Neurokinin1 - cholinergic receptor mechanisms in the medial Septum-Dorsal hippocampus axis mediates experimental neuropathic pain.

The neurokinin-1 receptors (NK1Rs) in the forebrain medial septum (MS) region are localized exclusively on cholinergic neurons that partly project to the hippocampus and the cingulate cortex (Cg), regions implicated in nociception. In the present study, we explored the hypothesis that neurotransmission at septal NK1R and hippocampal cholinergic mechanisms mediate experimental neuropathic pain in the rodent chronic constriction injury model (CCI). Our investigations showed that intraseptal microinjection of substance P (SP) in rat evoked a peripheral hypersensitivity (PH)-like response in uninjured animals that was attenuated by systemic atropine sulphate, a muscarinic-cholinergic receptor antagonist. Conversely, pre-emptive destruction of septal cholinergic neurons attenuated the development of PH in the CCI model that also prevented the expression of cellular markers of nociception in the spinal cord and the forebrain. Likewise, anti-nociception was evoked on intraseptal microinjection of L-733,060, an antagonist at NK1Rs, and on bilateral or unilateral microinjection of the cholinergic receptor antagonists, atropine or mecamylamine, into the different regions of the dorsal hippocampus (dH) or on bilateral microinjection into the Cg. Interestingly, the effect of L-733,060 was accompanied with a widespread decreased in levels of CCI-induced nociceptive cellular markers in forebrain that was not secondary to behaviour, suggesting an active modulation of nociceptive processing by transmission at NK1R in the medial septum. The preceding suggest that the development and maintenance of neuropathic nociception is facilitated by septal NK1R-dH cholinergic mechanisms which co-ordinately affect nociceptive processing in the dH and the Cg. Additionally, the data points to a potential strategy for pain modulation that combines anticholinergics and anti-NKRs.

The protective effects of orexin B in neuropathic pain by suppressing inflammatory response.

Chronic pain induced by pathological insults to the sensorimotor system is a typical form of neuropathic pain (NP), and the underlying mechanism is complex. Currently, there are no successful therapeutic interventions for NP. Orexin B is a neuropeptide with a wide range of biological functions. However, the pharmacological function of orexin B in chronic neuropathic pain has been less studied. Here, we aim to examine the neuroprotective effects of orexin B in chronic constriction injury (CCI)- induced NP. Firstly, we found that orexin type 2 receptor (OX2R) but not orexin type 1 receptor (OX1R) was reduced in the spinal cord (SC) of CCI-treated rats. Mechanical withdrawal threshold and thermal withdrawal latency assays display that administration of orexin B clearly ameliorated CCI-evoked neuropathic pain dose-dependently. Notably, orexin B treatment also effectively prevented microglia activation by reducing the levels of IBA1. Additionally, orexin B was also found to suppress the inflammatory response in the SC tissue by reducing the levels of IL-6, TNF-α, iNOS, and COX-2 as well as the production of NO and PGE2 in CCI-treated rats. Furthermore, orexin B administration attenuated oxidative stress (OS) by increasing the activity of SOD and the levels of GSH. Mechanically, orexin B prevented activation of JNK/NF-κB signaling in the SC of CCI-treated rats. Based on these findings, we conclude that orexin B might have a promising role in ameliorating CCI-evoked neuropathic pain through the inhibition of microglial activation and inflammatory response.

Nicorandil antiallodynic activity in a model of neuropathic pain is associated with the activation of ATP-dependent potassium channels and opioidergic pathways, and reduced production of cytokines and neutrophils recruitment in paw, sciatic nerve, and dorsal root ganglia.

