Animal Studies

Glycosphingolipids (GSLs) are abundant, ceramide-containing lipids in the nervous system that play key functional roles in pain and inflammation. We measured gene expression (Ugcg, St3gal5, St8sia1, B4galNT1, Ugt8a, and Gal3st1) of glycosyltransferases involved in GSL synthesis in murine dorsal root ganglion (DRG) and spinal cord after complete Freund's adjuvant (CFA)-induced unilateral hind-paw inflammation (1 day vs. 15 days). Chronic inflammation (15 days) sensitized both ipsilateral and contralateral paws to pain. One day of induced unilateral hind-paw inflammation (1d-IUHI) increased Ugcg, St8sia1, B4galnt1, and Gal3st1 expression in ipsilateral cord, suggesting that sulfatide and b-series gangliosides were also elevated. In addition, 1d-IUHI increased Ugcg, st3gal5 and B4galnt1 expression in contralateral cord, suggesting that sulfatide and a-/b-series gangliosides were elevated. By contrast, 1d-IUHI decreased Ugcg, St3gal5, and St8sia1 expression bilaterally in the DRG, suggesting that b-series gangliosides were depressed. Since intrathecal injection of b-series ganglioside induced mechanical allodynia in naïve mice, it seems reasonable that b-series gangliosides synthesized from upregulated St8sia1 in the ipsilateral spinal cord are involved in mechanical allodynia. By contrast, chronic inflammation led to a decrease of Ugcg, St3gal5, B4galnt1, and Gal3st1 expression in spinal cord bilaterally and an increase of St8sia1 expression in the ipsilateral DRG, suggesting that a-/b-series gangliosides in the spinal cord decreased and b-series gangliosides in ipsilateral DRG increased. These changes in glycosyltransferase gene expression in the DRG and the spinal cord may contribute to the modification of pain sensitivity in both inflamed and non-inflamed tissues and the transition from early to chronic inflammatory pain.

T lymphocytes are increasingly implicated in pain signaling. A subset of T lymphocytes, termed TChAT, express the rate-limiting enzyme for acetylcholine (ACh) production, choline acetyltransferase (ChAT), and mediate numerous physiological functions. Given that cholinergic signaling has long been known to modulate pain processing and is the basis for several analgesics used clinically, we asked whether TChAT could be the intersection between T lymphocyte and cholinergic mediation of pain signaling. In this study, we used a mouse gene knockout strategy to ablate ChAT specifically from T lymphocytes and examined the development and expression of mechanical and thermal hypersensitivity in a spared nerve injury (SNI) mouse model of neuropathic pain. We found that mice with ChAT knockout in T cells (floxed plus CD4-Cre recombinase) did not differ from control mice with intact ChAT (floxed , but no Cre recombinase) in their expression of mechanical sensitivity before or after injury. Similarly, thermal sensitivity was unaffected after injury, with control mice expressing similar patterns of thermal preference to mice whose T cells do not express ChAT. Our experiments demonstrate that cholinergic signaling initiated by T lymphocytes neither dampens nor exacerbates the expression of mechanical or thermal sensitivity in neuropathic mice. Thus, while both cholinergic signaling and T lymphocytes have established roles in modulating pain phenotypes, it is not cholinergic signaling initiated by T lymphocytes that drive this. Our findings will help to narrow in on which aspects of T-cell modulation may prove useful as therapies.

Adult rats that experienced neonatal limited bedding (NLB), a form of early-life stress, experience persistent muscle mechanical hyperalgesia. Since there is a growing recognition that the gut microbiome regulates pain and nociception, and that early-life stress produces a long-lasting impact on the gut microbiome, we tested the hypothesis that persistent muscle hyperalgesia seen in adult NLB rats could be ameliorated by interventions that modify the gut microbiome. Adult NLB rats received probiotics, either GG (10 billion CFU/150 ml) or De Simone Formulation (DSF) (112.5 billion CFU/150 ml mixture of 8 bacterial species), in their drinking water, or non-absorbable antibiotics, rifaximin or neomycin, admixed with cookie dough, to provide 50 mg/kg. Mechanical nociceptive threshold in the gastrocnemius muscle was evaluated before and at several time points after administration of probiotics or antibiotics. Adult NLB rats fed probiotics or DSF, antibiotics, as well as rats fed non-absorbable antibiotics rifaximin or neomycin, had markedly attenuated muscle mechanical hyperalgesia. We hypothesize that persistent skeletal muscle hyperalgesia produced by NLB stress may be, at least in part, due to a contribution of the gut microbiome, and that modulation of gut microbiome using probiotics or non-absorbable antibiotics, may be novel therapeutic approaches for the treatment of chronic musculoskeletal pain.

Osteoarthritis of the knee impairs activities of daily living of those affected. Its irreversible degenerative changes to the knee joint induce functional disturbance and unpleasant arthralgia. The pain has inflammatory components and often is manifested with mechanical allodynia and hyperalgesia. Sustained weight bearing and joint movements increase pain sensitivity in knee osteoarthritis. Understanding the mechanisms underlying the mechanical allodynia and hyperalgesia might provide a therapeutical target for pain relief in patients with such symptoms. Piezo channel is a mechanically activated ion channel that may be involved in mechanical transduction in the articular cartilage. Although it has been shown that inflammation potentiates Piezo channel current induced by mechanical stimulation, whether Piezo expression levels are influenced by knee osteoarthritis has remained unknown. We measured Piezo mRNA in knee joints and dorsal root ganglia after establishing a model of knee osteoarthritis in rats using monosodium iodoacetate and found Piezo mRNA level is not upregulated. This finding raises a question as whether and how Piezo channels may be involved in mechanically induced pain in osteoarthritis.

Central amygdala (CeA) neurons expressing protein kinase Cδ (PKCδ) or somatostatin (Som) differentially modulate diverse behaviors. The underlying features supporting cell-type-specific function in the CeA, however, remain unknown. Using whole-cell patch-clamp electrophysiology in acute mouse brain slices and biocytin-based neuronal reconstructions, we demonstrate that neuronal morphology and relative excitability are two distinguishing features between Som and PKCδ neurons in the laterocapsular subdivision of the CeA (CeLC). Som neurons, for example, are more excitable, compact, and with more complex dendritic arborizations than PKCδ neurons. Cell size, intrinsic membrane properties, and anatomic localization were further shown to correlate with cell-type-specific differences in excitability. Lastly, in the context of neuropathic pain, we show a shift in the excitability equilibrium between PKCδ and Som neurons, suggesting that imbalances in the relative output of these cells underlie maladaptive changes in behaviors. Together, our results identify fundamentally important distinguishing features of PKCδ and Som cells that support cell-type-specific function in the CeA.

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2) receive direct Aβ low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2 neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.