Nerve Chronic Constriction Injury Research Articles

CHOP-Mediated Disruption of Hippocampal Synaptic Plasticity and Neuronal Activity Contributes to Chronic Pain-Related Cognitive Deficits.

Endoplasmic reticulum (ER) stress-induced protein homeostasis perturbation is a core pathological element in the pathogenesis of neurodegenerative diseases. This study aims to clarify the unique role played by C/EBP homologous protein (CHOP) as a biomarker of the unfolded protein response (UPR) in the etiology of chronic pain and related cognitive impairments following chronic constrictive nerve injury (CCI). The memory capability following CCI was assessed utilizing the Morris water maze (MWM) and fear conditioning test (FCT). Activation of the UPR was quantified by assessing levels of CHOP and key ER stress sensors. The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay and the levels of cleaved caspase-3 were utilized to assess apoptosis level. Synaptic plasticity was assessed via a modified Golgi-Cox staining method, and long-term potentiation (LTP) measurements were taken. Neuronal activity was determined by immunofluorescence and fiber photometry. Knockdown of CHOP and alleviation of ER stress were selectively induced by LV-Ddit3-shRNAs and the chemical chaperone 4-phenylbutyric acid (4-PBA), respectively. Mice subjected to CCI displayed enduring pain and cognitive impairments evident on Days 21-28 post-surgery. Following CCI, changes in the dorsal CA1 (dCA1) manifested as ER dilation, upregulation of CHOP and upstream signaling molecules, reduced dendritic spine density, and PSD95 levels, and impaired LTP. Additionally, the co-localization of CaMKIIα/c-Fos and CaMKIIαdCA1-mediated calcium signaling was significantly reduced, while the activation of CaMKIIα was found to mitigate cognitive impairments in CCI mice. Selective knockdown of CHOP enhanced synaptic plasticity and CaMKIIα neuron activity, while 4-PBA treatment alleviated ER stress, synergistically improving cognitive deficits associated with chronic pain. CCI-induced CHOP upregulation impairs dCA1 synaptic plasticity and neuronal activity, leading to chronic pain-related cognitive deficits.

Disrupted stress-induced analgesia in a neuropathic pain state is rescued by the endocannabinoid degradation inhibitor JZL195.

Acute stress normally engages descending brain pathways to produce an antinociceptive response, known as stress-induced analgesia. Paradoxically, these descending pain modulatory pathways are also involved in the maintenance of the abnormal pain associated with chronic neuropathic pain. It remains unclear how stress-induced analgesia is affected by neuropathic pain states. We therefore examined the impact of a chronic constriction nerve-injury (CCI) model of neuropathic pain on restraint stress-induced analgesia in C57BL/6 mice. Thirty minutes of restraint stress produced analgesia in the hotplate thermal nociceptive assay that was less in CCI compared to control mice who underwent a sham-surgery. In sham but not CCI mice, stress-induced analgesia was reduced by the opioid receptor antagonist naltrexone. The cannabinoid CB1 receptor antagonist AM281 did not affect stress-induced analgesia in either sham or CCI mice. Low-dose pre-treatment with the dual fatty acid amide hydrolase and monoacylglycerol lipase inhibitor JZL195 increased stress-induced analgesia in CCI but not sham mice. The JZL195 enhancement of stress-induced analgesia in CCI mice was abolished by AM281 but was unaffected by naltrexone. These findings indicate that the acute opioid-mediated analgesic response to a psychological stressor is disrupted in a nerve-injury model of neuropathic pain. Importantly, this impairment of stress-induced analgesia was rescued by blockade of endocannabinoid breakdown via a cannabinoid CB1 receptor dependent mechanism. These findings suggest that subthreshold treatment with endocannabinoid degradation blockers could be used to alleviate the disruption of endogenous pain control systems in a neuropathic pain state.

