Animal Studies

Nerve growth factor (NGF) is thought to play a key role in chronic pain felt by bladder pain syndrome/interstitial cystitis (BPS/IC) patients by activating its high affinity receptor tropomyosin-related kinase subtype A (Trk A). Whether this pathway is also involved in the aggravation of pain sensation during stress events was here investigated. The levels of plasmatic NGF were increased in rats submitted to Water Avoidance Stress test (WAS), compared to controls. The administration of the alpha1A adrenoceptors blocker silodosin prevented the increase of plasmatic NGF. Urinary NGF levels were also moderately increased in animals submitted to WAS. WAS increased pain behaviour score, lowered abdominal mechanical pain threshold and increase voiding bladder reflex activity. These changes were prevented by the administration of TrkA antagonist GW441756. These findings prompt the use of plasmatic NGF as diagnosis tool for chronic visceral painful conditions and opens therapeutic opportunities for TrkA receptors antagonist/NGF sequestration.

Spinal cord injury (SCI) leads to sensorimotor deficits and autonomic changes. Macrophages and microglia could be polarized into the classically activated pro-inflammatory M1 phenotype or the alternatively activated anti-inflammatory M2 phenotype. Transmembrane protein with unknown function 16F (TMEM16F) exhibits functional diversity and may contribute to microglial function. However, the effects of TMEM16F on the modulation of macrophage/microglial polarization are still not fully understood. In the study, TMEM16F up-regulation was detected after SCI in mice, and TMEM16F protein was found in macrophages/microglia in injured spinal cord sections. Depletion of TMEM16F improved motor function in male mice with SCI. M1-type macrophages/microglia accumulated in lower numbers in the injured spinal cord of TMEM16F-knockout (KO) mice. M2 polarization inhibited by SCI was improved in mice with TMEM16F deficiency. TMEM16F deletion also attenuated microglial/macrophage pro-inflammatory response. Furthermore, significant down-regulation of A disintegrin and metalloprotease 17 (ADAM17) was observed in TMEM16F-KO mice. Importantly, TMEM16F-promoted M1 polarization and -inhibited M1 polarization were largely associated with the suppression of ADAM17. Overall, our findings provided new insights into the regulatory mechanisms of macrophage/microglial polarization, thereby possibly facilitating the development of new therapeutic strategies for SCI through the regulation of TMEM16F/ADAM17 signaling.

The mesolimbic dopaminergic signaling, such as that originating from the ventral tegmental area (VTA) neurons in the medial part of the nucleus accumbens (mNAc), plays a role in complex sensory and affective components of pain. To date, we have demonstrated that optogenetic sensory nerve stimulation rapidly alters the dopamine (DA) content within the mNAc. However, the physiological role and biochemical processes underlying such rapid and regional dynamics of DA remain unclear. In this study, using imaging mass spectrometry (IMS), we observed that sensitized pain stimulation by optogenetic sensory nerve activation increased DA and 3-Methoxytyramine (3-MT; a post-synaptic metabolite obtained following DA degradation) in the mNAc of the experimental mice. To delineate the mechanism associated with elevation of DA and 3-MT, the de novo synthesized DA in the VTA/substantia nigra terminal areas was evaluated using IMS by visualizing the metabolic conversion of stable isotope-labeled tyrosine (CN-Tyr) to DA. Our approach revealed that at steady state, the de novo synthesized DA occupied >10% of the non-labeled DA pool in the NAc within 1.5 h of isotope-labeled Tyr administration, despite no significant increase following pain stimulation. These results suggested that sensitized pain triggered an increase in the release and postsynaptic intake of DA in the mNAc, followed by its degradation, and likely delayed de novo DA synthesis. In conclusion, we demonstrated that short, peripheral nerve excitation with mechanical stimulation accelerates the mNAc-specific DA signaling and metabolism which might be associated with the development of mechanical allodynia.

