensive neuropathic pain and scored 7 on the 11-point Numerical Rating Scale (NRS) for his left upper limb. The patient was treated with neuromodulation by magnetic field exposure using the AT-04. Baseline NRS scores were obtained three times daily during the baseline period (days 1–5). Magnetic field exposure was then performed for 30 min three times daily (morning, noon, and evening) at home for 36 days, which was termed the intervention period (days 6–41). During the baseline period, the median NRS score was 7 and the baseline NRS score for calculating the percentage of nonoverlap data (PND) was 6. During the intervention period, the median NRS score was 4 and the PND value of the NRS score was 77.8% (28/36). Neuromodulation by magnetic field exposure using the AT-04 effectively decreased the patient’s NRS score. The patient had no adverse effects during the intervention period.ConclusionsNeuromodulation by magnetic field exposure using the AT-04 was effective in decreasing the NRS score in a patient with neuropathic pain. The AT-04 portable magnetic field-generating device shows potential as a therapeutic option for refractory neuropathic pain.

Sex differences in carbamazepine effects in a rat model of trigeminal neuropathic pain

Carbamazepine (CBZ) represents the first-line treatment for trigeminal neuralgia, a condition of facial pain that affects mainly women. The chronic constriction of the infraorbital nerve (CCI-ION) is a widely used model to study this condition, but most studies do not include females. Thus, this study aimed to characterize sensory and affective changes in female rats after CCI-ION and compare the effect of CBZ in both sexes. Mechanical allodynia was assessed 15 days after CCI-ION surgery in rats treated with CBZ (10 and 30 mg/kg, i.p.) or vehicle, together with the open-field test. Independent groups were tested on the Conditioned Place Preference (CPP) paradigm and ultrasonic vocalization (USV) analysis. Blood samples were collected for dosage of the main CBZ metabolite. CBZ at 30 mg/kg impaired locomotion of CCI-ION male and sham and CCI-ION female rats and resulted in significantly higher plasma concentrations of 10-11-EPX-CBZ in the latter. Only male CCI-ION rats showed increased facial grooming which was significantly reduced by CBZ at 10 mg/kg. CBZ at 10 mg/kg significantly reduced mechanical allodynia and induced CPP only in female CCI-ION rats. Also, female CCI-ION showed reduced emission of appetitive USV but did not show anxiety-like behavior. In conclusion, male and female CCI-ION rats presented differences in the expression of the affective-motivational pain component and CBZ was more effective in females than males. Further studies using both sexes in trigeminal neuropathic pain models are warranted for a better understanding of potential differences in the pathophysiological mechanisms and efficacy of pharmacological treatments.

J-2156, a small molecule somatostatin type 4 receptor agonist, alleviated hindpaw hypersensitivity in the streptozotocin-induced rat model of painful diabetic neuropathy but with a 2-fold decrease in potency at an advanced stage in the model, mimicking morphine.

There is a large unmet need for novel pain-killers to improve relief of painful diabetic neuropathy (PDN). Herein, we assessed the efficacy of the somatostatin type 4 (SST4) receptor agonist, J-2156, for relief of PDN in rats. Diabetes was induced with streptozotocin (STZ; 70mg/kg) and bilateral hindpaw hypersensitivity was fully developed by 8-week post-STZ. In the intervals, 8-12-weeks (morphine-sensitive phase; Phase 1) and 16-18-weeks (morphine-hyposensitive phase; Phase 2) post-STZ, rats received a single dose of intraperitoneal (i.p.) J-2156 (10, 20, 30mg/kg), gabapentin (100mg/kg i.p.), subcutaneous morphine (1mg/kg) or vehicle. Hindpaw withdrawal thresholds (PWTs) were assessed using von Frey filaments pre-dose and at regular intervals over 3-h post-dose. In Phase 1, J-2156at 30mg/kg evoked significant anti-allodynia in the hindpaws with maximal effect at 1.5h compared with 1h for gabapentin and morphine. The durations of action for all three compounds were greater than 3h. The corresponding mean (±SEM) extent and duration of anti-allodynia (ΔPWT AUC) for gabapentin did not differ significantly from that for J-2156 (30mg/kg) or morphine. However, in Phase 2, the ΔPWT AUC for morphine was reduced to approximately 25% of that in Phase 1, mirroring our previous work. Similarly, the mean (±SEM) ΔPWT AUC for J-2156 (30mg/kg) in Phase 2 was approximately 45% of that for Phase 1 whereas for gabapentin the mean (±SEM) ΔPWT AUCs did not differ significantly (p > 0.05) between the two phases. Our findings further describe the preclinical pain relief profile of J-2156 and complement previous work in rat models of inflammatory pain, neuropathic pain and low back pain. SST4 receptor agonists hold promise as novel therapeutics for the relief of PDN, a type of peripheral neuropathic pain that is often intractable to relief with clinically used drug treatment options.