BACKGROUND: Recently, we demonstrated that nicorandil inhibits mechanical allodynia induced by paclitaxel. In the present study, we evaluated the effect induced by nicorandil in a model of neuropathic pain induced by chronic constriction injury (CCI) in mice. We also investigated putative mechanisms underlying such an effect. METHODS: CCI was induced by three ligatures of the left sciatic nerve. Mechanical allodynia was evaluated by measuring the paw withdrawal threshold with an electronic von Frey apparatus. Concentrations of cytokines and myeloperoxidase activity were determined in the paw tissue, sciatic nerve, and dorsal root ganglia (DRG). RESULTS: Oral administration of two doses of nicorandil (150 mg/kg po), but not equimolar doses of nicotinamide or nicotinic acid, attenuated mechanical allodynia induced by CCI. Nicorandil activity was reduced by previous administration of glibenclamide (40 mg/kg) or naltrexone (5 mg/kg or 10 mg/kg). Two doses of nicorandil (150 mg/kg, po) reduced tumor necrosis factor-α, interleukin-1ß and interleukin-6, but not CXCL-1, concentrations in the paw tissue of CCI mice. Two doses of nicorandil (150 mg/kg, po) reduced concentrations of all these mediators in the sciatic nerve and DRG. Two doses of nicorandil (150 mg/kg, po) also reduced the myeloperoxidase activity in the paw tissue, sciatic nerve, and DRG. CONCLUSIONS: Nicorandil exhibits antiallodynic activity in a model of neuropathic pain induced by CCI. Inhibition of cytokines production and reduction of neutrophils recruitment in paw tissue, sciatic nerve, and DRG as well as activation of ATP-dependent potassium channels and opioidergic pathways, underlie nicorandil antiallodynic activity.

Prevention and reversal of neuropathic pain by near-infrared photobiomodulation therapy in male and female rats.

Pathological nociception arising from peripheral nerve injury impacts quality of life. Current therapeutics are generally ineffective. However, photobiomodulation therapy (PBMT) has shown promise in addressing this issue. We aimed to assess the potential anti-allodynic effects of 2 p.m. protocols, each applied transcutaneously over the peripheral nerve injury. In addition to evaluating nociceptive behavior, we also conducted morphological analysis using electron microscopy (EM) to investigate potential ultrastructural changes at the cellular level. We sought to determine, using the chronic constriction injury (CCI) model, whether our parameters could alleviate established allodynia and/or dampen allodynia development. Adult male and female rats with CCI or sham were treated with PBMT (850-nm wavelength) for 2 min, 3 times a week over three or four weeks across three studies, where PBMT began either before or after CCI. Allodynia was assessed prior to surgery and across weeks and, at the conclusion of the third study, sciatic nerve was processed for EM and histomorphometrically evaluated. The results showed that PBMT before versus after CCI injury yielded similar behaviors, effectively decreasing allodynia. Interestingly, these positive effects of PBMT do not appear to be accounted by protection of the sciatic injury site, based on EM. CCI reliably decreased axon size and the number of myelinated axons present in both PBMT and control groups. While PBMT reduced the number of C-fibers in CCI samples, no improvement in any measure was observed in response to PBMT.

Octanol alleviates chronic constriction injury of sciatic nerve-induced peripheral neuropathy by regulating AKT/mTOR signaling.

OBJECTIVE: Activation of gap junction channels can induce neuropathic pain. Octanol can limit the conductance of gap junctions containing connexin 43 proteins. Thus, this study focused on the roles of octanol in chronic constriction injury (CCI)-induced peripheral neuropathy in mice and its mechanisms of action. METHODS: Male mice were assigned into control, sham, CCI, CCI + Octanol-20 mg/kg, CCI + Octanol-40 mg/kg and CCI + Octanol-80 mg/kg groups. CCI was performed by applying three loose ligations to mouse sciatic nerve, and the mice with CCI was administered with 20 mg/kg, 40 mg/kg, or 80 mg/kg octanol. The neuropathic pain development was examined by assessing thermal withdrawal latency, paw withdrawal mechanical threshold, and sciatic functional index. Histopathological changes were evaluated by hematoxylin and eosin staining. The phosphorylation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) was examined by western blotting. The expression of Akt and mTOR was also evaluated by immunofluorescence staining. RESULTS: Octanol alleviated the CCI-induced mechanical and thermal hyperalgesia and sciatic functional loss. Additionally, octanol relieved the CCI-induced abnormal histopathological changes. Mechanistically, octanol inactivated the Akt/mTOR pathway in the mice with CCI. CONCLUSION: In conclusion, octanol can alleviate CCI-induced peripheral neuropathic by regulating the Akt/mTOR pathway and might be a novel pharmacological intervention for neuropathic pain.