Examination of the effects of vitexin and vitexin-loaded solid lipid nanoparticles on neuropathic pain and possible mechanisms of action

This research aims to investigate the possible antiallodynic and antihyperalgesic effects of pure vitexin and vitexin-loaded solid lipid nanoparticles (SLN) on neuropathic pain and the pathways mediating these effects. Chronic constriction nerve injury was induced in female rats, and the effects of vitexin at the doses of 5, 10, 20, 40 mg/kg were evaluated. Ketanserin, ondansetron, WAY-100635, yohimbine and bicuculin, which are antagonists of receptors on pain pathways. were used to examine the mechanisms of the effects of vitexin. Pure vitexin exhibited antiallodynic activity at all administered doses, whereas antihyperalgesic activity was not observed at 5 mg/kg vitexin dose. SLN formulation was prepared with 5 mg/kg vitexin, the lowest dose. Vitexin-loaded formulation significantly increased antiallodynic and antihyperalgesic effects. Ondansetron, WAY-100635, yohimbine, and bicuculine antagonized the antiallodynic and antihyperalgesic effects of vitexin. So, it was concluded that serotonin (5-hydroxtryptamine, 5-HT) receptor subtypes 5-HT3 and 5-HT1A, alpha-2 adrenergic, and γ-Aminobutyric acid type A (GABA-A) receptors are involved in the antiallodynic and antihyperalgesic activity of vitexin. In conclusion, vitexin and vitexin-loaded formulation have the potential for clinical use in neuropathic pain management, and different pain pathways contributed to this effect. And also, it is thought that vitexin-loaded SLN formulation is more effective than pure vitexin, which will provide an advantage in treatment.

Astrocyte senescence-like response related to peripheral nerve injury-induced neuropathic pain

BackgroundPeripheral nerve damage causes neuroinflammation, which plays a critical role in establishing and maintaining neuropathic pain (NeP). The mechanisms contributing to neuroinflammation remain poorly elucidated, and pharmacological strategies for NeP are limited. Thus, in this study, we planned to explore the possible link between astrocyte senescence and NeP disorders following chronic sciatic nerve injury.MethodsAn NeP animal model was established by inducing chronic constrictive injury (CCI) to the sciatic nerve in adult rats. A senolytic drug combination of dasatinib and quercetin was gavaged daily from the first postoperative day until the end of the study. Paw mechanical withdrawal threshold (PMWT) and paw thermal withdrawal latency (PTWL) were evaluated to assess behaviors in response to pain in the experimental rats. Senescence-associated β-galactosidase staining, western blot analysis, and immunofluorescence were applied to examine the levels of proinflammatory factors and severity of the senescence-like response in the spinal cord. Lipopolysaccharide (LPS) was administered to induce senescence of spinal astrocytes in primary cultures in vitro, to explore the potential impacts of senescence on the secretion of proinflammatory factors. Furthermore, single-cell RNA sequencing (scRNA-seq) was conducted to identify senescence-related molecular responses in spinal astrocytes under neuropathic pain.ResultsFollowing sciatic nerve CCI, rats exhibited reduced PMWT and PTWL, increased levels of spinal proinflammatory factors, and an enhanced degree of senescence in spinal astrocytes. Treatment with dasatinib and quercetin effectively attenuated spinal neuroinflammation and mitigated the hypersensitivities of the rats subjected to sciatic nerve CCI. Mechanistically, the dasatinib-quercetin combination reversed senescence in LPS-stimulated primary cultured astrocytes and decreased the levels of proinflammatory factors. The scRNA-seq data revealed four potential senescence-related genes in the spinal astrocyte population, and the expression of clusterin (CLU) protein was validated via in vitro experiments.ConclusionThe findings indicate the potential role of astrocyte senescence in neuroinflammation following peripheral nerve injury, and suggest that targeting CLU activation in astrocytes might provide a novel therapeutic strategy to treat NeP.Graphical abstract

Curcumin promotes microglial M2 polarization and suppresses chronic constriction: Injury-induced neuropathic pain in a rat model of peripheral neuropathy