Peripheral mu-opioid receptor activation by dermorphin [D-Arg2, Lys4] (1–4) amide alleviates behavioral and neurobiological aberrations in rat model of chemotherapy-induced neuropathic pain

Paclitaxel, a frequently utilized chemotherapeutic agent, often gives rise to severe and distressing sensory neuropathy in patients undergoing chemotherapy. Unfortunately, current therapeutics for chemotherapy-induced neuropathic pain (CINP) demonstrate limited effectiveness and are burdened with the potential for central side effects such as sedation, respiratory depression, cognitive impairment, and addiction, posing substantial clinical challenges. In light of these limitations, present study is designed to investigate the therapeutic potential of Dermorphin [D-Arg2, Lys4] (1–4) amide (DALDA), a preferential peripherally acting mu-opioid receptor agonist, in rat model of CINP. The primary objective was to assess the analgesic properties of DALDA and elucidate the underlying mechanisms governing its therapeutic activity. Our findings revealed that DALDA treatment significantly ameliorated paclitaxel-induced evoked and spontaneous ongoing pain in rats without causing drug addiction and other central side effects. Molecular analyses further unveiled that paclitaxel administration resulted in increased expression of TRP channels, NR2B, voltage-gated sodium channels (VGSCs) and neuroinflammatory markers in both the dorsal root ganglion (DRG) and the spinal cord (L4-L5 region) of rats. DALDA treatment significantly downregulated ion channels (TRPs, VGSCs) and NR2B expressions, concomitant with the inhibition of microglial activation, resulting in the suppression of oxido-nitrosative stress and neuroinflammatory cascade. Findings from the current study suggests that peripheral mu-opioid receptors may offer a potential target for the treatment of patients suffering from CINP, offering new avenues for improved pain relief while minimizing central side effects.

Increase of glutamate in satellite glial cells of the trigeminal ganglion in a rat model of craniofacial neuropathic pain.

Satellite glial cells (SGCs) that envelop the cell bodies of neurons in sensory ganglia have been shown to both release glutamate, and be activated by glutamate in the context of nociceptive signaling. However, little is known about the subpopulations of SGCs that are activated following nerve injury and whether glutamate mechanisms in the SGCs are involved in the pathologic pain. To address this issue, we used light and electron microscopic immunohistochemistry to examine the change in the glutamate levels in the SGCs and the structural relationship between neighboring neurons in the trigeminal ganglion (TG) in a rat model of craniofacial neuropathic pain, CCI-ION. Administration of ionomycin, ATP and Bz-ATP induced an increase of extracellular glutamate concentration in cultured trigeminal SGCs, indicating a release of glutamate from SGCs. The level of glutamate immunostaining in the SGCs that envelop neurons of all sizes in the TG was significantly higher in rats with CCI-ION than in control rats, suggesting that SGCs enveloping nociceptive as well as non-nociceptive mechanosensitive neurons are activated following nerve injury, and that the glutamate release from SGCs increases in pathologic pain state. Close appositions between substance-P (SP)-immunopositive (+) or calcitonin gene-related peptide (CGRP)+, likely nociceptive neurons, between Piezo1+, likely non-nociceptive, mechanosensitive neurons and SP+ or CGRP+ neurons, and between SGCs of neighboring neurons were frequently observed. These findings suggest that glutamate in the trigeminal SGCs that envelop all types of neurons may play a role in the mechanisms of neuropathic pain, possibly via paracrine signaling.

Exploring Serum Biomarkers for Neuropathic Pain in Rat Models of Chemotherapy-Induced Peripheral Neuropathy: A Comparative Pilot Study with Oxaliplatin, Paclitaxel, Bortezomib, and Vincristine.

