Animal Studies

Transient receptor potential melastatin member 8 (TRPM8) is a Ca-permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo-electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP, and Ca and in complex with the menthol analog WS-12 and PIP Our structures reveal the binding sites for cooling agonists and PIP in TRPM8. Notably, PIP binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.

Muscle pain is a common condition in many diseases and is induced by muscle overuse. Muscle overuse induces an increase in uric acid, which stimulates the nucleotide-binding oligomerization domain-like receptor (NLR). This receptor contains the pyrin domain NLRP-3 inflammasome which when activated, results in the secretion of potent pro-inflammatory cytokines such as interleukin-1β (IL-1β). The aim of this study was to investigate the involvement of inflammasome activation via the elevation of uric acid level in nociception in a mouse model of muscle pain. The right hind leg muscles of BALB/c mice were stimulated electrically to induce excessive muscle contraction. The left hind leg muscles were not stimulated as a control. Mechanical withdrawal thresholds (MWTs), levels of uric acid, IL-1β, and NLRP3, caspase-1 activity, and the number of macrophages were investigated. Furthermore, the effects of xanthine oxidase inhibitors, such as Brilliant Blue G, caspase-1 inhibitor, and clodronate liposome, on pain were investigated. In the stimulated muscles, MWTs decreased, and the levels of uric acid, NLRP3, and IL-1β, caspase-1 activity, and the number of macrophages increased compared to that in the non-stimulated muscles. Administration of the inhibitors attenuated hyperalgesia caused by excessive muscle contraction. These results suggested that IL-1β secretion and NLRP3 inflammasome activation in macrophages produced mechanical hyperalgesia by elevating uric acid level, and xanthine oxidase inhibitors may potentially reduce over-exercised muscle pain.

Heme oxygenase 1 (HO-1) and carbon monoxide were shown to normalize oxidative stress and inflammatory reactions induced by neuropathic pain in the central nervous system, but their effects in the locus coeruleus (LC) of animals with peripheral inflammation and their interaction with nitric oxide are unknown. In wild-type (WT) and knockout mice for neuronal (NOS1-KO) or inducible (NOS2-KO) nitric oxide synthases with inflammatory pain induced by complete Freund's adjuvant (CFA), we assessed: 1) antinociceptive actions of cobalt protoporphyrin IX (CoPP), an HO-1 inducer; 2) effects of CoPP and tricarbonyldichlororuthenium(II)dimer (CORM-2), a carbon monoxide-liberating compound, on the expression of HO-1, NOS1, NOS2, CD11b/c, GFAP,and mitogen-activated protein kinases (MAPK)in the LC. CoPP reduced inflammatory pain in different time-dependent manners in WT and KO mice. Peripheral inflammation activated astroglia in the LC of all genotypes and increased the levels of NOS1 and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK 1/2) in WT mice. CoPP and CORM-2 enhanced HO-1 and inhibited astroglial activationin all genotypes. Both treatments blocked NOS1 overexpression,and CoPP normalized ERK 1/2 activation. This study reveals an interaction between HO-1 and NOS1/NOS2 during peripheral inflammation andshows that CoPP and CORM-2 improved HO-1 expression and modulated the inflammatory and/or plasticity changes caused by peripheral inflammation in the LC.

In mice, spared nerve injury replicates symptoms of human neuropathic pain and induces upregulation of many genes in somatosensory neurons. Here we used single cell transcriptomics to probe the effects of partial infraorbital transection of the trigeminal nerve at the cellular level. Uninjured neurons were unaffected by transection of major nerve branches, segregating into many different classes. In marked contrast, axotomy rapidly transformed damaged neurons into just two new and closely-related classes where almost all original identity was lost. Remarkably, sensory neurons also adopted this transcriptomic state following various minor peripheral injuries. By genetically marking injured neurons, we showed that the injury-induced transformation was reversible, with damaged cells slowly reacquiring normal gene expression profiles. Thus, our data expose transcriptomic plasticity, previously thought of as a driver of chronic pain, as a programed response to many types of injury and a potential mechanism for regulating sensation during wound healing.

