Animal Studies

Mesocorticolimbic dopaminergic (DA) neurons have been implicated in regulating nociception in chronic pain, yet the mechanisms are barely understood. Here, we found that chronic constructive injury (CCI) in mice increased the firing activity and decreased the KCNQ channel-mediated M-currents in ventral tegmental area (VTA) DA neurons projecting to the nucleus accumbens (NAc). Chemogenetic inhibition of the VTA-to-NAc DA neurons alleviated CCI-induced thermal nociception. Opposite changes in the firing activity and M-currents were recorded in VTA DA neurons projecting to the medial prefrontal cortex (mPFC) but did not affect nociception. In addition, intra-VTA injection of retigabine, a KCNQ opener, while reversing the changes of the VTA-to-NAc DA neurons, alleviated CCI-induced nociception, and this was abolished by injecting exogenous BDNF into the NAc. Taken together, these findings highlight a vital role of KCNQ channel-mediated modulation of mesolimbic DA activity in regulating thermal nociception in the chronic pain state.

The mechanism of pain chronicity is largely unknown in lumbar radiculopathy (LR). The anatomical location of nerve injury is one of the important factors associated with pain chronicity of LR. Accumulating evidence has shown constriction distal to the dorsal root ganglion (DRG) caused more severe radiculopathy than constriction proximal to the DRG; thereby, the mechanism of pain chronicity in LR could be revealed by comparing the differences in pathological changes of DRGs between nerve constriction distal and proximal to the DRG. Here, we used 2 rat models of LR with nerve constriction distal or proximal to the DRG to probe how the different nerve injury sites could differentially affect pain chronicity and the pathological changes of DRG neuron subpopulations. As expected, rats with nerve constriction distal to the DRG showed more persistent pain behaviors than those with nerve constriction proximal to the DRG in 50% paw withdraw threshold, weight-bearing test, and acetone test. One day after the operation, distal and proximal nerve constriction showed differential pathological changes of DRG. The ratios of activating transcription factor3 (ATF3)-positive DRG neurons were significantly higher in rats with nerve constriction distal to DRG than those with nerve constriction proximal to DRG. In subpopulation analysis, the ratios of ATF3-immunoreactivity (IR) in neurofilament heavy chain (NFH)-positive DRG neurons significantly increased in distal nerve constriction compared to proximal nerve constriction; although, both distal and proximal nerve constriction presented increased ratios of ATF3-IR in calcitonin gene-related peptide (CGRP)-positive DRG neurons. Moreover, the nerve constriction proximal to DRG caused more hypoxia than did that distal to DRG. Together, ATF3 expression in NHF-positive DRG neurons at the acute stage is a potential bio-signature of persistent pain in rat models of LR.

During persistent pain, the dorsal spinal cord responds to painful inputs from the site of injury, but the molecular modulatory processes have not been comprehensively examined. Using transcriptomics and multiplex in situ hybridization, we identified the most highly regulated receptors and signaling molecules in rat dorsal spinal cord in peripheral inflammatory and post-surgical incisional pain models. We examined a time course of the response including acute (2 hrs) and longer term (2 day) time points after peripheral injury representing the early onset and instantiation of hyperalgesic processes. From this analysis, we identify a key population of superficial dorsal spinal cord neurons marked by somatotopic upregulation of the opioid neuropeptide precursor prodynorphin, and two receptors: the neurokinin 1 receptor, and anaplastic lymphoma kinase. These alterations occur specifically in the glutamatergic subpopulation of superficial dynorphinergic neurons. In addition to specific neuronal gene regulation, both models showed induction of broad transcriptional signatures for tissue remodeling, synaptic rearrangement, and immune signaling defined by complement and interferon induction. These signatures were predominantly induced ipsilateral to tissue injury, implying linkage to primary afferent drive. We present a comprehensive set of gene regulatory events across two models that can be targeted for the development of non-opioid analgesics. PERSPECTIVE: The deadly impact of the opioid crisis and the need to replace morphine and other opioids in clinical practice is well recognized. Embedded within this research is an overarching goal of obtaining foundational knowledge from transcriptomics to search for non-opioid analgesic targets. Developing such analgesics would address unmet clinical needs.