Blood biomarkers, including neurofilament light chain (NfL), have garnered attention as potential indicators for chemotherapy-induced peripheral neuropathy (CIPN), a dose-limiting adverse effect of neurotoxic anticancer drugs. However, no blood biomarker has been established for routine application or translational research. This pilot study aimed to evaluate a limited panel of blood biomarkers in rat models of CIPN and their correlations with neuropathic pain. CIPN models were induced through repeated injections of oxaliplatin, paclitaxel, bortezomib, and vincristine. Electronic von Frey testing was used to assess tactile allodynia. Post anticancer injections, serum concentrations of 31 proteins were measured. Allodynia thresholds decreased in anticancer-treated animals compared to controls. No consistent modifications were observed in the biomarkers across CIPN models. The most noteworthy biomarkers with increased concentrations in at least two CIPN models were NfL (paclitaxel, vincristine), MCP-1, and RANTES (oxaliplatin, vincristine). Vincristine-treated animals exhibited strong correlations between LIX, MCP-1, NfL, and VEGF concentrations and tactile allodynia thresholds. No single biomarker can be recommended as a unique indicator of CIPN-related pain. Because of the study limitations (single dose of each anticancer drug, young animals, and single time measurement of biomarkers), further investigations are necessary to define the kinetics, specificities, and sensitivities of MCP-1, RANTES, and NfL.

MiR-99b-3p/Mmp13 axis regulates NLRP3 inflammasome-dependent microglial pyroptosis and alleviates neuropathic pain via the promotion of autophagy

BackgroundNeuropathic pain significantly impairs quality of life, and effective interventions are limited. NOD-like receptor thermal protein domain associated protein 3 (NLRP3)-mediated microglial pyroptosis and the subsequent proinflammatory cytokine production are critical in exacerbating pain. Considering microglial pyroptosis as a potential target for developing specific analgesic interventions for neuropathic pain, our study investigated the pathogenesis and therapeutic targets in this condition. MethodsIn vitro experiments involved the co-culture of the immortalized BV-2 microglia cell line with lipopolysaccharide (LPS) to induce microglial pyroptosis. Differentially expressed microRNAs (miRNAs) were identified using high-throughput sequencing analysis. The downstream target genes of these miRNAs were determined through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, and the downstream target genes, combined with miRNAs, were predicted and verified through dual luciferase reporter gene assays. In vivo experiments were conducted to construct a chronic constriction injury (CCI) neuropathic pain model in rats and evaluate the analgesic effects of intrathecal injection of an adeno-associated virus vector (AAV) carrying miR-99b-3p. Gene expression was modulated through mimic or siRNA transfection. Western blot analysis assessed the expression of microglial pyroptosis and autophagy-related proteins, whereas RT-qPCR measured changes in proinflammatory cytokines expression. ResultsLPS-stimulated up-regulation of proinflammatory cytokines in microglia, accompanied by NLRP3-dependent pyroptosis, including increased NLRP3, GSDMD-N, Caspase1-p20, and mature-IL-1β expression. High-throughput sequencing analysis revealed 16 upregulated and 10 downregulated miRNAs in LPS-stimulated microglia, with miR-99b-3p being the most downregulated. KEGG analysis revealed that the target genes of these miRNAs are primarily enriched in calcium, FoxO, and mitogen-activated protein kinase (MAPK) signal pathways. Furthermore, overexpression of miR-99b-3p through mimic transfection significantly inhibited the inflammatory response and NLRP3-mediated pyroptosis by promoting autophagy levels in activated microglia. In addition, we predicted that the 3′ untranslated region (UTR) of matrix metalloproteinase-13 (Mmp13) could bind to miR-99b-3p, and knockdown of Mmp13 expression through siRNA transfection similarly ameliorated enhanced proinflammatory cytokines expression and microglial pyroptosis by enhancing autophagy. In vivo, Mmp13 was co-localized with spinal dorsal horn microglia and was suppressed by intrathecal injection of the AAV-miR-99b-3p vector. Moreover, overpressed miR-99b-3p alleviated CCI-induced mechanical allodynia and neuroinflammation while suppressing pyroptosis by enhancing autophagy in the spinal cord of CCI rats. ConclusionmiR-99b-3p exerts analgesic effects on neuropathic pain by targeting Mmp13. These antinociceptive effects are, at least in part, attributed to the promotion of autophagy, thereby inhibiting neuroinflammation and NLRP3-mediated pyroptosis in activated microglia.

Mechanism of analgesic effect of fire needle on peripheral sensitization in rats with neuropathic pain induced by chemotherapy.