ObjectivesGlia (i.e., astrocyte and microglia) activation in the central nervous system plays a critical role in developing neuropathic pain. Microglia can be activated into proinflammatory (M1) and anti-inflammatory (M2) phenotypes. Switching microglial polarization from M1 to M2 phenotypes represents a novel therapeutic strategy for neuropathic pain. Curcumin has been widely used for its anti-inflammatory and immunomodulatory effects. This study investigated effects of curcumin on astrocyte activation and microglia polarization in the cuneate nucleus (CN) and development of neuropathic pain behavior after chronic constriction injury (CCI) of the median nerve. MethodsRats were fed with curcumin once daily at a dose of 40, 80, or 120 mg/kg 30 min before and until 7 d after median nerve CCI. Subsequently, mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filaments and plantar tests, respectively. The levels of astrocyte marker, monoclonal glial fibrillary acidic protein; microglia marker, ionized calcium-binding adapter molecule 1; M1 marker, CD86; and M2 marker, CD206 in the cuneate nucleus were determined. Enzyme-linked immunosorbent assay was applied to measure cytokine concentrations. ResultsCurcumin administration dose-dependently reduced mechanical allodynia and thermal hyperalgesia and decreased monoclonal glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 immunoreactivity in the ipsilateral cuneate nucleus after CCI. On ultrastructural observation, curcumin treatment was associated with fewer features of activated astrocytes and microglia. Furthermore, CCI rats given curcumin exhibited a decline in CD86 immunoreactivity and proinflammatory cytokine levels but an increase in CD206 immunoreactivity and release of anti-inflammatory cytokines. ConclusionsIn our findings, curcumin switches microglial phenotypes from M1 to M2 by suppressing astrocytic activation, reducing proinflammatory cytokine release, promoting anti-inflammatory cytokine production, and contributing to relief of neuropathic pain.

Progesterone modulates the expression of spinal ephrin-B2 after peripheral nerve injury: New insights into progesterone mechanisms

Recent studies have shown that the ephrin/Eph signaling pathway may contribute to the pathology of neuropathic pain. Drugs like progesterone may be used to counteract both thermal hyperalgesia and mechanical allodynia in different models of neuropathic pain. The present study was designed to determine progesterone’s modulatory role on neuropathic pain and spinal expression of ephrin-B2 following chronic constriction nerve injury (CCI).Thirty-six adult male Wistar rats were used. The sciatic nerve was chronically constricted. Progesterone (5 mg/kg and 15 mg/kg) was administrated for 10 days (from day 1 up to day10) following sciatic constriction. Behavioral tests were performed before surgery (day 0) and on days 1, 3, 7, and 14 after CCI and before progesterone administration on the same days. Western blotting was performed on days 3, 7, and 14th post-surgery. The findings showed that after CCI, the expression of spinal cord ephrin-B2 increased significantly in parallel with mechanical allodynia and thermal hyperalgesia. Post-injury administration of progesterone (15 mg/kg but not 5) decreased mechanical allodynia, thermal hyperalgesia, and the expression of spinal ephrin-B2.It is concluded that post-injury repeated administration of progesterone could be an effective way of alleviating neuropathic pain by suppressing ephrin-B2 activation and helps to make the better design of steroid-based therapies to inhibit pain after peripheral injury.

RgIA4 Prevention of Acute Oxaliplatin-Induced Cold Allodynia Requires α9-Containing Nicotinic Acetylcholine Receptors and CD3+ T-Cells.

Chemotherapy-induced neuropathic pain is a debilitating and dose-limiting side effect. Oxaliplatin is a third-generation platinum and antineoplastic compound that is commonly used to treat colorectal cancer and commonly yields neuropathic side effects. Available drugs such as duloxetine provide only modest benefits against oxaliplatin-induced neuropathy. A particularly disruptive symptom of oxaliplatin is painful cold sensitivity, known as cold allodynia. Previous studies of the Conus regius peptide, RgIA, and its analogs have demonstrated relief from oxaliplatin-induced cold allodynia, yielding improvement that persists even after treatment cessation. Moreover, underlying inflammatory and neuronal protection were shown at the cellular level in chronic constriction nerve injury models, consistent with disease-modifying effects. Despite these promising preclinical outcomes, the underlying molecular mechanism of action of RgIA4 remains an area of active investigation. This study aimed to determine the necessity of the α9 nAChR subunit and potential T-cell mechanisms in RgIA4 efficacy against acute oxaliplatin-induced cold allodynia. A single dose of oxaliplatin (10 mg/kg) was utilized followed by four daily doses of RgIA4. Subcutaneous administration of RgIA4 (40 µg/kg) prevented cold allodynia in wildtype mice but not in mice lacking the α9 nAChR-encoding gene, chrna9. RgIA4 also failed to reverse allodynia in mice depleted of CD3+ T-cells. In wildtype mice treated with oxaliplatin, quantitated circulating T-cells remained unaffected by RgIA4. Together, these results show that RgIA4 requires both chrna9 and CD3+ T-cells to exert its protective effects against acute cold-allodynia produced by oxaliplatin.

