Animal Studies

Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the NMDA receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, LSOS or the D-serine degrading enzyme, DAAO on post-operative days 0-3 significantly reduced the CCI-induced increase in NO levels and NADPH-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and PKC-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, SIN-1. In naïve mice, exogenouse D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechnical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1, and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.

Sensitivity to different pain modalities has a genetic basis that remains largely unknown. Employing closely related inbred mouse substrains can facilitate gene mapping of nociceptive behaviors in preclinical pain models. We previously reported enhanced sensitivity to acute thermal nociception in C57BL/6J (B6J) versus C57BL/6N (B6N) substrains. Here, we expanded on nociceptive phenotypes and observed an increase in formalin-induced inflammatory nociceptive behaviors and paw diameter in B6J versus B6N mice (Charles River Laboratories). No strain differences were observed in mechanical or thermal hypersensitivity or in edema following the Complete Freund's Adjuvant (CFA) model of inflammatory pain, indicating specificity in the inflammatory nociceptive stimulus. In the chronic nerve constriction injury (CCI), a model of neuropathic pain, no strain differences were observed in baseline mechanical threshold or in mechanical hypersensitivity up to one month post-CCI. We replicated the enhanced thermal nociception in the 52.5°C hot plate test in B6J versus B6N mice from The Jackson Laboratory. Using a B6J x B6N-F2 cross (N=164), we mapped a major quantitative trait locus (QTL) underlying hot plate sensitivity to chromosome 7 that peaked at 26 Mb (LOD=3.81, p<0.01; 8.74 Mb-36.50 Mb) that was more pronounced in males. Genes containing expression QTLs (eQTLs) associated with the peak nociceptive marker that are implicated in pain and inflammation include Ryr1, Cyp2a5, Pou2f2, Clip3, Sirt2, Actn4, and Ltbp4 (FDR < 0.05). Future studies involving positional cloning and gene editing will determine the quantitative trait gene(s) and potential pleiotropy of this locus across pain modalities.

Bone cancer pain (BCP) is refractory to currently available clinical treatment owing to its complicated underlying mechanisms. Studies found that extracellular matrix molecules can participate in the regulation of chronic pain. Decorin is one of the most abundant extracellular matrix molecules, the present study evaluated the effect of decorin on the development of BCP. We found that decorin was upregulated in the L4-6 spinal dorsal horn of the BCP rats. Spinal microinjection of a decorin-targeting RNAi lentivirus alleviated BCP-induced mechanical allodynia and reduced the expression of pGluR1-Ser831 in the BCP rats. Meanwhile, decorin knockdown impaired the excitatory synaptogenesis in cultured neurons and prevented the clustering and insertion of pGluR1-Ser831 into postsynaptic membranes. Taken together, the results of our study suggested that decorin contributes to the development of BCP possibly by regulating the activity of excitatory synaptic molecules in the spinal cord. Our findings provide a better understanding of the function of decorin as a possible therapeutic target for alleviating BCP.

We examined the effect of immobilization, low-intensity muscle contraction exercise, and transcutaneous electrical nerve stimulation (TENS) on tissue inflammation and acute pain following the onset of arthritis in a rat model. Sixty Wistar rats were divided into five groups: (1) Arthritis group, (2) arthritis and immobilization (Immobilization group), (3) arthritis and low intensity muscle contraction (Exercise group), (4) arthritis and TENS (TENS group), and (5) sham arthritis (Sham group). Arthritis was induced in the right knee joints by single injection of 3% kaolin and carrageenan. Immobilization of the right hindlimb was conducted by full extension of the right knee joints and full plantar flexion of the ankle joints using a plaster cast for 7 days after injection. The right quadriceps muscles were subjected to electrical stimulation (frequency: 50 Hz; intensity: 2-3 mA) for 20 min/day as contraction exercise for one week. TENS was delivered at 20 min/day for one week (frequency: 50 Hz; intensity: 1 mA). The pressure pain threshold (PPT) and paw withdrawal response (PWR) were evaluated at 1 and 7 days after injection. We also analyzed the number of CD68-positive cells in the synovium by immunohistochemistry and determined the expression level of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn with immunofluorescence. Improvements of both PPT and PWR were observed in the Exercise group at 7 days after injection compared to those of the Arthritis and Immobilization groups, although only improvement of PPT was observed in the TENS group. The number of CD68-positive cells in the synovium and CGRP expression in the dorsal horn decreased only in the Exercise group. These results suggested that low-intensity muscle contraction exercise might be a better treatment for reduction of arthritis-induced inflammation and acute pain compared to immobilization and TENS.

