Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-genechip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade either by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naïve rats is sufficient to enhance the frequency of excitatory postsynaptic currents in spinal dorsal horn neurons, suggesting an increased excitatory pre-synaptic input, and cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal pre-synaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Unilateral ligation of the tendon of anterior superficial part of rat masseter muscle (TASM) leads to long-lasting allodynia. Sex differences in peripheral mu-opioid receptor (MOR)-mediated analgesia under persistent myogenic pain are not well understood. In this study, we examined (1) whether locally applied MOR agonists attenuate persistent pain following TASM ligation in a sex dependent manner, (2) whether there are sex differences of MOR expression changes in rat trigeminal ganglia (TG). The effects of MOR agonist, D-Ala2, N–Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO), were assessed 14 days after TASM ligation in male, female and orchidectomized (GDX) male rats. MOR mRNA and protein levels in TG 14 days following tendon ligation were also determined. The mechanical thresholds of the injured side were significantly decreased in both male and female rats, from 3 days to 28 days after TASM ligation. A10 μg DAMGO significantly attenuated allodynia in male rats. A 10-fold higher dose of DAMGO was required in female and GDX male rats to produce the level of anti- allodynia achieved in male rats. The level of MOR mRNA in TG from male rats was significantly greater 14 days after TASM ligation compared with the sham-operated male rats, but not from female and GDX male rats. After TASM ligation, males had significantly more MOR immunoreactivity in TG compared to sham-operated males. The MOR levels increased to 181.8% of the sham level in male rats receiving tendon injury. But there was no significant change in female rats receiving tendon injury compared to the sham female rats. Taken together, our data suggest that there were sex differences in the effects of peripheral MOR agonists between male and female rats under TASM ligation developing long-lasting pain condition, which is partly mediated by sex differences in the changes of MOR expressions and testosterone is an important factor in the regulation of MOR.

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Recent clinical studies have revealed sex differences in response to transient receptor potential vanilloid 1 (TRPV1) agonist-induced pain. However, the mechanism of these differences in TRPV1-related chronic pain remains unclear. In the present study, we investigate the effects of inflammation and gonadal hormones on TRPV1 expression in trigeminal ganglia. Inflammatory pain was modeled by injecting complete Freund's adjuvant (CFA) into the left masseter muscle in rats. TRPV1 mRNA and protein levels in the trigeminal ganglia of male and female rats following CFA injection were assessed. CFA-induced changes in TRPV1 mRNA and protein expression in the trigeminal ganglia from orchidectomized (ODX) male rats and testosterone-replaced ODX rats were examined. Additionally, TRPV1 mRNA levels in the trigeminal ganglia from ovariectomized (OVX) female and ODX male rats treated with tamoxifen were assessed. We found that the levels of TRPV1 mRNA and protein in the trigeminal ganglia from female rats following CFA injection were significantly higher than in the ganglia from naïve female rats. CFA-induced inflammatory hyperalgesia did not alter TRPV1 expression in the trigeminal ganglia from male rats. The TRPV1 mRNA and protein expression levels in the ODX male trigeminal ganglia were significantly upregulated on day 3 following the initiation of inflammation. However, CFA-induced inflammatory pain had no significant effect on TRPV1 mRNA or protein expression in testosterone-replaced ODX rats. Furthermore, tamoxifen was unable to inhibit the upregulation of TRPV1 expression in OVX female and ODX male rats after CFA injection. In summary, these data indicate that gender differences in TRPV1 function may be, in part, mediated by sex-dependent TRPV1 expression in sensory ganglia. Testosterone plays a key role in the inhibition of TRPV1 expression in this rat chronic inflammatory pain model.

Background Although the efficacy of peripherally administered opioid has been demonstrated in preclinical and clinical studies, the underlying mechanisms of its anti-hyperalgesic effects are poorly understood. G protein-coupled inwardly rectifying potassium (GIRK) channels are linked to opioid receptors in the brain. However, the role of peripheral GIRK channels in analgesia induced by peripherally administered opioid, especially in trigeminal system, is not clear. Methods Expression of GIRK subunits in rat trigeminal ganglia (TG) was examined with RT-PCR, western blot and immunohistochemistry. Chemical profiles of GIRK expressing neurons in TG were further characterized. Behavioral and Fos experiments were performed to examine the functional involvement of GIRK channels in delta opioid receptor (DOR)-mediated anti-hyperalgesia under an acute myositis condition. Results TG expressed mRNA and proteins for GIRK1 and GIRK2 subunits. Majority of GIRK1- and GIRK2-expressing neurons were non-peptidergic afferents. Inhibition of peripheral GIRK using Tertiapin-Q (TPQ) attenuated anti-nociceptive effects of peripherally administered DOR agonist, DPDPE, on mechanical hypersensitivity in masseter muscle. Furthermore, TPQ attenuated the suppressive effects of peripheral DPDPE on neuronal activation in the subnucleus caudalis of the trigeminal nucleus (Vc) following masseteric injection of capsaicin. Conclusions Our data indicate that peripheral DOR agonist-induced suppression of mechanical hypersensitivity in the masseter muscle involves the activity of peripheral GIRK channels. These results could provide a rationale for developing a novel therapeutic approach using peripheral GIRK channel openers to mimic or supplement the effects of peripheral opioid agonist.