Ameliorative effects of probiotic Limosilactobacillus fermentum and Enterococcus lactis isolated from cameroonian traditionally processed milk and palm wine against chronic constriction injury induced neuropathic pain in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Fermented milk and palm wine are regularly used by several ethnic groups in Cameroon in traditional treatment rituals for infections, inflammatory, cardiovascular disorders, and even metabolic diseases such as diabetes, hypercholesterolemia etc. Reports from many studies have demonstrated that fermented milk and palm wine are potential sources of probiotic bacteria. However, the capacity of probiotics isolated from these natural sources to alleviate neuropathic pain has not been experimentally tested. AIM OF THE STUDY: This study aimed at investigating the ameliorative potential of lactic acid bacteria isolated from palm wine and traditional fermented cow milk on the chronic constriction injury (CCI) induced neuropathic pain in mice. MATERIALS AND METHODS: Pour plating technique on De Man Rogasa (MRS) agar was utilised for isolation of lactic acid bacteria from fermented cow milk and palm wine, and identified using the 16S r RNA gene sequencing. Neuropathic pain was induced by chronic constriction injury of the sciatic nerve. These bacteria were orally administered at different concentrations to Balb/c mice by gavage for 14 consecutive days. Cold allodynia, mechanical hyperalgesia and exploratory behaviour were evaluated on day 0, 7th and 14th respectively. The total level of calcium, oxidative stress markers and myeloperoxidase were also quantified in the sciatic nerve homogenate. Cyclooxygenase-2(COX-2) and cytokine profile were determined from serum. RESULTS: Lactic acid bacteria were isolated from fermented cow milk and palm wine and two isolates were chosen according to their probiotic potentials and identified as strain of Limolactobacillus fermentum and Enterococcus lactis. Their 16 S rRNA gene sequences were deposited in NCBI genbank with accession number of OP896078 and OR619545, respectively. Pretreatment with Limosilactobacillus fermentum and Enterococcus lactis significantly alleviated mechanical hyperalgesia and cold allodynia with similar effect to the reference drug, morphine. These two isolates ameliorated CCI induced neuropathic pain by increasing antioxida776nts (GSH, CAT and SOD, P < 0.01) and decreasing pro-oxidants (MDA and NO, P < 0.01). Also, they inhibited the release of proinflammatory cytokines (IL-1ß, TNF-α, IFN-γ, and IL-6; P < 0.01) and IL-10 level was significantly (P < 0.01) increased when compared to the negative control. Treatment with these bacteria significantly dropped the level of total calcium (P < 0.01), COX-2 (P < 0.01) and MPO (P < 0.01) when compared with the negative control. CONCLUSION: The neuroprotective potentials of these selected lactic acid bacteria against CCI induced neuropathic pain may be attributed to their anti-oxidant, anti-inflammatory properties and reduced calcium deposition in sciatic nerve.

Sevoflurane impedes neuropathic pain by maintaining endoplasmic reticulum stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.

OBJECTIVE: To investigate the effect of sevoflurane on neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve in mice, and to elucidate its mechanism by animal experiments. METHODS AND RESULTS: Thirty-two C57BL/6 mice were randomly divided into four groups: Sham group, Model group, Control group and Sevoflurane group. First, a mouse model of neuropathic pain was established. Then, the mice in each group were killed on Day 14 after operation to harvest the enlarged lumbosacral spinal cord. In contrast with the Model group, the Sevoflurane group displayed a significantly increased paw withdrawal mechanical threshold (PWMT) and significantly prolonged paw withdrawal thermal latency (PWTL) from Day 5 after operation. The morphological changes of lumbosacral spinal cord were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy. Pathological results showed that sevoflurane reduced nuclear pyknosis in lumbosacral spinal cord tissue, with a large number of mitochondrial crista disappearance and mitochondrial swelling. The results of Western blotting showed that sevoflurane significantly decreased the protein expressions of phosphorylated phospholipase Cγ (p-PLCγ), phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) and phosphorylated inositol 1,4,5-triphosphate receptor (p-IP3R), and reduced the protein expressions of endoplasmic reticulum (ER) stress proteins glucose-regulated protein 78 (GRP78) and GRP94, oxidative stress-related proteins P22 and P47 and inflammatory factors nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), interleukin-1 ß (IL-1ß), and tumor necrosis factor-α (TNF-α). CONCLUSIONS: Sevoflurane inhibits neuropathic pain by maintaining ER stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.

Fu's subcutaneous needling promotes axonal regeneration and remyelination by inhibiting inflammation and endoplasmic reticulum stress.