IRG1/itaconate increases IL-10 release to alleviate mechanical and thermal hypersensitivity in mice after nerve injury.

Inflammation plays an important role in the occurrence and development of neuropathic pain. Immune-responsive gene 1 (IRG1) decarboxylates cis-aconitate to produce itaconate in the mitochondria. Itaconate serves as an immunomodulator of macrophages and represses inflammation in infectious diseases. Recently, a study showed that an itaconate derivative inhibits neuroinflammation and reduces chronic pain in mice. However, the function and molecular mechanisms of endogenous itaconate in neuropathic pain have not been fullyelucidated. In this study, the content of itaconate in the ipsilateral spinal cord after nerve-injured mice was detected with mass spectrometry. The Irg1-/- mouse was constructed to determine the role of endogenous itaconate in the chronic constriction nerve injury (CCI) model. The analgesic effect of exogenous itaconate was assessed with intraperitoneal and intrathecal administration in both male and female CCI mice. The spinal application of 4-OI also reduced the evoked responses of wide dynamic range neurons in CCI mice. The potential analgesic mechanism of itaconate was explored through molecular biology experiments and verified in Interleukin (IL)-10-/- mice. We found the levels of itaconate and IRG1 in the spinal cord significantly increased after CCI. Irg1 deficiency aggravated the mechanical and heat hypersensitivity, while the exogenous administration of the itaconate derivative 4-OI alleviated the neuropathic pain in male and female CCI mice. Mechanistically, the treatment of 4-OI increased the level of IL-10 and activates STAT3/β-endorphin pathway in the spinal cord, and the analgesia effect of itaconate was impaired in IL-10-/- mice. Finally, we showed that the upregulation of IL-10 induced by 4-OI was mainly from spinal neurons through Nrf2 pathway. This study demonstrated the analgesic effect of endogenous and exogenous itaconate in the neuropathic pain model, suggesting that the spinal IL-10/STAT3/β-endorphin pathway might mediate the analgesia effect of itaconate.

Modulation of Aryl Hydrocarbon Receptor Expression Alleviated Neuropathic Pain in a Chronic Constriction Nerve Injury Animal Model.

Neuropathic pain is well known to occur after damage to the somatosensory system. Aryl hydrocarbon receptor (AhR) has neuroprotective effects when the central nervous system is subjected to internal and external stimulations. However, the exact mechanism by which AhR regulates neuropathic pain is poorly understood. Nerve explant culture and the chronic constrictive nerve injury (CCI) model in wild or AhR-knockout mice were used in this study. In the nerve explant culture, the ovoid number increased in the AhR−/− condition and was decreased by omeprazole (AhR agonist) in a dose-dependent manner. Increased nerve degeneration and the associated inflammation response appeared in the AhR−/− condition, and these changes were attenuated by omeprazole. High expression of AhR in the injured nerve was noted after CCI. Deletion of AhR aggravated nerve damages and this was restored by omeprazole. Deletion of AhR increased NGF expression and reduced axon number in the paw skin, but this was attenuated by omeprazole. A highly expressed inflammation reaction over the dorsal spinal cord, somatosensory cortex, and hippocampus was noted in the AhR-deleted animals. Administration of omeprazole attenuated not only the inflammatory response, but also the amplitude of somatosensory evoked potential. Deletion of AhR further aggravated the neurobehavior compared with the wild type, but such behavior was attenuated by omeprazole. Chronic constrictive nerve injury augmented AhR expression of the injured nerve, and AhR deletion worsened the damage, while AhR agonist omeprazole counteracted such changes. AhR agonists could be potential candidates for neuropathic pain treatment.

Analgesic effects of Terminalia chebula extract are mediated by the suppression of the protein expression of nerve growth factor and nuclear factor-κB in the brain and oxidative markers following neuropathic pain in rats.