Radiotherapy-related pain is a common adverse reaction with a high incidence among cancer patients undergoing radiotherapy and remarkably reduces the quality of life. However, the mechanisms of ionizing radiation (IR)-induced pain are largely unknown. In present study, mice were treated with 20 Gy X-ray to establish IR-induced pain model. X-ray evoked a prolonged mechanical, heat and cold allodynia in mice. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) were significantly upregulated in lumbar dorsal root ganglion (DRG). The mechanical and heat allodynia could be transiently reverted by intrathecal injection of TRPV1 antagonist capsazepine and TRPA1 antagonist HC-030031. Additionally, the phosphorylated ERK and JNK in pain neural pathway were induced by X-ray treatment. Our findings indicated activation of TRPA1 and TRPV1 is essential for the development of X-ray-induced allodynia. Furthermore, our findings suggest targeting on TRPV1 and TRPA1 may be promising prevention strategies for X-ray-induced allodynia in clinical practice.

The size and modular structure of versican and its gene suggests the existence of multiple splice variants. We have identified, cloned and sequenced a previously unknown exon located within the non-coding gene sequence downstream of exon 8. This exon, which we have named exon 8 specifies two stop-codons. mRNAs of the versican gene with exon 8 are predicted to be constitutively degraded by nonsense mediated RNA decay. Here we tested the hypothesis that these transcripts become expressed in a model of neuropathic pain.

The study reported here investigated the role of the central vascular endothelial growth factor-A (VEGF-A) pathway in the development of trigeminal neuropathic pain following nerve injury. A Sprague-Dawley rat model of trigeminal neuropathic pain was produced using malpositioned dental implants. The left mandibular second molar was extracted under anesthesia and replaced with a miniature dental implant to induce injury to the inferior alveolar nerve. The inferior alveolar nerve injury produced a significant upregulation of astrocytic VEGF-A expression in the medullary dorsal horn. The nerve injury-induced mechanical allodynia was inhibited by an intracisternal infusion of VEGF-A164 antibody. Although both VEGF-A Receptor 1 (VEGF-A R1; colocalized with the blood-brain barrier) and VEGF-A Receptor 2 (VEGF-A R2; colocalized with astrocytes) participated in the development of trigeminal neuropathic pain following nerve injury, only the intracisternal infusion of a VEGF-A R1 antibody, and not that of a VEGF-A R2 antibody, inhibited the increased blood-brain barrier (BBB) permeability produced by nerve injury. Finally, we confirmed the participation of the central VEGF-A pathway in the development of trigeminal neuropathic pain by reducing VEGF-A expression using VEGF-A164 siRNA. This suppression of VEGF-A produced significant prolonged anti-allodynic effects. These results suggest that the central VEGF-A pathway plays a key role in the development of trigeminal neuropathic pain following nerve injury through two separate pathways: VEGF-A R1 and VEGF-A R2. Hence, a blockade of the central VEGF-A pathway provides a new therapeutic avenue for the treatment of trigeminal neuropathic pain.

As a protective mechanism, the cornea is sensitive to noxious stimuli. Here, we show that in mice, a high proportion of corneal TRPM8 cold-sensing fibers express the heat-sensitive TRPV1 channel. Despite its insensitivity to cold, TRPV1 enhances membrane potential changes and electrical firing of TRPM8 neurons in response to cold stimulation. This elevated neuronal excitability leads to augmented ocular cold nociception in mice. In a model of dry eye disease, the expression of TRPV1 in TRPM8 cold-sensing fibers is increased, and results in severe cold allodynia. Overexpression of TRPV1 in TRPM8 sensory neurons leads to cold allodynia in both corneal and non-corneal tissues without affecting their thermal sensitivity. TRPV1-dependent neuronal sensitization facilitates the release of the neuropeptide substance P from TRPM8 cold-sensing neurons to signal nociception in response to cold. Our study identifies a mechanism underlying corneal cold nociception and suggests a potential target for the treatment of ocular pain.