Fu's subcutaneous needling (FSN) is a traditional Chinese acupuncture procedure used to treat pain-related neurological disorders. Moreover, the regulation of inflammatory cytokines may provide a favorable environment for peripheral nerve regeneration. In light of this, FSN may be an important novel therapeutic strategy to alleviate pain associated with peripheral neuropathy; however, the underlying molecular mechanisms remain unclear. This study revealed that patients who had osteoarthritis with peripheral neuropathic pain significantly recovered after 1 to 2 weeks of FSN treatment according to the visual analog scale, Western Ontario and McMaster Universities Osteoarthritis Index, Lequesne index, walking speed, and passive range of motion. Similarly, we demonstrated that FSN treatment in an animal model of chronic constriction injury (CCI) significantly improved sciatic nerve pain using paw withdrawal thresholds, sciatic functional index scores, and compound muscle action potential amplitude tests. In addition, transmission electron microscopy images of sciatic nerve tissue showed that FSN effectively reduced axonal swelling, abnormal myelin sheaths, and the number of organelle vacuoles in CCI-induced animals. Mechanistically, RNA sequencing and gene set enrichment analysis revealed significantly reduced inflammatory pathways, neurotransmitters, and endoplasmic reticulum stress pathways and increased nerve regeneration factors in the FSN+CCI group, compared with that in the CCI group. Finally, immunohistochemistry, immunoblotting and enzyme-linked immunosorbent assay showed similar results in the dorsal root ganglia and sciatic nerve. Our findings suggest that FSN can effectively ameliorate peripheral neuropathic pain by regulate inflammation and endoplasmic reticulum stress, thereby determine its beneficial application in patients with peripheral nerve injuries.

Intravenous application of human umbilical cord mesenchymal stem cells alleviate neuropathic pain by suppressing microglia activation in rats.

Objective: Neuropathic pain has been considered as one of the most serious chronic pain subtypes and causes intolerable suffering to patients physically and mentally. This study aimed to verify the analgesic effect of intravenous administration of human umbilical cord mesenchymal stem cells (HUC-MSCs) upon rats with chronic constriction injury (CCI)-induced neuropathic pain and the concomitant mechanism via modulating microglia. Methods: 30 male SD rats were randomized divided into three groups (n = 10 per group): Sham + Saline group (S&S group), CCI + Saline group (C&S group) and CCI + HUC-MSCs group (C&U group). Rats were injected with either saline or HUC-MSCs via the caudal vein on the 7th day after modelling. The paw mechanical withdrawal threshold (PMWT) and thermal withdrawal latency (TWL) of the ligation side were measured before (day 0) and after (day 1, 3, 5, 7, 9, 11, 13, and 15) modelling. On day 15 after modelling, western-blotting and immunofluorescent staining were used to assess the expressive abundance of Iba-1 (a typical biomarker of activated microglia) in the ligation side of the spinal cord dorsal horn, and ultrastructural changes of the ligation of sciatic nerve were evaluated by transmission electron microscope (TEM). Results: Compared with the S&S group, PMWT and TWL in the C&S group were significantly decreased on day 5 and then persisted to day 15 after modelling (C&S vs S&S, P < 0.05), while a significant amelioration of mechanical hyperalgesia (day 13, day 15) and thermal allodynia (day 9, day 11, day 15) was observed in the C&U group (C&U vs C&S, P < 0.05). Meanwhile, the expression of Iba-1 was significantly suppressed by systemic infusion of HUC-MSCs in the C&U group according to western-blotting and immunofluorescent staining analyses (P < 0.05). With the aid of TEM detection, we intuitively noticed the efficacious reconstruction of the laminate structure of the sciatic nerve ligation, elimination of mitochondrial swelling, and formation of new myelination were noted on day 15 after modelling in the C&U group. Conclusions: Overall, intravenous administration of HUC-MSCs systemically revealed an ameliorative effect upon CCI-induced neuropathic pain in SD rats by inhibiting microglia activation in the dorsal horn of the impaired spinal cord and alleviating sciatic nerve injury. Our findings supply new references for the further development of HUC-MSCs-based cytotherapy for neuropathic pain administration.

Minocycline Abrogates Individual Differences in Nerve Injury-Evoked Affective Disturbances in Male Rats and Prevents Associated Supraspinal Neuroinflammation.