Due to the complications related to the use of the current pharmacological approach for the alleviation of neuropathic pain, searching for effective compound with fewer complications is a requirement of the present era. It is well known that the pathophysiological mechanism of neuropathic pain is related to excessive inflammation in the nervous system. Hence, the present study focuses on whether the potential analgesic effects of Terminalia chebula (TC) extract are mediated by the changes in the protein expression of nerve growth factor (NGF) and nuclear factor-kappa B (NF-κB) in the brain in a rat model of sciatic nerve chronic constriction injury (CCI). Neuropathic pain was induced by the left sciatic nerve CCI. Male Wistar rats were assigned to three groups: sham, CCI, and CCI + TC (40mg/kg). Animals received either normal saline (1 mL) or the aqueous-alcoholic extract of TC (40mg/kg) for 30 days via gavage needles once a day. Cold allodynia and anxiety-like behaviors were examined one day before CCI surgery (day - 1), as well as days 2, 7, 14, and 30 following CCI. We also assessed the effects of the TC extract oxidative stress markers on day 30 following CCI. Moreover, a western blot analysis was performed on day 30 following CCI to evaluate the effects of the TC extract on the protein expression of NGF and NF-κB in the brain. Oral gavage of the TC extract significantly decreased cold allodynia on days 2 and 14 following CCI. Additionally, the CCI model of chronic pain significantly increased the protein expression of NGF and NF-κB in the brain on day 30 following CCI. Furthermore, the TC extract significantly decreased the protein expression of NGF and NF-κB in the brain. The TC extract also significantly increased the brain glutathione (GSH) content and decreased the malondialdehyde (MDA) content. It is suggested that the analgesic effects of the TC extract are mediated by the suppression of brain NGF, NF-κB, and by its antioxidant activity in the brain following neuropathic pain in rats.

Frozen vein wrapping for chronic nerve constriction injury reduces sciatic nerve allodynia in a rat model

BackgroundAutologous vein wrapping (VW) is used in the treatment of recurrent chronic constriction neuropathy and traumatic peripheral nerve injury. However, use of autologous veins is limited by the inability to obtain longer veins of sufficient length for larger sites. Frozen allograft tissue has several advantages, including its availability for large grafts, avoidance of donor-site morbidity, and shorter operation time. Here, we investigated the effect of frozen vein wrapping (FVW) in Wistar rats as a model of sciatic nerve injury.ResultsThe rats were grouped by treatment as (i) untreated after chronic constriction injury surgery (CCI; control group), (ii) treated with vein wrapping using freshly isolated vein (VW), and (iii) treated with vein wrapping using frozen vein (FVW). Mechanical allodynia was assessed with von Frey filaments on postoperative days (PODs) 1, 3, 5, 7, and 14. Gene expression of HO-1 was evaluated by quantitative polymerase chain reaction (qPCR). The response of heme oxygenase-1 gene, Hmox-1, expression to VW and FVW was assessed by RT-PCR. Both VW and FVW significantly increased withdrawal threshold levels compared to the untreated control group on POD 1, 3, and 5. Both VW and FVW also showed increased HO-1 expression compared to the CCI group.ConclusionsFVW increased the withdrawal threshold similar to VW in a rat CCI model for short periods. Frozen vein wrapping using vein allograft without donor site morbidity may be an alternative therapeutic option.

Characterization of CM-398, a Novel Selective Sigma-2 Receptor Ligand, as a Potential Therapeutic for Neuropathic Pain.

Sigma receptors modulate nociception, offering a potential therapeutic target to treat pain, but relatively little is known regarding the role of sigma-2 receptors (S2R) in nociception. The purpose of this study was to investigate the in vivo analgesic and anti-allodynic activity and liabilities of a novel S2R selective ligand, 1-[4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-2-yl)butyl]-3-methyl-1,3-dihydro-1,3-benzimidazol-2-one (CM-398). The inhibition of thermal, induced chemical, or inflammatory pain as well as the allodynia resulting from chronic nerve constriction injury (CCI) model of neuropathic pain were assessed in male mice. CM-398 dose-dependently (10–45 mg/kg i.p.) reduced mechanical allodynia in the CCI neuropathic pain model, equivalent at the higher dose to the effect of the control analgesic gabapentin (50 mg/kg i.p.). Likewise, pretreatment (i.p.) with CM-398 dose-dependently produced antinociception in the acetic acid writhing test (ED50 (and 95% C.I.) = 14.7 (10.6–20) mg/kg, i.p.) and the formalin assay (ED50 (and 95% C.I.) = 0.86 (0.44–1.81) mg/kg, i.p.) but was without effect in the 55 °C warm-water tail-withdrawal assay. A high dose of CM-398 (45 mg/kg, i.p.) exhibited modest locomotor impairment in a rotarod assay and conditioned place aversion, potentially complicating the interpretation of nociceptive testing. However, in an operant pain model resistant to these confounds, mice experiencing CCI and treated with CM-398 demonstrated robust conditioned place preference. Overall, these results demonstrate the S2R selective antagonist CM-398 produces antinociception and anti-allodynia with fewer liabilities than established therapeutics, adding to emerging data suggesting possible mediation of nociception by S2R, and the development of S2R ligands as potential treatments for chronic pain.