A number of cellular systems work in concert to modulate nociceptive processing in the periphery, but the mechanisms that regulate neonatal nociception may be distinct compared with adults. Our previous work indicated a relationship between neonatal hypersensitivity and growth hormone (GH) signaling. Here, we explored the peripheral mechanisms by which GH modulated neonatal nociception under normal and injury conditions (incision) in male and female mice. We found that GH receptor (GHr) signaling in primary afferents maintains a tonic inhibition of peripheral hypersensitivity. After injury, a macrophage dependent displacement of injury-site GH was found to modulate neuronal transcription at least in part via serum response factor (SRF) regulation. A single GH injection into the injured hindpaw muscle effectively restored available GH signaling to neurons and prevented acute pain-like behaviors, primary afferent sensitization, and neuronal gene expression changes. GH treatment also inhibited long-term somatosensory changes observed after repeated peripheral insult. Results may indicate a novel mechanism of neonatal nociception.Although it is noted that mechanisms of pain development in early life are unique compared with adults, little research focuses on neonatal-specific peripheral mechanisms of nociception. This gap is evident in the lack of specialized care for infants following an injury including surgeries. This report evaluates how distinct cellular systems in the periphery including the endocrine, immune and nervous systems work together to modulate neonatal-specific nociception. We uncovered a novel mechanism by which muscle injury induces a macrophage-dependent sequestration of peripheral growth hormone (GH) that effectively removes its normal tonic inhibition of neonatal nociceptors to promote acute pain-like behaviors. Results indicate a possible new strategy for treatment of neonatal postsurgical pain.

Acute pain is prevalent following burn injury and can often transition to chronic pain. Prolonged acute pain is an important risk factor for chronic pain and there is little preclinical research to address this problem. Using a mouse model of second-degree burn, we investigated whether pre-existing stress influences pain(sensitivity) after a burn injury. We introduced a contribution of stress in two different ways: (1) the use of foot-shock as a pre-injury stressor or (2) the use of A/J mice to represent higher pre-existing stress compared to C57Bl/6 mice. C57Bl/6 and A/J mice were exposed to repeated mild foot shock to induce stress for 10 continuous days and mice underwent either burn injury or sham burn injury of the plantar surface of the right hind paw. Assessments of mechanical and thermal sensitivities of the injured and uninjured paw were conducted during the shock protocol and at intervals up to 82-day post-burn injury. In both strains of mice that underwent burn injury, thermal hypersensitivity and mechanical allodynia appeared rapidly in the ipsilateral paw. Mice that were stressed took much longer to recover their hind paw mechanical thresholds to baseline compared to non-stressed mice in both burn and non-burn groups. Analysis of the two mouse strains revealed that the recovery of mechanical thresholds in A/J mice which display higher levels of baseline anxiety was shorter than C57Bl/6 mice. No differences were observed regarding thermal sensitivities between strains. Our results support the view that stress exposure prior to burn injury affects mechanical and thermal thresholds and may be relevant to as a risk factor for the transition from acute to chronic pain. Finally, genetic differences may play a key role in modality-specific recovery following burn injury.

High molecular weight hyaluronan (HMWH), a prominent component of the extracellular matrix binds to and signals via multiple receptors, including cluster of differentiation 44 (CD44), and toll-like receptor 4 (TLR4). We tested the hypothesis that, in the setting of inflammation, HMWH acts at TLR4 to attenuate hyperalgesia. We found that the attenuation of prostaglandin E (PGE)-induced hyperalgesia by HMWH was attenuated by a TLR4 antagonist (NBP2-26245), but only in male and ovariectomized female rats. In this study we sought to evaluated the role of the TLR4 signaling pathway in anti-hyperalgesia induced by HMWH in male rats. Decreasing expression of TLR4 in nociceptors, by intrathecal administration of an oligodeoxynucleotide (ODN) antisense to TLR4 mRNA, also attenuated HMWH-induced anti-hyperalgesia, in male and ovariectomized female rats. Estrogen replacement in ovariectomized females reconstituted the gonad-intact phenotype. The administration of an inhibitor of myeloid differentiation factor 88 (MyD88), a TLR4 second messenger, attenuated HMWH-induced anti-hyperalgesia, while an inhibitor of the MyD88-independent TLR4 signaling pathway did not. Since it has previously been shown that HMWH-induced anti-hyperalgesia is also mediated, in part by CD44 we evaluated the effect of the combination of ODN antisense to TLR4 and CD44 mRNA. This treatment completely reversed HMWH-induced anti-hyperalgesia in male rats. Our results demonstrate a sex hormone-dependent, sexually dimorphic involvement of TLR4 in HMWH-induced anti-hyperalgesia, that is MyD88 dependent. PERSPECTIVE: The role of TLR4 in anti-hyperalgesia induced by HMWH is a sexually dimorphic, TLR4 dependent inhibition of inflammatory hyperalgesia that provides a novel molecular target for the treatment of inflammatory pain.