Chronic neuropathic pain precipitates a complex range of affective and behavioural disturbances that differ markedly between individuals. While the reasons for differences in pain-related disability are not well understood, supraspinal neuroimmune interactions are implicated. Minocycline has antidepressant effects in humans and attenuates affective disturbances in rodent models of pain, and acts by reducing neuroinflammation in both the spinal cord and brain. Previous studies, however, tend not to investigate how minocycline modulates individual affective responses to nerve injury, or rely on non-naturalistic behavioural paradigms that fail to capture the complexity of rodent behaviour. We investigated the development and resolution of pain-related affective disturbances in nerve-injured male rats by measuring multiple spontaneous ethological endpoints on a longitudinal naturalistic foraging paradigm, and the effect of chronic oral minocycline administration on these changes. Disrupted foraging behaviours appeared in 22% of nerve-injured rats - termed 'affected' rats - and were present at day 14 but partially resolved by day 21 post-injury. Minocycline completely prevented the emergence of an affected subgroup while only partly attenuating mechanical allodynia, dissociating the relationship between pain and affect. This was associated with a lasting downregulation of ΔFosB expression in ventral hippocampal neurons at day 21 post-injury. Markers of microglia-mediated neuroinflammation were not present by day 21, however proinflammatory microglial polarisation was apparent in the medial prefrontal cortex of affected rats and not in CCI minocycline rats. Individual differences in affective disturbances following nerve injury are therefore temporally related to altered microglial morphology and hippocampal neuronal activation, and are abrogated by minocycline.

Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway.

BACKGROUND: Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity. OBJECTIVE: To investigate whether TLR4 can regulate Nav1.3 through the TRAF6/NF-κB p65 pathway after infraorbital nerve chronic constriction injury (ION-CCI). STUDY DESIGN: ION-CCI modeling was performed on SD (Sprague Dawley) rats. To verify the success of the modeling, we need to detect the mechanical pain threshold and ATF3. Then, detecting the expression of TLR4, TRAF6, NF-κB p65, p-p65, and Nav1.3 in rat TG. Subsequently, investigate the role of TLR4/TRAF6/NF-κB pathway in ION-CCI model by intrathecal injections of LPS-rs (TLR4 antagonist), C25-140 (TRAF6 inhibitor), and PDTC (NF-κB p65 inhibitor). RESULTS: ION-CCI surgery decreased the mechanical pain threshold of rats and increased the expression of ATF3, TLR4, TRAF6, NF-κB p-p65 and Nav1.3, but there was no difference in NF-κB p65 expression. After inject antagonist or inhibitor of the TLR4/TRAF6/NF-κB pathway, the expression of Nav1.3 was decreased and mechanical pain threshold was increased. CONCLUSION: In the rat model of ION-CCI, TLR4 in the rat trigeminal ganglion regulates Nav1.3 through the TRAF6/NF-κB p65 pathway, and TLR4 antagonist alleviates neuropathic pain in ION-CCI rats.

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)-4­chloro-2-iodobenzamine (KB 09) and (E)-N-(4-methylbenzylidene)-4­chloro-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.

Neuroinflammation signatures in dorsal root ganglia following chronic constriction injury.

Neuropathic pain (NP) is a common debilitating chronic pain condition with limited effective therapeutics. Further investigating mechanisms underlying NP is therefore of great importance for discovering more promising therapeutic targets. In the current study, we employed high-throughput RNA sequencing to explore transcriptome profiles of mRNAs and microRNAs in the dorsal root ganglia (DRG) following chronic constriction injury (CCI) and also integrated published datasets for comprehensive analysis. First, we established CCI rat model confirmed by behavioral testings, and excavated 467 differentially expressed mRNAs (DEGs) and 16 differentially expressed microRNAs (DEmiRNAs) in the ipsilateral lumbar 4-6 DRG of CCI rats 11 days after surgery. Functional enrichment analysis of 337 upregulated DEGs showed that most of the DEGs were enriched in inflammation- and immune-associated biological processes and signaling pathways. The protein-protein interaction networks were constructed and hub DEGs were screened. Besides hub DEGs, we also identified 113 overlapped DEGs by intersecting our dataset with dataset GSE100122. Subsequently, we predicted potential miRNA-mRNA regulatory pairs using DEmiRNAs and a given set of key DEGs (including hub and overlapped DEGs). By integrative analysis, we found commonly differentially expressed mRNAs and miRNAs following CCI of different time points and different nerve injury types. Highlighted mRNAs include Atf3, Vip, Gal, Npy, Adcyap1, Reg3b, Jun, Cd74, Gadd45a, Tgm1, Csrp3, Sprr1a, Serpina3n, Gap43, Serpinb2 and Vtcn1, while miRNAs include miR-21-5p, miR-34a-5p, miR-200a-3p, miR-130a-5p, miR-216b-5p, miR-217-5p, and miR-541-5p. Additionally, 15 DEGs, including macrophages-specific (Cx3cr1, Arg1, Cd68, Csf1r) and the ones related to macrophages' involvement in NP (Ccl2, Fcgr3a, Bdnf, Ctss, Tyrobp) were verified by qRT-PCR. By functional experiments in future studies, promising therapeutic targets for NP treatment may be identified among these mRNAs and miRNAs.