MiR-223-3p alleviates trigeminal neuropathic pain in the male mouse by targeting MKNK2 and MAPK/ERK signaling.

BackgroundTrigeminal neuralgia (TN) is a neuropathic pain that occurs in branches of the trigeminal nerve. MicroRNAs (miRNAs) have been considered key mediators of neuropathic pain. This study was aimed to elucidate the pathophysiological function and mechanisms of miR‐223‐3p in mouse models of TN.MethodsInfraorbital nerve chronic constriction injury (CCI‐ION) was applied in male C57BL/6J mice to establish mouse models of TN. Pain responses were assessed utilizing Von Frey method. The expression of miR‐223‐3p, MKNK2, and MAPK/ERK pathway protein in trigeminal ganglions (TGs) of CCI‐ION mice was measured using RT‐qPCR and Western blotting. The concentrations of inflammatory cytokines were evaluated using Western blotting. The relationship between miR‐223‐3p and MKNK2 was tested by a luciferase reporter assay.ResultsWe found that miR‐223‐3p was downregulated, while MKNK2 was upregulated in TGs of CCI‐ION mice. MiR‐223‐3p overexpression by an intracerebroventricular injection of Lv‐miR‐223‐3p attenuated trigeminal neuropathic pain in CCI‐ION mice, as well as reduced the protein levels of pro‐inflammatory cytokines in TGs of CCI‐ION mice. MKNK2 was verified to be targeted by miR‐223‐3p. Additionally, miR‐223‐3p overexpression decreased the phosphorylation levels of ERK1/2, JNK, and p38 protein in TGs of CCI‐ION mice to inhibit MAPK/ERK signaling.ConclusionsOverall, miR‐223‐3p attenuates the development of TN by targeting MKNK2 to suppress MAPK/ERK signaling.

A 4/8 Subtype α-Conotoxin Vt1.27 Inhibits N-Type Calcium Channels With Potent Anti-Allodynic Effect.

A novel 4/8 subtype α-conotoxin, Vt1.27 (NCCMFHTCPIDYSRFNC-NH2), was identified from Conus vitulinus in the South China Sea by RACE methods. The peptide was synthesized and structurally characterized. Similar to other α-conotoxins that target neuronal nicotinic acetylcholine receptor (nAChR) subtypes, Vt1.27 inhibited the rat α3β2 nAChR subtype (IC50 = 1160 nM) and was inactive at voltage-gated sodium and potassium channels in rat sensory neurons. However, Vt1.27 inhibited high voltage-activated N-type (CaV2.2) calcium channels expressed in HEK293T cells with an IC50 of 398 nM. An alanine scan of the peptide showed that residues Phe5, Pro9, Ile10, and Ser13 contribute significantly to the inhibitory activity of Vt1.27. The molecular dockings indicate that Vt1.27 inhibits the transmembrane region of CaV2.2, which is different from that of ω-conotoxins. Furthermore, Vt1.27 exhibited potent anti-allodynic effect in rat partial sciatic nerve injury (PNL) and chronic constriction injury (CCI) pain models at 10 nmol/kg level with the intramuscular injection. The pain threshold elevation of Vt1.27 groups was higher than that of α-conotoxin Vc1.1 in CCI rat models. These findings expand our knowledge of targets of α-conotoxins and potentially provide a potent, anti-allodynic peptide for the treatment of neuropathic pain.

