Animal Studies

The noradrenergic neurons of locus coeruleus (LC) project to the spinal dorsal horn (SDH), and release norepinephrine (NE) to inhibit pain transmission. However, its effect on pathological pain and the cellular mechanism in the SDH remains unclear. This study aimed to explore the analgesic effects and the anti-neuroinflammation mechanism of LC-spinal cord noradrenergic pathway (LC:SC) in neuropathic pain (NP) mice with sciatic chronic constriction injury.

Oxaliplatin (OXA) is a key platinum-based chemotherapeutic agent for treatment of metastatic colorectal cancer, but the side effects of acute and chronic neuropathies limit its clinical application. Duloxetine has been found to have the potential to prevent OXA-induced peripheral neuropathy in several studies, but the underlying mechanisms remain unclear. The purpose of this study was to evaluate the effects of duloxetine on OXA-induced peripheral neuropathy and to find the potential mechanisms. The neuropathic pain mice model was used to explore the role of duloxetine on OXA-induced peripheral neuropathy by measuring the change of thermal withdrawal latency (TWL), paw withdrawal threshold (PWT), and intraepidermal nerve fiber density (IENFD). Moreover, to explore molecular mechanisms, effects of duloxetine on OXA-induced changes in mRNA and protein expression of components of the p53-related pathways in cultured rat dorsal root ganglion (DRG) neurons were measured. In vivo, we found duloxetine treatment could significantly prevent the changes in the TWL, PWT to mechanical stimulation, and the IENFD of mice caused by OXA. In vitro, we found duloxetine notably inhibits the relative mRNA and protein expression levels of p53, Bax/Bcl2, caspase-3, and caspase-9 in DRG neurons, which may indicate duloxetine protected the DRG neuron by inhibiting p53-related pathways. These results suggest that duloxetine could alleviate the OXA-induced peripheral neuropathy. Duloxetine deserves further consideration as a potential protective agent against peripheral neuropathy.

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

Nerve injury leads to chronic pain and exaggerated sensitivity to gentle touch (allodynia) as well as a loss of sensation in the areas in which injured and non-injured nerves come together. The mechanisms that disambiguate these mixed and paradoxical symptoms are unknown. Here we longitudinally and non-invasively imaged genetically labelled populations of fibres that sense noxious stimuli (nociceptors) and gentle touch (low-threshold afferents) peripherally in the skin for longer than 10 months after nerve injury, while simultaneously tracking pain-related behaviour in the same mice. Fully denervated areas of skin initially lost sensation, gradually recovered normal sensitivity and developed marked allodynia and aversion to gentle touch several months after injury. This reinnervation-induced neuropathic pain involved nociceptors that sprouted into denervated territories precisely reproducing the initial pattern of innervation, were guided by blood vessels and showed irregular terminal connectivity in the skin and lowered activation thresholds mimicking low-threshold afferents. By contrast, low-threshold afferents-which normally mediate touch sensation as well as allodynia in intact nerve territories after injury-did not reinnervate, leading to an aberrant innervation of tactile end organs such as Meissner corpuscles with nociceptors alone. Genetic ablation of nociceptors fully abrogated reinnervation allodynia. Our results thus reveal the emergence of a form of chronic neuropathic pain that is driven by structural plasticity, abnormal terminal connectivity and malfunction of nociceptors during reinnervation, and provide a mechanistic framework for the paradoxical sensory manifestations that are observed clinically and can impose a heavy burden on patients.

Targeted muscle reinnervation (TMR) is a clinical intervention that is rapidly becoming common in major limb amputation to prevent or reduce amputation-related pain. However, TMR is much less effective when applied long after injury compared to acute TMR. Since the mechanisms governing pain relief in TMR of amputated nerves are unknown, we developed a preclinical model as a platform for mechanistic examination. Following spared nerve injury (SNI), rats underwent either TMR, simple neuroma excision, or a sham manipulation of the injury site. These interventions were performed immediately or delayed (3 or 12 weeks) following SNI. Pain behavior was measured as sensitivity to mechanical stimuli (pin, von Frey, dynamic brush) and thermal stimuli (acetone, radiant heat). SNI produced hypersensitivity to all mechanical stimuli and cold, which persisted following sham surgery. TMR at the time of SNI prevented the development of pain behaviors and performing TMR 3 weeks after SNI reversed pain behaviors to baseline. In contrast, TMR performed at 12 weeks after SNI had no effect on pain behaviors. Neuroma excision resulted in significantly less reduction in hyperalgesia compared to TMR when performed 3 weeks following SNI but had no effect at 12 weeks post SNI. In this model, the pain phenotype induced by nerve transection is reduced by TMR when performed within three weeks after injury. However, TMR delayed 12 weeks after injury fails to reduce pain behaviors. This replicates clinical experience with limb amputation, supporting validity of this model for examining the mechanisms of TMR analgesia.

Chemotherapy-induced peripheral neuropathy (CIPN) and hypersensitivity reactions (HSRs) are among the most frequent and impairing side effects of the antineoplastic agent paclitaxel. Here, we demonstrated that paclitaxel can bind and activate complement component 5a receptor 1 (C5aR1) and that this binding is crucial in the etiology of paclitaxel-induced CIPN and anaphylaxis. Starting from our previous data demonstrating the role of interleukin (IL)-8 in paclitaxel-induced neuronal toxicity, we searched for proteins that activate IL-8 expression and, by using the Exscalate platform for molecular docking simulations, we predicted the high affinity of C5aR1 with paclitaxel. By in vitro studies, we confirmed the specific and competitive nature of the C5aR1-paclitaxel binding and found that it triggers intracellularly the NFkB/P38 pathway and c-Fos. In F11 neuronal cells and rat dorsal root ganglia, C5aR1 inhibition protected from paclitaxel-induced neuropathological effects, while in paclitaxel-treated mice, the absence (knock-out mice) or the inhibition of C5aR1 significantly ameliorated CIPN symptoms-in terms of cold and mechanical allodynia-and reduced the chronic pathological state in the paw. Finally, we found that C5aR1 inhibition can counteract paclitaxel-induced anaphylactic cytokine release in macrophages in vitro, as well as the onset of HSRs in mice. Altogether these data identified C5aR1 as a key mediator and a new potential pharmacological target for the prevention and treatment of CIPN and HSRs induced by paclitaxel.