This study aims to explore the analgesic mechanism of fire needle on peripheral sensitization in rats with neuropathic pain(NP) induced by oxaliplatin, so as to investigate its mechanism in improving peri-pheral sensitization. Male SD rats aged 8 weeks were randomly divided into 4 groups：normal group(n=6), model group(n=6), fire needle group(n=6), and medication group(n=6). NP rat model was established by intraperitoneal injection of oxaliplatin(4 mg/kg) on days 1, 2, 8, 9, 15, 16, 22, and 23. For rats in the fire needle group, fire needle treatment was performed at the "Jiaji"(EX-B2) acupoints of the L4-L6 segments on days 24, 26, and 28, ie. 1 day, 3 and 5 days after modeling. The medication group received intraperitoneal injection of pregabalin(100 mg/kg). Mechanical pain thresholds of the rats were measured before modeling, after modeling and intervention. Serum contents of tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and chemokine ligand 12(CXCL12) were detected by ELISA. Skin histopathology changes in the acupoint area were observed using HE staining. The number of mast cells in the skin of the acupoints was observed using toluidine blue staining. Immunohistochemical staining was performed to detect the postive expressions of transient receptor potential vanilloid 1(TRPV1), protease-activated receptor 2(PAR2) and tryptase(TPS) in the skin of the acupoint area. Western blot was used to detect the protein expressions of TRPV1 and PAR2 in the dorsal root ganglia(DRG). Compared with the normal group, the model group had decreased paw withdrawal threshold(PWT) after modeling(P<0.05), increased serum contents of IL-6, TNF-α, and CXCL12(P<0.05), increased number of mast cells in the acupoint area(P<0.05), and increased positive protein expressions of TPS, TRPV1, and PAR2 in the skin of the acupoint area(P<0.05). Compared with the model group, the fire needle group and medication group had increased PWT after intervention(P<0.05), decreased serum contents of IL-6, TNF-α, and CXCL12, and postive protein expressions of TPS, TRPV1, and PAR2 in the skin of the acupoint area(P<0.05)；while the medication group had decreased protein expressions of TRPV1 and PAR2 in DRG(P<0.05). HE staining showed thickened epidermis, disordered cellular arrangement, significant intercellular edema, and inflammatory cell infiltration in the model group. In the medication and fire needle groups, the epidermis was thinner, cellular arrangement was clearer, and the extent of tissue edema and inflammatory cell infiltration was reduced compared to the model group. Fire needle can improve mechanical pain threshold and reduce the contents of peripheral inflammatory factors in rats with oxaliplatin-induced NP. This effect may be related to the inhibition of mast cell activation and the inhibition of TPS, TRPV1 and PAR2 protein expressions, in the local areas of acupoints.

Liquid Crystal Polymer-Based Miniaturized Fully Implantable Deep Brain Stimulator.

A significant challenge in improving the deep brain stimulation (DBS) system is the miniaturization of the device, aiming to integrate both the stimulator and the electrode into a compact unit with a wireless charging capability to reduce invasiveness. We present a miniaturized, fully implantable, and battery-free DBS system designed for rats, using a liquid crystal polymer (LCP), a biocompatible and long-term reliable material. The system integrates the simulator circuit, the receiver coil, and a 20 mm long depth-type microelectrode array in a dome-shaped LCP package that is 13 mm in diameter and 5 mm in height. Wireless powering and control via an inductive link enable device miniaturization, allowing for full implantation and, thus, the free behavior of untethered animals. The eight-channel stimulation electrode array was microfabricated on an LCP substrate to form a multilayered system substrate, which was monolithically encapsulated by a domed LCP lid using a specialized spot-welding process. The device functionality was validated via an in vivo animal experiment using a neuropathic pain model in rats. This experiment demonstrated an increase in the mechanical withdrawal threshold of the rats with microelectrical stimulation delivered using the fully implanted device, highlighting the effectiveness of the system.