Examination of the Novel Sigma-1 Receptor Antagonist, SI 1/28, for Antinociceptive and Anti-allodynic Efficacy against Multiple Types of Nociception with Fewer Liabilities of Use.

Neuropathic pain is a significant problem with few effective treatments lacking adverse effects. The sigma-1 receptor (S1R) is a potential therapeutic target for neuropathic pain, as antagonists for this receptor effectively ameliorate pain in both preclinical and clinical studies. The current research examines the antinociceptive and anti-allodynic efficacy of SI 1/28, a recently reported benzylpiperazine derivative and analog of the S1R antagonist SI 1/13, that was 423-fold more selective for S1R over the sigma-2 receptor (S2R). In addition, possible liabilities of respiration, sedation, and drug reinforcement caused by SI 1/28 have been evaluated. Inflammatory and chemical nociception, chronic nerve constriction injury (CCI) induced mechanical allodynia, and adverse effects of sedation in a rotarod assay, conditioned place preference (CPP), and changes in breath rate and locomotor activity were assessed after i.p. administration of SI 1/28. Pretreatment with SI 1/28 produced dose-dependent antinociception in the formalin test, with an ED50 (and 95% C.I.) value of 13.2 (7.42–28.3) mg/kg, i.p. Likewise, SI 1/28 produced dose-dependent antinociception against visceral nociception and anti-allodynia against CCI-induced neuropathic pain. SI 1/28 demonstrated no impairment of locomotor activity, conditioned place preference, or respiratory depression. In summary, SI 1/28 proved efficacious in the treatment of acute inflammatory pain and chronic neuropathy without liabilities at therapeutic doses, supporting the development of S1R antagonists as therapeutics for chronic pain.

Knock-In Mice Expressing a 15-Lipoxygenating Alox5 Mutant Respond Differently to Experimental Inflammation Than Reported Alox5−/− Mice

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5−/− mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5−/− animals tested previously in similar experimental setups.

Autofluorescent imprint of chronic constriction nerve injury identified by deep learning

Our understanding of chronic pain and the underlying molecular mechanisms remains limited due to a lack of tools to identify the complex phenomena responsible for exaggerated pain behaviours. Furthermore, currently there is no objective measure of pain with current assessment relying on patient self-scoring. Here, we applied a fully biologically unsupervised technique of hyperspectral autofluorescence imaging to identify a complex signature associated with chronic constriction nerve injury known to cause allodynia. The analysis was carried out using deep learning/artificial intelligence methods. The central element was a deep learning autoencoder we developed to condense the hyperspectral channel images into a four- colour image, such that spinal cord tissue based on nerve injury status could be differentiated from control tissue.This study provides the first validation of hyperspectral imaging as a tool to differentiate tissues from nerve injured vs non-injured mice. The auto-fluorescent signals associated with nerve injury were not diffuse throughout the tissue but formed specific microscopic size regions. Furthermore, we identified a unique fluorescent signal that could differentiate spinal cord tissue isolated from nerve injured male and female animals. The identification of a specific global autofluorescence fingerprint associated with nerve injury and resultant neuropathic pain opens up the exciting opportunity to develop a diagnostic tool for identifying novel contributors to pain in individuals.

Mechanisms Underlying the Selective Therapeutic Efficacy of Carbamazepine for Attenuation of Trigeminal Nerve Injury Pain.

