Animal Studies

Neuropathic pain (NP) is one of the most common and debilitating comorbidities of spinal cord injury (SCI). Current therapies are often ineffective due in part to an incomplete understanding of underlying pathogenic mechanisms. In particular, it remains unclear how SCI leads to dysfunction in the excitability of nociceptive circuitry. The immediate early gene c-Fos has long been used in pain processing locations as a marker of neuronal activation. We employed a mouse reporter line with fos-promoter driven Cre-recombinase to define neuronal activity changes in relevant pain circuitry locations following C5/6 contusion (using both females and males), a SCI model that results in multiple forms of persistent NP-related behavior. SCI significantly increased activation of cervical dorsal horn (DH) projection neurons, as well as induced a selective reduction in the activation of a specific DH projection neuron subpopulation that innervates the periaqueductal gray (PAG), an important brain region involved in descending inhibitory modulation of DH pain transmission. SCI also increased the activation of both PKCγ and calretinin excitatory DH interneuron populations. Interestingly, SCI promoted a significant decrease in the activation selectively of nNOS expressing inhibitory interneurons of cervical DH. In addition, SCI altered activation of various supraspinal neuron populations associated with pain processing, including a large increase in thalamus and a significant decrease in PAG. These findings reveal a complex and diverse set of SCI-induced neuron activity changes across the pain circuitry neuraxis. Moving forward, these results can be used to inform therapeutic targeting of defined neuronal populations in NP.NP is one of the most common and highly debilitating comorbidities of SCI. Unfortunately, current therapies are often ineffective due in part to an incomplete understanding of underlying pathogenic mechanisms. In particular, it remains unclear how SCI leads to dysfunction in excitability of nociceptive circuitry. Using a FosTRAP2 reporter mouse line in a model of SCI-induced NP, we show SCI alters activation of a number of important interneuron and projection neuron populations across relevant spinal cord and brain locations of the pain circuitry neuraxis. These data suggest a role for maladaptive plasticity involving specific subpopulations of neurons and circuits in driving SCI-induced chronic pain. Moving forward, these results can be used to inform therapeutic targeting of defined neuronal populations in NP.

Brainstem to spinal cord noradrenergic pathways include a locus coeruleus origin projection and diffuse noxious inhibitory controls. While both pathways are traditionally viewed as exerting an inhibitory effect on spinal neuronal activity, the locus coeruleus was previously shown to have a facilitatory influence on thermal nociception according to the subpopulation of coerulean neurons activated. Coupled with knowledge of its functional modular organisation and the fact that diffuse noxious inhibitory controls are not expressed in varied animal models of chronicity, we hypothesised a regulatory role for the locus coeruleus on non-coerulean, discrete noradrenergic cell group(s). We implemented locus coeruleus targeting strategies by microinjecting canine adenovirus encoding for channelrhodopsin-2 under a noradrenaline-specific promoter in the spinal cord (retrogradely labelling a coeruleospinal module) or the locus coeruleus itself (labelling the entire coerulean module). Coeruleospinal module optoactivation abolished diffuse noxious inhibitory controls (Two-Way ANOVA, P < 0.0001), which were still expressed following locus coeruleus neuronal ablation. We propose that the cerulean system interacts with, but does not directly govern, diffuse noxious inhibitory controls. This mechanism may underlie the role of the locus coeruleus as a 'chronic pain generator'. Pinpointing the functionality of discrete top-down pathways is crucial for understanding sensorimotor modulation in health and disease.

Post-traumatic trigeminal neuropathy (PTTN) is a chronic sensory disorder that afflicts patients with nerve injury caused by orofacial and dental surgery or cervicofacial trauma. Currently, effective treatment strategies for PTTN are lacking, and patients treated with conventional drugs for PTTN experience adverse effects such as drowsiness and drug addiction. In the present study, we investigated whether mirogabalin, a novel gabapentinoid, could be an effective treatment for PTTN induced by distal infraorbital nerve chronic constriction injury (dIoN-CCI) in the mouse. Increased facial grooming time and hyper-responsiveness to acetone were observed in dIoN-CCI mice. These pain-related behaviors were attenuated by intraperitoneal injection of mirogabalin. In particular, mirogabalin significantly diminished the increase in facial grooming time. The analgesic effect of mirogabalin injection started 45minutes after the injection and persisted for 6hours. Additionally, 10mg/kg mirogabalin did not affect locomotor activity in the open field test, suggesting that it does not cause sedation. Together, the current findings suggest that mirogabalin could be a valuable therapeutic drug for PTTN following orofacial surgeries without sedative side effects.

Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, and it is a typical disease which can develop chronic pain. Our previous study has proved that endocannabinoid (2-AG)-CB1R-GABA-5-HT pathway is involved in electroacupuncture (EA) mediated inhibition of chronic pain. However, it is still unclear which among the 5-HT receptor subtype is involved in EA evoked 5-HT mediated inhibition of chronic pain in the dorsal spinal cord. 5-HT2A is a G protein-coupled receptor and it is involved in 5-HT descending pain modulation system. We found that EA treatment at frequency of 2 Hz +1 mA significantly increased the expression of 5-HT2A receptor in the dorsal spinal cord and intrathecal injection of 5-HT2A receptor antagonist or agonist reversed or mimicked the analgesic effect of EA in each case respectively. Intrathecal injection of a selective GABAA receptor antagonist Bicuculline also reversed the EA effect on pain hypersensitivity. Additionally, EA treatment reversed the reduced expression of GABAA receptor and KCC2 in the dorsal spinal cord of KOA mice. Furthermore, we demonstrated that intrathecal 5-HT2A receptor antagonist/agonist reversed or mimicked the effect of EA up-regulate of KCC2 expression respectively. Similarly, intrathecal injection of PLC and PKC inhibitors prevented both anti-allodynic effect and up-regulation of KCC2 expression by EA treatment. Our data suggest that EA treatment up-regulated KCC2 expression through activating 5-HT2A-Gq-PLC-PKC pathway and enhanced the inhibitory function of GABAA receptor, thereby inhibiting chronic pain in a mouse model of KOA.