Cancer pain induced by pancreatic carcinoma is one of the most common symptoms and is difficult to endure, especially in the advanced stage. Evidence suggests that mast cells are recruited and degranulate in enteric disease-related visceral hypersensitivity. However, whether mast cells promote the visceral pain induced by pancreatic carcinoma remains unclear. Here, using toluidine blue staining and western blotting, we observed that mast cells were dramatically recruited to tissues surrounding pancreatic carcinoma, but not inside the carcinoma in patients with severe visceral pain. The levels of mast cell degranulation products, including tryptase, histamine, and nerve growth factor, were significantly increased in pericarcinoma tissues relative to their levels in normal controls, as evidenced by enzyme-linked immunosorbent assay. We determined that systemic administration of mast cell secretagogue compound 48/80 exacerbated pancreatic carcinoma-induced visceral hypersensitivity in a male BALB/c nude mouse model as assessed by measuring the hunching behavior scores and mechanical withdrawal response frequency evoked by von Frey stimulation. In contrast, the mast cell stabilizer ketotifen dose-dependently alleviated pancreatic cancer pain. In addition, we observed incomplete development of abdominal mechanical hyperalgesia and hunching behavior in mast cell-deficient mice with pancreatic carcinoma. However, ketotifen did not further attenuate visceral hypersensitivity in mast cell-deficient mice with carcinoma. Finally, we confirmed that intraplantar injection of pericarcinoma supernatants from BALB/c nude mice but not mast cell-deficient mice caused acute somatic nociception. In conclusion, our findings suggest that mast cells contribute to pancreatic carcinoma-induced visceral hypersensitivity through enrichment and degranulation in pericarcinoma tissues. The inhibition of mast cell degranulation may be a potential strategy for the therapeutic treatment of pancreatic carcinoma-induced chronic visceral pain.

Mounting evidence suggests that the spinal dorsal horn (SDH) contains multiple subpopulations of inhibitory interneurons that play distinct roles in somatosensory processing, as exemplified by the importance of spinal dynorphin-expressing neurons for the suppression of mechanical pain and chemical itch. While it is clear that GABAergic transmission in the SDH undergoes significant alterations during early postnatal development, little is known about the maturation of discrete inhibitory "microcircuits" within the region. As a result, the goal of the present study was to elucidate the gene expression profile of spinal dynorphin (pDyn)-lineage neurons throughout life. We isolated nuclear RNA specifically from pDyn-lineage SDH interneurons at postnatal days 7, 21, and 80 using the Isolation of Nuclei Tagged in Specific Cell Types (INTACT) technique, followed by RNA-seq analysis. Over 650 genes were ≥2-fold enriched in adult pDyn nuclei compared to non-pDyn spinal cord nuclei, including targets with known relevance to pain such as galanin (Gal), prepronociceptin (Pnoc), and nitric oxide synthase 1 (Nos1). In addition, the gene encoding a membrane-bound guanylate cyclase, Gucy2d, was identified as a novel and highly selective marker of the pDyn population within the SDH. Differential gene expression analysis comparing pDyn nuclei across the three ages revealed sets of genes that were significantly upregulated (such as Cartpt, encoding cocaine- and amphetamine-regulated transcript peptide) or downregulated (including Npbwr1, encoding the receptor for neuropeptides B/W) during postnatal development. Collectively, these results provide new insight into the potential molecular mechanisms underlying the known age-dependent changes in spinal nociceptive processing and pain sensitivity.

Sodium channel Na1.7 controls firing of nociceptors, and its role in human pain has been validated by genetic and functional studies. However, little is known about Na1.7 trafficking or membrane distribution along sensory axons, which can be a meter or more in length. We show here with single-molecule resolution the first live visualization of Na1.7 channels in dorsal root ganglia neurons, including long-distance microtubule-dependent vesicular transport in Rab6A-containing vesicles. We demonstrate nanoclusters that contain a median of 12.5 channels at the plasma membrane on axon termini. We also demonstrate that inflammatory mediators trigger an increase in the number of Na1.7-carrying vesicles per axon, a threefold increase in the median number of Na1.7 channels per vesicle and a ~50% increase in forward velocity. This remarkable enhancement of Na1.7 vesicular trafficking and surface delivery under conditions that mimic a disease state provides new insights into the contribution of Na1.7 to inflammatory pain.

Neuropathic pain can be a debilitating condition with both sensory and affective components, the underlying brain circuitry of which remains poorly understood. In the present study, a basolateral amygdala (BLA)-prefrontal cortex (PFC)-periaqueductal gray (PAG)-spinal cord pathway was identified that is critical for the development of mechanical and thermal hypersensitivity after peripheral nerve injury. It was shown that nerve injury strengthens synaptic input from the BLA onto inhibitory interneurons located in the prelimbic medial PFC, by virtue of reduced endocannabinoid modulation. These augmented synaptic connections mediate a feedforward inhibition of projections from the PFC to the ventrolateral PAG region and its downstream targets. Optogenetic approaches combined with in vivo pharmacology reveal that these BLA-PFC-PAG connections alter pain behaviors by reducing descending noradrenergic and serotoninergic modulation of spinal pain signals. Thus, a long-range brain circuit was identified that is crucial for pain processing and that can potentially be exploited toward targeting neuropathic pain.