Different peripheral nerve injuries cause neuropathic pain through distinct mechanisms. Even the site of injury may impact underlying mechanisms, as indicated by the clinical finding that the antiseizure drug carbamazepine (CBZ) relieves pain because of compression injuries of trigeminal but not somatic nerves. We leveraged this observation in the present study hypothesizing that because CBZ blocks voltage-gated sodium channels (VGSCs), its therapeutic selectivity reflects differences between trigeminal and somatic nerves with respect to injury-induced changes in VGSCs. CBZ diminished ongoing and evoked pain behavior in rats with chronic constriction injury (CCI) to the infraorbital nerve (ION) but had minimal effect in rats with sciatic nerve CCI. This difference in behavior was associated with a selective increase in the potency of CBZ-induced inhibition of compound action potentials in the ION, an effect mirrored in human trigeminal versus somatic nerves. The increase in potency was associated with a selective increase in the efficacy of the NaV1.1 channel blocker ICA-121431 and NaV1.1 protein in the ION, but no change in NaV1.1 mRNA in trigeminal ganglia. Importantly, local ICA-121431 administration reversed ION CCI-induced hypersensitivity. Our results suggest a novel therapeutic target for the treatment of trigeminal neuropathic pain.SIGNIFICANCE STATEMENT This study is based on evidence of differences in pain and its treatment depending on whether the pain is above (trigeminal) or below (somatic) the neck, as well as evidence that voltage-gated sodium channels (VGSCs) may contribute to these differences. The focus of the present study was on channels underlying action potential propagation in peripheral nerves. There were differences between somatic and trigeminal nerves in VGSC subtypes underlying action potential propagation both in the absence and presence of injury. Importantly, because the local block of NaV1.1 in the trigeminal nerve reverses nerve injury-induced mechanical hypersensitivity, the selective upregulation of NaV1.1 in trigeminal nerves suggests a novel therapeutic target for the treatment of pain associated with trigeminal nerve injury.

Development of New Benzylpiperazine Derivatives as σ1 Receptor Ligands with in Vivo Antinociceptive and Anti-Allodynic Effects.

σ-1 receptors (σ1R) modulate nociceptive signaling, driving the search for selective antagonists to take advantage of this promising target to treat pain. In this study, a new series of benzylpiperazinyl derivatives has been designed, synthesized, and characterized for their affinities toward σ1R and selectivity over the σ-2 receptor (σ2R). Notably, 3-cyclohexyl-1-{4-[(4-methoxyphenyl)methyl]piperazin-1-yl}propan-1-one (15) showed the highest σ1R receptor affinity (Ki σ1 = 1.6 nM) among the series with a significant improvement of the σ1R selectivity (Ki σ2/Ki σ1= 886) compared to the lead compound 8 (Ki σ2/Ki σ1= 432). Compound 15 was further tested in a mouse formalin assay of inflammatory pain and chronic nerve constriction injury (CCI) of neuropathic pain, where it produced dose-dependent (3–60 mg/kg, i.p.) antinociception and anti-allodynic effects. Moreover, compound 15 demonstrated no significant effects in a rotarod assay, suggesting that this σ1R antagonist did not produce sedation or impair locomotor responses. Overall, these results encourage the further development of our benzylpiperazine-based σ1R antagonists as potential therapeutics for chronic pain.

PDTC ameliorates neuropathic pain by inhibiting microglial activation via blockage of the TNFα-CX3CR1 pathway.

Previous studies have suggested that pyrrolidine dithiocarbamate (PDTC), a nuclear factor κB (NF-κB) inhibitor, plays a role in deterring nerve injury-induced neuropathic pain (NP). The activation of NF-κB pathway may contribute to spinal microglial activation, CX3CR1 and tumor necrosis factor-alpha (TNF-a) up-regulation. The aim of this study was to clarify whether PDTC could inhibit the development of neuropathic pain via decreasing TNF-a-induced CX3CR1 up-regulation. Sprague-Dawley rats were randomly divided into sham group and NP group. Rats in each group were treated with intrathecal infusion of PDTC (100 or 1000 pmol/d) or saline. The sciatic nerve chronic constriction injury (CCI) model was used to induce NP in rats. Mechanical stimuli and radiant heat were used to evaluate mechanical allodynia and thermal hyperalgesia. Spinal microglial marker OX42 and TNF-a were detected by immunohistochemistry. In vitro BV-2 microglia activation was induced by TNF-a incubation, and the levels of CX3CR1 were assessed by western blot and reverse transcription- polymerase chain reaction. Pain behavior and immunohistochemistry results showed that intrathecal infusion of PDTC at 100 or 1000 pmol/d prevented the development of mechanical and thermal hyperalgesia, spinal microglial activation and TNF-a expression induced by sciatic nerve CCI in rats. In vitro experiment results showed that PDTC inhibited the TNF-a-induced CX3CR1 up-regulation in BV-2 microglial cells. In conclusion, intrathecal infusion of PDTC could attenuate the pain-related behaviors induced by sciatic nerve CCI through suppressing the spinal microglia activation and TNF-a up-regulation in rats. The NF-κB activation might be responsible for TNF-a-induced CX3CR1 up-regulation in microglia.