LPAR6 Participates in Neuropathic Pain by Mediating Astrocyte Cells via ROCK2/NF-κB Signal Pathway.

Neuropathic pain (NPP) is a common type of chronic pain. Glial cells, including astrocytes (AS), are believed to play an important role in the progression of NPP. AS cells can be divided into various types based on their expression profiles, among which A1 and A2 types have clear functions. A1-type AS cells are neurotoxic, while A2-type AS cells exert neuroprotective functions. Some types of lysophosphatidic acid receptors (LPAR) have been shown to play a role in NPP. However, it remains unclear how AS cells and LPAR6 affect the occurrence and progression of NPP. In this study, we established a mouse model of chronic constriction injury (CCI) to simulate NPP. It was found that the expression of LPAR6 in AS cells of the spinal dorsal horn was increased in the CCI model, and the thresholds of mechanical and thermal pain were elevated after knocking out LPAR6, indicating that LPAR6 and AS cells participated in the occurrence of NPP. The experiment involved culturing primary AS cells and knocking down LPAR6 by Lentivirus. The results showed that the NF-κB signal pathway was activated and the number of A1-type AS cells increased in the CCI model. However, LPAR6 knockdown inhibited the NF-κB signal pathway and A1-type AS cells. The results of the mRNA sequencing and immunoprecipitation test indicate an interaction between LPAR6 and ROCK2. Inhibiting ROCK2 by Y-27632 increased mechanical and thermal pain thresholds and alleviated NPP at the molecular level. The study presents evidence that LPAR6 activates the NF-κB pathway through ROCK2 and contributes to the progression of NPP by increasing A1-type AS and decreasing A2-type AS. This suggests that LPAR6 could be a potential therapeutic target for alleviating NPP. Clinical applications that are successful can offer new therapeutic options, enhance the quality of life for patients, and potentially uncover new mechanisms for pain modulation.

Identification of Regulatory Elements in Primary Sensory Neurons Involved in Trauma-Induced Neuropathic Pain.

Chronic pain is a significant public health issue that is often refractory to existing therapies. Here we use a multiomic approach to identify cis-regulatory elements that show differential chromatin accessibility and reveal transcription factor (TF) binding motifs with functional regulation in the rat dorsal root ganglion (DRG), which contain cell bodies of primary sensory neurons, after nerve injury. We integrated RNA-seq to understand how differential chromatin accessibility after nerve injury may influence gene expression. Using TF protein arrays and chromatin immunoprecipitation-qPCR, we confirmed C/EBPγ binding to a differentially accessible sequence and used RNA-seq to identify processes in which C/EBPγ plays an important role. Our findings offer insights into TF motifs that are associated with chronic pain. These data show how interactions between chromatin landscapes and TF expression patterns may work together to determine gene expression programs in rat DRG neurons after nerve injury.

Time-Course Progression of Whole Transcriptome Expression Changes of Trigeminal Ganglia Compared to Dorsal Root Ganglia in Rats Exposed to Nerve Injury.