Influence of the descending pain-inhibiting serotonergic pathway on the antihyperalgesic effect of gabapentin in neuropathic pain model rats

Gabapentinoids are used worldwide as first-line agents for the treatment of neuropathic pain. Accumulating evidence indicates that one of the antihyperalgesic mechanisms of gabapentinoids is through activation of the noradrenergic pathway of the descending pain inhibition system. However, the involvement of the serotonin pathway is unclear. We investigated the effects of gabapentin (GBP) on the serotonergic pathway of the descending inhibitory system using the spinal nerve ligation (SNL) rat model. As in previous reports, administration of GBP to SNL rats improved paw withdrawal thresholds (PWT). Intrathecally administered serotonin receptor antagonists abolished GBP's amelioration in PWT. GBP did not ameliorate PWT in noradrenaline-depleted SNL rats by DSP-4. However, GBP ameliorated PWT in serotonin-depleted SNL rats by para-chlorophenylalanine, which was not inhibited by intrathecal administration of a serotonin receptor antagonist. Immunohistochemical analysis of serotonin in the spinal dorsal horn revealed a slight, albeit statistically insignificant, increase in 5-HT levels in SNL rats compared to naive rats. However, no apparent changes were observed before or after GBP administration in naive and SNL rats. In conclusion, the involvement of the serotonergic pathway in the antihyperalgesic effects of GBP on the spinal cord is secondary, although it cooperates with the noradrenergic system to produce analgesia.

Neuromodulation Through Magnetic Fields Irradiation with AT-04 Improves Hyperalgesia in a Rat Model of Neuropathic Pain via Descending Pain Modulatory Systems and Opioid Analgesia

Neuromodulation through magnetic fields irradiation with ait® (AT-04), a device that irradiates a mixed alternating magnetic fields (2 kHz and 83.3 MHz), has been shown to have high efficacy for fibromyalgia and low back pain in our previous clinical trials. The aim of this study was to elucidate the underlying analgesic mechanism of the AT-04 using the partial sciatic nerve ligation (PSL) model as an animal model of neuropathic pain. AT-04 was applied to PSL model rats with hyperalgesia and its pain-improving effect was verified by examining mechanical allodynia using the von Frey method. The results demonstrated a significant improvement in hyperalgesia in PSL model rats. We also examined the involvement of descending pain modulatory systems in the analgesic effects of AT-04 using antagonism by serotonin and noradrenergic receptor antagonists. These antagonists significantly reduced the analgesic effect of AT-04 on pain in PSL model rats by approximately 50%. We also measured the amount of serotonin and noradrenaline in the spinal fluid of PSL model rats using microdialysis during AT-04 treatment. Both monoamines were significantly increased by magnetic fields irradiation with AT-04. Furthermore, we evaluated the involvement of opioid analgesia in the analgesic effects of AT-04 using naloxone, the main antagonist of the opioid receptor, and found that it significantly antagonized the effects by approximately 60%. Therefore, the analgesic effects of AT-04 in PSL model rats involve both the endogenous pain modulation systems, including the descending pain modulatory system and the opioid analgesic system.Graphical

MiR‑488‑3p alleviates neuropathic pain by regulating target gene ROCK1.

The function of microRNA (miRNA) in neuropathic pain (NP) has received widespread attention. The current research sought to address the contribution of miR‑488‑3p in NP and its downstream mechanisms. The NP rat model was constructed by chronic constriction injury (CCI) surgery in rats. Regulation of miR‑488‑3p or Rho‑associated coiled‑coil‑containing protein kinase 1 (ROCK1) in rats by intrathecal injection of lentivirus or plasmid. Real‑time quantitative reverse transcription polymerase chain reaction (RT‑qPCR) to examine the levels of miR‑488‑3p and ROCK1 in the dorsal root ganglion (DRG). Enzyme‑linked immunosorbent assay (ELISA) to monitor the secretion of pro‑inflammatory and anti‑inflammatory factors. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) for the evaluation of mechanosensitive and thermal nociceptive hypersensitivity of NP behaviors. Validation of molecular mechanism between miR‑488‑3p and ROCK1 using RNA immunoprecipitation assay and dual‑luciferase reporter (DLR) assay. miR‑488‑3p was vigorously less expressed in the DRGs of CCI rats, while ROCK1 was upregulated. Elevated miR‑488‑3p alleviated the decrease of PWL and PWT in CCI rats, inhibited the secretion of pro‑inflammatory factors, and enhanced anti‑inflammatory factors levels. Mechanistically, ROCK1 was the target of miR‑488‑3p. Raised ROCK1 partially attenuated the mitigating effect of miR‑488‑3p on NP behavior and the suppression of inflammatory responses in rats. Current research demonstrated that miR‑488‑3p may be a novel therapeutic target for NP.