The purpose of this study was to determine effects and mechanisms of sacral nerve stimulation (SNS) on visceral hypersensitivity in rodent models of colonic hypersensitivity. SNS was performed with different sets of parameters for 30 minutes in six regular rats. Visceral sensitivity was assessed by the measurement of electromyogram (EMG) and abdominal withdrawal reflex (AWR) before and after SNS. Real/sham SNS with optimized parameters was performed in eight restraint stress-induced visceral hypersensitivity rats and 10 neonatal acetic acid treated colonic hypersensitivity rats, acute effect of SNS was assessed by comparing EMG and heart rate variability (HRV). Neonatal acetic acid treated rats were treated by SNS (n=10) or sham-SNS (n=10) daily for 7 days for the assessment of the chronic effect of SNS. (1)When the stimulation amplitude was reduced from 90% of motor threshold (MT) to 65% or 40% MT, SNS with certain parameters showed an inhibitory effect on AWR. The best stimulation parameters for SNS was "14Hz, 330μs, and 40% MT". (2)SNS significantly reduced visceral hypersensitivity and improved autonomic function in restraint stress-induced rats. The inhibitory effect was blocked by naloxone.(3)Acute and Chronic SNS significantly reduced visceral hypersensitivity and improved autonomic function in acetic acid treated rats. SNS with reduced stimulation strength may be used to treat colonic hypersensitivity and the best stimulation parameters seem to be"14Hz, 330μs and 40% MT". SNS with optimized parameters improved visceral hypersensitivity in rodent models of colonic hypersensitivity mediated via the autonomic and opioid mechanisms.

We investigated the calcium dynamics of dorsal root ganglion (DRG) neurons using transgenic mice to target expression of the genetically encoded calcium indicator (GECI), GCaMP6s, to a subset of neurons containing parvalbumin (PV), a calcium-binding protein present in proprioceptors and low-threshold mechanoreceptors. This study provides the first analysis of GECI calcium transient parameters from large-diameter DRG neurons. Our approach generated calcium transients of consistent shape and time-course, with quantifiable characteristics. Four parameters of calcium transients were determined to vary independently from each other and thus are likely influenced by different calcium-regulating mechanisms: peak amplitude, rise time (RT), decay time, and recovery time. Pooled analysis of 188 neurons demonstrated unimodal distributions, providing evidence that PV+ DRG neurons regulate calcium similarly as a population despite their differences in size, electrical properties, and functional sensitivities. Calcium transients increased in size with elevated extracellular calcium, longer trains of action potentials, and higher stimulation frequencies. RT and decay time increased with the addition of the selective sarco/endoplasmic reticulum calcium ATPases (SERCA) blocker, thapsigargin (TG), while peak amplitude and recovery time remained the same. When elevating bath pH to 8.8 to block plasma-membrane calcium ATPases (PMCA), all measured parameters significantly increased. These results illustrate that GECI calcium transients provide sufficient resolution to detect changes in electrical activity and intracellular calcium concentration, as well as discern information about the activity of specific subclasses of calcium regulatory mechanisms.

Pain is more prevalent in women for reasons that remain unclear. We have identified a mechanism of injury-free nociceptor sensitization and opioid-induced hyperalgesia (OIH) promoted by prolactin (PRL) in females. PRL signals through mutually inhibitory long (PRLR-L) and short (PRLR-S) receptor isoforms, and PRLR-S activation induces neuronal excitability. PRL and PRLR expression were higher in females. CRISPR-mediated editing of PRLR-L promoted nociceptor sensitization and allodynia in naïve, uninjured female mice that depended on circulating PRL. Opioids, but not trauma-induced nerve injury, decreased PRLR-L promoting OIH through activation of PRLR-S in female mice. Deletion of both PRLR-L and PRLR-S (total PRLR) prevented, whereas PRLR-L overexpression rescued established OIH selectively in females. Inhibition of circulating PRL with cabergoline, a dopamine D2 agonist, up-regulated PRLR-L and prevented OIH only in females. The PRLR-L isoform therefore confers protection against PRL-promoted pain in females. Limiting PRL/PRLR-S signaling pharmacologically or with gene therapies targeting the PRLR may be effective for reducing pain in a female-selective manner.