Chronic pain is among the most burdensome and devastating disorders affecting millions of people worldwide. Recent studies suggest the role of kinesin nanomotors in development and maintenance of chronic pain. KIF17 is a member of kinesin superfamily that binds to NR2B cargo system via mLin10 scaffolding protein and makes the NMDARs functional at cell surface. NMDA receptor activation is known to induce the central sensitization and excitotoxicity which can be recognized by the glial cells followed by the release of cytokine storm at spinal and supraspinal level leading to chronic pain. In this study, we have investigated the role of aurora kinase in the regulation of KIF17 and NR2B trafficking in the animal model of chronic inflammatory pain. Tozasertib (10, 20, and 40 mg/kg i.p.), a pan aurora kinase inhibitor, significantly attenuates acute inflammatory pain and suppresses enhanced pain hypersensitivity to heat, cold, and mechanical stimuli in CFA-injected rats. Molecular investigations suggest enhanced expression of KIF17/mLin10/NR2B in L4-L5 dorsal root ganglion (DRG) and spinal cord of CFA-injected rats which was significantly attenuated on treatment with tozasertib. Moreover, tozasertib treatment significantly attenuated CFA-induced oxido-nitrosative stress and macrophage activation in DRG and microglia activation in spinal cord of rats. Findings from the current study suggest that tozasertib mediates anti-nociceptive activity by inhibiting aurora kinase-mediated KIF17/mLin10/NR2B signaling.

Fentanyl derivatives (FENS) belongs to the class of Novel Synthetic Opioids that emerged in the illegal drug market of New Psychoactive Substances (NPS). These substances have been implicated in many cases of intoxication and death with overdose worldwide. Therefore, the aim of this study is to investigate the pharmaco-dynamic profiles of three fentanyl (FENT) analogues: Acrylfentanyl (ACRYLF), Ocfentanyl (OCF) and Furanylfentanyl (FUF). In vitro, we measured FENS opioid receptor efficacy, potency, and selectivity in calcium mobilization studies performed in cells coexpressing opioid receptors and chimeric G proteins and their capability to promote the interaction of the mu receptor with G protein and β-arrestin 2 in bioluminescence resonance energy transfer (BRET) studies. In vivo, we investigated the acute effects of the systemic administration of ACRYLF, OCF and FUF (0.01-15 mg/kg i.p.) on mechanical and thermal analgesia, motor impairment, grip strength and cardiorespiratory changes in CD-1 male mice. Opioid receptor specificity was investigated in vivo using naloxone (NLX; 6 mg/kg i.p) pre-treatment. In vitro, the three FENS were able to activate the mu opioid receptor in a concentration dependent manner with following rank order potency: FUF > FENT=OCF > ACRYLF. All compounds were able to elicit maximal effects similar to that of dermorphin, with the exception of FUF which displayed lower maximal effects thus behaving as a partial agonist. In the BRET G-protein assay, all compounds behaved as partial agonists for the β-arrestin 2 pathway in comparison with dermorphin, whereas FUF did not promote β-arrestin 2 recruitment, behaving as an antagonist. In vivo, all the compounds increased mechanical and thermal analgesia with following rank order potency ACRYLF = FENT > FUF > OCF and impaired motor and cardiorespiratory parameters. Among the substances tested, FUF showed lower potency for cardiorespiratory and motor effects. These findings reveal the risks associated with the use of FENS and the importance of studying the pharmaco-dynamic properties of these drugs to better understand possible therapeutic interventions in the case of toxicity.

Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a β-lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases.

The etiology of neuropathic pain is complex, and the patients are distressed. In order to master more accurate information in the treatment of human nerve tissue and improve the efficiency of treatment, this paper discusses the antagonistic effect of gabapentin on neuropathic pain in rats through the p38 MAPK signal pathway. Thirty-six female Sprague Dawley (SD) rats were randomly divided into three groups, 12 in each group. One group was spinal nerve ligation group (SNL group), gabapentin Group (GBP group, spinal nerve ligation and intraperitoneal injection of gabapentin (50 mg/kg)) and sham operation group (sham group, no spinal nerve ligation, other surgical procedures were the same as SNL group), At 1, 3, 5 and 7 days after operation, the paw contraction latency (TWL) and mechanical paw contraction threshold (MWT) were detected. Then, the expression of Toll-like receptor 4 (TLR4) in dorsal root ganglia was detected by SPSS statistical analysis. Compared with the sham group, MWT and TWL in the SNL group and GBP group were lower at each time point after the operation (all P < 0.05). MWT and TWL in the GBP group were higher than those in the SNL group at 5 and 7 days after the operation (all P < 0.05). In addition, compared with the sham group, the expression of TLR4 in the dorsal root ganglia of the SNL group was significantly increased (P < 0.05), while the expression of TLR4 in the GBP group was not significantly increased (P > 0.05). Compared with the SNL group, the expression of TLR4 in the dorsal root ganglion of the GBP group was significantly decreased (P < 0.05). Thus, gabapentin combined therapy can effectively reduce the degree of pain in patients with significant efficacy, high safety and fewer adverse reactions.