Accepted

Irritable Bowel Syndrome is a functional gastrointestinal (GI) disorder that leads to chronic abdominal pain. The perception of this visceral pain involves peripheral mechanisms, such as nociceptors with cell bodies in dorsal root ganglia (DRG) and axons in the gut wall, and central mechanisms, such as neuroplasticity within the spinal cord and brain. Lysophosphatidylcholine (LPC), which has recently been shown to bind to GPR55 in human prostate carcinoma cells, is released from cell membranes and is elevated in patients with IBS. Dorsal root ganglion (DRG) neurons express GPR55, and activation of GPR55 has been implicated in inflammatory pain. Therefore, we hypothesised that elevated LPC during IBS contributes to pain due to its activation of GPR55 on dorsal root ganglion (DRG) neurons, leading to the excitation of visceral pain pathways. Current clamp recordings revealed that application of LPC (10 µM) to murine DRG neurons depolarised the resting membrane potential (p = 0.0001) by approximately 8 mV and decreased the rheobase (p < 0.05) by approximately 20%. Using ratiometric Ca2+ imaging using FURA-2 AM, LPC (10 µM) doubled intracellular [Ca2+]i. This effect was significantly reduced by the selective GPR55 antagonist CID16020046 (10 µM) (p < 0.05), suggesting the response to LPC is at least partially mediated by GPR55. The source of the [Ca2+] elevation following LPC application was elucidated using cyclopiazonic acid (CPA; 10 µM), which depletes intracellular Ca2+ stores, and a 0-Ca2+ external solution to remove the contribution of Ca2+ influx from extracellular sources. While both significantly decreased the Ca2+ influx elicited by LPC, the 0-Ca2+ external solution almost abolished the effect (p < 0.0001) of LPC. Together, these data suggest that the increased [Ca2+] elicited by LPC activation of GPR55 is partially mediated through the release of Ca2+ from intracellular stores but is mostly due to influx of extracellular Ca2+.

Neuropathic pain is a debilitating chronic condition that remains difficult to treat. There is a high priority to identify novel non-opioid-based therapeutic targets as long-term use of opioids is problematic due to its severe side effects and strong abuse potential. Our lab recently discovered G-protein coupled receptor 160 (GPR160) has a role in neuropathic pain. Gpr160 was upregulated in the dorsal horn of the spinal cord (DH-SC) on the side of nerve injury relative to the uninjured side in mice. Blocking GPR160 using siRNA or a neutralizing antibody (Ab) reversed and prevented pain hypersensitivity. In addition, our lab deorphanized GPR160 as the receptor for cocaine- and amphetamine-regulated transcript peptide (CARTp). We showed that an intrathecal (i.th.) injection of CARTp in naive mice caused mechano-hypersensitivity that was dependent on GPR160. CARTp-induced gene expression is relatively unexplored and the mechanism by which CARTp/GPR160 signaling promotes chronic pain is not well known. Therefore, our objective was to perform an unbiased RNA transcriptomics analysis to identify which genes were altered at the time of CARTp-mediated peak pain in the DH-SC. We found that Nucleotide-binding oligomerization domain-containing protein 2 (Nod2) expression was increased upon i.th. injection of CARTp and its expression decreased after CARTp was co-administered with GPR160 Ab, pointing to a potential interaction between CARTp/GPR160 and NOD2. NOD2 is a cytosolic pattern recognition receptor that is involved in activating the immune system in response to pathogens. Moreover, a recent study linked NOD2 to the development of neuropathic pain. This led to our hypothesis that CARTp/GPR160 causes mechano-hypersensitivity through NOD2 and NOD2 has a functional role in neuropathic pain. C57BL/6 (WT) and NOD2 mice received an i.th. injection of CARTp and mechano-allodynia was assessed. The WT mice developed mechano-allodynia by 30min and persisted for 5hrs. In contrast, mechano-allodynia was attenuated for 4hrs in NOD2 mice, suggesting CARTp/GPR160 induces mechano-hypersensitivity through NOD2. Furthermore, we found that NOD2 has a functional role in a chronic constriction injury (CCI) model of neuropathic pain. WT mice started developing mechano-allodynia on day 3 (D3) after CCI, reached a maximum by D7, and persisted until D14. However, NOD2 mice did not develop mechano-allodynia until D10, indicating that NOD2 is involved in the development of CCI neuropathic pain. Overall, our results provide a potential mechanistic insight on how CARTp causes mechano-sensitivity and NOD2 has a functional role in CCI-mediated neuropathic pain.