Mechanisms underlying neuropathic pain (NP) are complex with multiple genes, their interactions, environmental and epigenetic factors being implicated. Transcriptional changes in the trigeminal (TG) and dorsal root (DRG) ganglia have been implicated in the development and maintenance of NP. Despite efforts to unravel molecular mechanisms of NP, many remain unknown. Also, most of the studies focused on the spinal system. Although the spinal and trigeminal systems share some of the molecular mechanisms, differences exist. We used RNA-sequencing technology to identify differentially expressed genes (DEGs) in the TG and DRG at baseline and 3 time points following the infraorbital or sciatic nerve injuries, respectively. Pathway analysis and comparison analysis were performed to identify differentially expressed pathways. Additionally, upstream regulator effects were investigated in the two systems. DEG (differentially expressed genes) analyses identified 3,225 genes to be differentially expressed between TG and DRG in naïve animals, 1,828 genes 4 days post injury, 5,644 at day 8 and 9,777 DEGs at 21 days postinjury. A comparison of top enriched canonical pathways revealed that a number of signaling pathway was significantly inhibited in the TG and activated in the DRG at 21 days postinjury. Finally, CORT upstream regulator was predicted to be inhibited in the TG while expression levels of the CSF1 upstream regulator were significantly elevated in the DRG at 21 days postinjury. This study provides a basis for further in-depth studies investigating transcriptional changes, pathways, and upstream regulation in TG and DRG in rats exposed to peripheral nerve injuries. PERSPECTIVE: Although trigeminal and dorsal root ganglia are homologs of each other, they respond differently to nerve injury and therefore treatment. Activation/inhibition of number of biological pathways appear to be ganglion/system specific suggesting that different approaches might be required to successfully treat neuropathies induced by injuries in spinal and trigeminal systems.

Evaluating the Effects of Probiotic Supplementation on Neuropathic Pain and Oxidative Stress Factors in an Animal Model of Chronic Constriction Injury of the Sciatic Nerve.

Introduction: Neuropathic pain is a common and painful somatosensory nervous system disease, and its treatment remains a medical challenge. Evidence demonstrates that gut microbiota alters in neuropathic pain and, therefore, improvement of the gut flora may affect the disease. The present study aimed to evaluate the antinociceptive effect of probiotics in neuropathic pain and oxidative biomarkers' responsiveness to the probiotic treatment. Methods: Using chronic constriction injury (CCI) of the rats' sciatic nerve, neuropathic pain was induced. Investigating the analgesic effect of the probiotics mixture, 40 male rats were randomly assigned to 4 groups (n=10 for each): Sham-operated (SM), and CCI model rats orally received 1 mL saline (CS), or 100 mg/kg gabapentin (CG) or 1 mL probiotics mixture (CP) Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Bifidobacterium bifidum (109 CFU of each) daily. Using behavioral tests, the pain was assessed on days 1, 4, 7, 14, and 21 of the study. Finally, the biochemical evaluation of sciatic nerve tissue was done. Results: Probiotics decreased cold and mechanic allodynia and thermal hyperalgesia. Reducing lipid peroxidation levels and increasing total antioxidant capacity, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were also significant in the probiotics group. Conclusion: These findings suggest that probiotics have analgesic effects on the CCI model of neuropathic pain via increasing the antioxidant capacity of the rats' sciatic nerve. Highlights: Oral probiotics mixture diminishes cold and mechanical allodynia in chronic constriction injury (CCI) rats.Probiotics mixture reduces thermal hyperalgesia in CCI rats as well as gabapentine.Balancing in oxidant/anti-oxidant system is key pathway in neuropathic pain reduction. Plain Language Summary: Currently, neuropathic pain is an underestimated socioeconomic health problem affecting millions of people worldwide. Neuropathic pain occurs when a health condition affects the nerves that carry sensations to the brain. With neuropathic pain, the pain isn't typically triggered by an event or injury. Instead, the body sends pain signals to the brain unprompted. Neuropathic pain tends to get worse over time. As neuropathic pain could result from an imbalance in the oxidative/antioxidative system, antioxidant supplementation may be a treatment option. Oxidative stress is known to result in the occurrence of molecular mechanisms of diabetes, atherosclerosis, inflammatory bowel disease, and damage to the heart, brain, or transplanted organs. Probiotics are made of good live bacteria and/or yeasts that naturally live in the body and have various health benefits. Consumption of probiotics alone or foods supplemented with probiotics may reduce cell oxidative damage. Incorporating probiotics in foods can provide an excellent strategy to supply dietary antioxidants. This study subjected rats to sciatic nerve ligation to induce neuropathic pain. The oral probiotics mixture was administered for 21 days post-surgery. The result showed that the probiotics mixture administration could reduce oxidative stress and pain in neuropathic pain rats. Therefore, probiotics can prevent and treat many systemic diseases in animal and human studies.

Neuropathy, its Profile and Experimental Nerve Injury Neuropathic Pain Models: A Review.

