Animal Studies

Advanced head and neck cancer (HNC) can invade facial bone and cause bone pain, thus posing a significant challenge to the quality of life of patients presenting with advanced HNC. The present study was designed to investigate HNC bone pain (HNC‑BP) in an intratibial mouse xenograft model that utilized an HNC cell line (SAS cells). These mice develop HNC‑BP that is associated with an expression of phosphorylated ERK1/2 (pERK1/2), which is a molecular indicator of neuron excitation in dorsal root ganglia (DRG) sensory neurons. Our experiments demonstrated that the inhibition of high mobility group box 1 (HMGB1) by short hairpin (shRNA) transduction, HMGB1 neutralizing antibody, and HMGB1 receptor antagonist suppressed the HNC‑BP and the pERK1/2 expression in DRG. It was also observed that HNC‑derived HMGB1 increased the expression of the acid‑sensing nociceptor, transient receptor potential vanilloid 1 (TRPV1), which is a major cause of osteoclastic HNC‑BP in DRG. Collectively, our results demonstrated that HMGB1 originating in HNC evokes HNC‑BP via direct HMGB1 signaling and hypersensitization for the acid environment in sensory neurons.

Global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues unabated. Binding of SARS-CoV-2's Spike protein to host angiotensin converting enzyme 2 triggers viral entry, but other proteins may participate, including neuropilin-1 receptor (NRP-1). As both Spike protein and vascular endothelial growth factor-A (VEGF-A) – a pro-nociceptive and angiogenic factor, bind NRP-1, we tested if Spike could block VEGF-A/NRP-1 signaling. VEGF-A-triggered sensory neuronal firing was blocked by Spike protein and NRP-1 inhibitor EG00229. Pro-nociceptive behaviors of VEGF-A were similarly blocked via suppression of spontaneous spinal synaptic activity and reduction of electrogenic currents in sensory neurons. Remarkably, preventing VEGF-A/NRP-1 signaling was antiallodynic in a neuropathic pain model. A 'silencing' of pain via subversion of VEGF-A/NRP-1 signaling may underlie increased disease transmission in asymptomatic individuals.

Chronic morphine-induced antinociceptive tolerance is a major unresolved issue in clinical practices, which is associated with microglia activation in the spinal cord. E3 ubiquitin ligase Pellino1 (Peli1) is known to be an important microglia-specific regulator. However, it is unclear whether Peli1 is involved in morphine tolerance. Here, we found that Peli1 levels in the spinal cord were significantly elevated in morphine tolerance mouse model. Notably, Peli1 was expressed in a great majority of microglia in the spinal dorsal horn, while downregulation of spinal Peli1 attenuated the development of morphine tolerance and associated hyperalgesia. Our biochemical data revealed that morphine tolerance-induced increase in Peli1 was accompanied by spinal microglia activation, activation of mitogen-activated protein kinase (MAPK) signaling, and production of proinflammatory cytokines. Peli1 additionally was found to promote K63-linked ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) in the spinal cord after repeated morphine treatment. Furthermore, knocking down Peli1 in cultured BV2 microglial cells significantly attenuated inflammatory reactions in response to morphine challenge. Therefore, we conclude that the upregulation of Peli1 in the spinal cord plays a curial role in the development of morphine tolerance via Peli1-dependent mobilization of spinal microglia, activation of MAPK signaling, and production of proinflammatory cytokines. Modulation of Peli1 may be a potential strategy for the prevention of morphine tolerance.

Adhesion-type GPCRs (aGPCRs) participate in a vast range of physiological processes. Their frequent association with mechanosensitive functions suggests that processing of mechanical stimuli may be a common feature of this receptor family. Previously, we reported that the aGPCR CIRL sensitizes sensory responses to gentle touch and sound by amplifying signal transduction in low-threshold mechanoreceptors (Scholz et al., 2017). Here, we show that is also expressed in high-threshold mechanical nociceptors where it adjusts nocifensive behaviour under physiological and pathological conditions. Optogenetic experiments indicate that CIRL lowers cAMP levels in both mechanosensory submodalities. However, contrasting its role in touch-sensitive neurons, CIRL dampens the response of nociceptors to mechanical stimulation. Consistent with this finding, rat nociceptors display decreased expression during allodynia. Thus, cAMP-downregulation by CIRL exerts opposing effects on low-threshold mechanosensors and high-threshold nociceptors. This intriguing bipolar action facilitates the separation of mechanosensory signals carrying different physiological information.

The noradrenergic locus coeruleus nucleus is an important station in both the ascending and descending pain regulatory pathways. These neurons discharge in tonic and phasic modes in response to sensory stimuli. However, few studies have set out to characterize the electrophysiological response of the locus coeruleus to noxious stimuli in conditions of neuropathic pain. Thus, the effects of mechanical nociceptive stimulation of the sciatic nerve area on spontaneous (tonic) and sensory-evoked (phasic) locus coeruleus discharge were studied by extracellular recording in anesthetized rats seven, fourteen and twenty-eight days after chronic constriction injury. Minor significant electrophysiological changes were found seven and fourteen days after nerve injury. However, alterations to the spontaneous activity in both the ipsilateral and contralateral locus coeruleus were found twenty-eight days after nerve constriction, as witnessed by an increase of burst firing incidence and irregular firing patterns. Furthermore, noxious-evoked responses were exacerbated in the contralateral and ipsilateral nucleus at twenty-eight days after injury, as were the responses evoked when stimulating the uninjured paw. In addition, mechanical stimulation of the hindpaw produced a significant sensitization of neuronal tonic activity after 28 days of neuropathy. In summary, long-term nerve injury led to higher spontaneous activity and exacerbated noxious-evoked responses in the locus coeruleus to stimulation of nerve-injured and even uninjured hindpaws, coinciding temporally with the development of depressive and anxiogenic-like behavior.

Orofacial pain or tenderness is a primary symptom associated with temporomandibular joint (TMJ) disorders (TMDs). To understand the pathological mechanisms underlying TMDs, several mouse models have been developed, including mechanical stimulus-induced TMD and genetic mouse models. However, a lack of feasible approaches for assessing TMD-related nociceptive behaviours in the orofacial region of mice has hindered the in-depth study of TMD-associated mechanisms. This study aimed to explore modifications of three existing methods to analyse nociceptive behaviours using two TMD mouse models: (1) mechanical allodynia was tested using von Frey filaments in the mouse TMJ region by placing mice in specially designed chambers; (2) bite force was measured using the Economical Load and Force (ELF) system; and (3) spontaneous feeding behaviour tests, including eating duration and frequency, were analysed using the Laboratory Animal Behaviour Observation Registration and Analysis System (LABORAS). We successfully assessed changes in nociceptive behaviours in two TMD mouse models, a unilateral anterior crossbite (UAC)-induced TMD mouse model and a β-catenin conditional activation mouse model. We found that the UAC model and β-catenin conditional activation mouse model were significantly associated with signs of increased mechanical allodynia, lower bite force, and decreased spontaneous feeding behaviour, indicating manifestations of TMD. These behavioural changes were consistent with the cartilage degradation phenotype observed in these mouse models. Our studies have shown reliable methods to analyse nociceptive behaviours in mice and may indicate that these methods are valid to assess signs of TMD in mice.