In most preclinical models of neuropathic pain, hypersensitivity to pain often resolves after a few weeks. However, spared nerve injury (SNI) produces a persistent pain state that lasts at least 18 months. The goal of the current study was to evaluate antihyperalgesic effects of mu-opioid receptor (MOR) agonists varying in potency and efficacy in the SNI model. We hypothesized that the MOR agonists, fentanyl, morphine, and nalbuphine, would produce antihyperalgesic effects in both male and female rats. Mechanical hypersensitivity was evaluated by measuring responses to increasing pressure (g) applied to each paw (paw pressure test or Randall-Selitto test) with a maximum cut-off of 300 g. Using a within-subjects design, responses were evaluated before and after SNI or sham surgery in male and female rats, and cumulative dose effects curves for fentanyl (0.01-0.1 mg/kg), morphine (0.3-10 mg/kg), and nalbuphine (0.3-10 mg/kg) were determined. Both male and female sham rats demonstrated an initial hypersensitivity on their ipsilateral paw following surgery that dissipated within 7 d. Following SNI surgery, paw pressure thresholds on the injured paw were lower as compared with the pre-surgical response and with the contralateral paw for at least 20 wks in male and female rats. In male rats, fentanyl, morphine, and nalbuphine dose-dependently alleviated SNI-induced hypersensitivity, with EC50 values of 0.03 (± 0.007), 3.9 (± 0.8), and 5.3 (± 0.8) mg/kg, respectively. In female rats, fentanyl, morphine, and nalbuphine also alleviated SNI-induced hypersensitivity in a dose-dependent manner, with EC50 values of 0.01 (± 0.002), 4.1 (± 0.8), and 5.5 (± 0.8) mg/kg, respectively. Naltrexone (0.3 mg/kg) produced rightward shifts in the fentanyl and morphine dose effect curves. Interestingly, fentanyl and morphine also increased paw pressure thresholds in the uninjured, contralateral paws and sham-treated ipsilateral paws in male and female rats. In contrast, gabapentin produced anti-hyperalgesic effects in both male and female rats at 180 mg/kg as demonstrated by a return to pre-surgical, paw pressure thresholds without altering thresholds in the non-injured paw. These results demonstrate MOR agonists produce dose-dependent increases in paw pressure thresholds following SNI surgery in both male and female rats. As expected, MOR agonists demonstrated a rank order in potency with fentanyl > morphine > nalbuphine in both sexes, and only fentanyl was slightly more potent in females. Fentanyl and morphine also increased paw pressure thresholds in non-injured paws, suggesting that large doses inhibited responses to mechanical stimulation perhaps due to sedation and/or behavioral suppression. Overall, these findings demonstrate that MOR agonists produce antihyperalgesic effects in male and female rats potentially with narrow therapeutic indices. Future studies will investigate other behavioral effects of MOR agonists, such as reinforcing and interoceptive effects, in this chronic neuropathic pain model.

Brain mechanisms linking chronic pain conditions and development of comorbid clinical depression are still largely unknown. Here, we used a genome-wide microarray analysis to examine the genetic profile of the hippocampus, a limbic region that regulates mood and stress responses, from male rats exposed to 21 days of inflammatory pain. Bioinformatic gene network/canonical pathways analyses have identified significantly dysregulated genes with known roles in either neuroinflammation or neurodegenerative processes. Akt (protein kinase B) was identified as the main network hub gene. Altered activity of Akt-related signaling pathways has been linked to both the development of depressive state and antidepressant treatment. Furthermore, lipocalin-2 (Lcn2) or NGAL was identified as one of the highest upregulated genes (~ 2-fold) within the hippocampus during chronic pain state. Lcn2/NGAL is an iron-related protein with roles in innate immune response and cell differentiation/maturation that was recently implicated in regulation of emotional behaviors and cognitive function through regulation of neuronal excitability and dendritic spine formation/maturation. Besides the hippocampus, robust increases in Lcn2/NGAL mRNA were also observed within the prefrontal cortex (PFC) and anterior cingulate cortex (ACC), as well as in the brains of female rats exposed to the same pain paradigm. Overall, the results of this study continue to strengthen the idea that dysregulation of genes involved in neuroinflammatory and neurodegenerative processes in the hippocampus and other limbic brain areas may be involved in the development of mood disorders during the chronic pain state.