Animal Studies

The nerve growth factor (NGF) plays an important role in the regulation of neuropathic pain. It has been demonstrated that calcitonin gene-related peptide (CGRP), a well-known contributor to neurogenic inflammation, increases neuroinflammatory pain induced by NGF. The inflammatory mediator that NGF most strongly induces is C-C chemokine ligand 19 (CCL19), which can recruit inflammatory cells by binding to the receptor CCR7 followed by promoting the response of neuroinflammation. However, the regulatory mechanism of NGF and CCL19 in tooth movement orofacial pain and the interaction between both are still unclear. In this study, male Sprague-Dawley rats were used to study the modulation of NGF on orofacial pain through CCL19 and the role of each in tooth movement pain in rats. The expression levels of CCL19 mRNA and protein were determined by real-time PCR and immunofluorescence, respectively. Pain levels were assessed by measuring the rats' bite force, which drops as pain rises. Meanwhile, by verifying the relationship between CGRP and CCL19, it was laterally confirmed that NGF could modulate tooth movement-induced mechanical hyperalgesia through CCL19. The results showed that the expression level of CCL19 rose with the increased NGF, and neurons expressing CGRP can express stronger CCL19. Compared with the baseline level, the bite force for all rats dropped sharply on day 1, reached its lowest level on day 3, and recovered gradually on day 5. All results indicated that NGF played an important role in tooth movement orofacial pain positively regulating CCL19 expression in the trigeminal ganglia of rats. Additionally, CCL19 increased the sensitivity to experimental tooth movement orofacial pain. NGF can regulate CCL19 expression, although it may regulate other inflammatory pathways as well. This is the first report on the interactions and modulations of tooth movement orofacial pain by NGF through CCL19 in rats.

Neuropathic pain refers to pain caused by lesions or diseases of the somatosensory nervous system that is characteristically different from nociceptive pain. Moreover, neuropathic pain occurs in the maxillofacial region due to various factors and is treated using tricyclic antidepressants and nerve block therapy; however, some cases do not fully recover. Netrin is a secreted protein crucially involved in neural circuit formation during development, including cell migration, cell death, neurite formation, and synapse formation. Recent studies show Netrin-4 expressed in the dorsal horn of the spinal cord is associated with chronic pain. Here we found involvement of Netrin-4 in neuropathic pain in the maxillofacial region. Netrin-4, along with one of its receptors, Unc5B, are expressed in the caudal subnucleus of the trigeminal spinal tract nucleus. Inhibition of its binding by anti-Netrin-4 antibodies not only shows a behavioral analgesic effect but also neuronal activity suppression. There was increased Netrin-4 expression at 14 days after infraorbital nerve injury. Our findings suggest that Netrin-4 induced by peripheral nerve injury causes neuropathic pain via Unc5B.

The voltage-gated potassium channel Kv3.4 is a crucial regulator of nociceptive signaling in the dorsal root ganglion (DRG) and the dorsal horn of the spinal cord. Moreover, Kv3.4 dysfunction has been linked to neuropathic pain. Although kinases and phosphatases can directly modulate Kv3.4 gating, the signaling mechanisms regulating the expression and stability of the Kv3.4 protein are generally unknown. We explored a potential role of PKCε and found an unexpected interaction that has a positive effect on Kv3.4 expression. Co-immunoprecipitation studies revealed a physical association between PKCε and Kv3.4 in both heterologous cells and rat DRG neurons. Furthermore, in contrast to the wild-type and constitutively active forms of PKCε, expression of a catalytically inactive form of the enzyme inhibits Kv3.4 expression and membrane localization through a dominant negative effect. Co-expression of Kv3.4 with the wild-type, constitutively active, or catalytically inactive forms of PKCε had no significant effects on Kv3.4 gating. These results suggest that a novel physical interaction of the Kv3.4 channel with functional PKCε primarily determines its stability and localization in DRG neurons. This interaction is akin to those of previously identified accessory ion channel proteins, which could be significant in neural tissues where Kv3.4 regulates electrical signaling.

von Frey hairs are important tools for the study of mechanisms of cutaneous stimulation-induced sensory input. Mechanical force is exerted via application of a particular hair to the cutaneous receptive field until buckling of the hair occurs. The most commonly used von Frey filaments are productive in evaluating behavioral responses of neuropathic pain in preclinical and clinical research. To reduce the potential experimenter bias, automated instruments are being developed for behavioral assessment.

To explore whether methotrexate (MTX) prevents joint destruction and improves pain-related behaviors in the acute phase of knee osteoarthritis (OA) induced by monosodium iodoacetate (MIA) in a rat model.

Visceral hypersensitivity (VH) is one of the underlying pathophysiologies of irritable bowel syndrome. Mast cell overactivation has been found to be one of the main causes of VH. We investigated the effects and mechanisms of actions of sacral nerve stimulation (SNS) on visceral pain in a rodent model of VH. The VH was established by an intrarectal infusion of AA in 10-day-old pups. Rats were chronically implanted with electrodes for SNS and recording electromyogram (EMG) and electrocardiogram. The acute study was performed in 2-randomized sessions with SNS (14 Hz, 330 μs, 40% motor threshold or MT, 30 min) or sham-SNS. Later on, rats were randomized into SNS/sham-SNS groups and a chronic study was performed with 2 h-daily SNS or sham-SNS for 21 days. Visceromotor reflexes were assessed by abdominal EMG and withdrawal reflex (AWR). Colon tissues were collected to study colonic acetylcholine (ACh), the enteric neurons (ChAT, nNOS, and PGP9.5), mast cells activity [Tryptase, prostaglandins E2 (PGE2), and cyclooxygenases-2 (COX2)] and pain markers [nerve growth factor (NGF) and Sub-P]. Sacral nerve stimulation significantly improved visceromotor reflexes assessed by the EMG and AWR, compared with sham-SNS. SNS normalized the protein expressions of ChAT and nNOS and regulated mast cells activity by downregulating Tryptase, COX2, and PGE2. Neonatal AA administration upregulated NGF and Sub-P; chronic SNS significantly decreased these pain biomarkers. Concurrently, chronic SNS increased ACh in colon tissues and vagal efferent activity. Sacral nerve stimulation reduces VH in rats and this ameliorating effect might be attributed to the suppression of mast cell overactivation in the colon tissue via the modulation of autonomic nervous system functions.

Patch clamp is an electrophysiological technique that allows to analyze the activity of ion channels in neurons. In this chapter, we provide a detailed description of patch clamp protocol to measure the effect of a μ-opioid receptor agonist on the activity of G protein-coupled inwardly rectifying potassium (GIRK or Kir3) channels. This is performed in peripheral sensory neurons isolated from dorsal root ganglia (DRG) of mice without or with a chronic constriction injury (CCI) of the sciatic nerve, which models neuropathic pain. We describe the induction of the CCI , isolation and culture of DRG neurons, performance of the patch clamp recordings, and identification of opioid-responding neurons.