Neuropathy is a terrible disorder that has a wide range of etiologies. Drug-induced neuropathy, which happens whenever a chemical agent damages the peripheral nerve system, has been linked here to the iatrogenic creation of some drugs. It is potentially permanent and causes sensory impairments and paresthesia that typically affects the hands, feet, and stockings; motor participation is uncommon. It might appear suddenly or over time, and the long-term outlook varies. The wide range of chronic pain conditions experienced by people has been one of the main obstacles to developing new, more effective medications for the treatment of neuropathic pain. Animal models can be used to examine various neuropathic pain etiologies and symptoms. Several models investigate the peripheral processes of neuropathic pain, whereas some even investigate the central mechanisms, such as drug induce models like vincristine, cisplatin, bortezomib, or thalidomide, etc., and surgical models like sciatic nerve chronic constriction injury (CCI), sciatic nerve ligation through spinal nerve ligation (SNL), sciatic nerve damage caused by a laser, SNI (spared nerve injury), etc. The more popular animal models relying on peripheral nerve ligatures are explained. In contrast to chronic sciatic nerve contraction, which results in behavioral symptoms of less reliable stressful neuropathies, (SNI) spared nerve injury generates behavioral irregularities that are more feasible over a longer period. This review summarizes the latest methods models as well as clinical ideas concerning this mechanism. Every strongest current information on neuropathy is discussed, along with several popular laboratory models for causing neuropathy.

A novel animal model of neuropathic corneal pain-the ciliary nerve constriction model.

Introduction: Neuropathic pain arises as a result of peripheral nerve injury or altered pain processing within the central nervous system. When this phenomenon affects the cornea, it is referred to as neuropathic corneal pain (NCP), resulting in pain, hyperalgesia, burning, and photoallodynia, severely affecting patients' quality of life. To date there is no suitable animal model for the study of NCP. Herein, we developed an NCP model by constriction of the long ciliary nerves innervating the eye. Methods: Mice underwent ciliary nerve constriction (CNC) or sham procedures. Safety was determined by corneal fluorescein staining to assess ocular surface damage, whereas Cochet-Bonnet esthesiometry and confocal microscopy assessed the function and structure of corneal nerves, respectively. Efficacy was assessed by paw wipe responses within 30 seconds of applying hyperosmolar (5M) saline at Days 3, 7, 10, and 14 post-constriction. Additionally, behavior was assessed in an open field test (OFT) at Days 7, 14, and 21. Results: CNC resulted in significantly increased response to hyperosmolar saline between groups (p < 0.0001), demonstrating hyperalgesia and induction of neuropathic pain. Further, animals that underwent CNC had increased anxiety-like behavior in an open field test compared to controls at the 14- and 21-Day time-points (p < 0.05). In contrast, CNC did not result in increased corneal fluorescein staining or decreased sensation as compared to sham controls (p > 0.05). Additionally, confocal microscopy of corneal whole-mounts revealed that constriction resulted in only a slight reduction in corneal nerve density (p < 0.05), compared to naïve and sham groups. Discussion: The CNC model induces a pure NCP phenotype and may be a useful model for the study of NCP, recapitulating features of NCP, including hyperalgesia in the absence of ocular surface damage, and anxiety-like behavior.

Synaptamide modulates glial and neurotransmitter activity in the spinal cord during neuropathic pain.

N-docosahexaenoylethanolamine, or synaptamide, is an endogenous metabolite of docosahexaenoic acid that is known for synaptogenic and neurogenic effects. In our previous studies we have shown that synaptamide attenuates neuropathic pain, facilitates remyelination, and reduces neuroinflammation after the chronic constriction injury (CCI) of the sciatic nerve in rats. In the current study, we show that daily synaptamide administration (4 mg/kg/day) within 14 days post-surgery: (1) decreases micro- and astroglia activity in the dorsal and ventral horns of the lumbar spinal cord; (2) modulates pro-inflammatory (IL1ß, IL6) and anti-inflammatory (IL4, IL10) cytokine level in the serum and spinal cord; (3) leads to a rise in synaptamide and anandamide concentration in the spinal cord; (4) enhances IL10, CD206 and N-acylethanolamine-hydrolyzing acid amidase synthesis in macrophage cell culture following LPS-induced inflammation. Thus, the ability of synaptamide to modulate glial and cytokine activity indicates its potential for implementation in the treatment peripheral nerve injury.