Animal Studies

NrCAM, a neuronal cell adhesion molecule in the L1 family of the immunoglobulin superfamily, is subjected to extensively alternative splicing and is involved in neural development and some disorders. The aim of this study was to explore the role of Nrcam mRNA alternative splicing in neuropathic pain. A next generation RNA sequencing analysis of dorsal root ganglions (DRGs) showed the differential expression of two splicing variants of Nrcam, Nrcam and Nrcam, in the injured DRG after the fourth lumbar spinal nerve ligation (SNL). SNL increased the exon 10 insertion, resulting in an increase in the amount of Nrcam and a corresponding decrease in the level of Nrcam in the injured DRG. An antisense oligonucleotide (ASO) that specifically targeted exon 10 of Nrcam gene (Nrcam ASO) repressed RNA expression of Nrcam while increased Nrcam in in vitro DRG cell culture. Either DRG microinjection or intrathecal injection of Nrcam ASO attenuated SNL-induced the development of mechanical allodynia, thermal hyperalgesia, or cold allodynia. Nrcam ASO also relieved SNL- or chronic compression of DRG (CCD)-induced the maintenance of pain hypersensitivities in male and female mice. PERSPECTIVE: We conclude that the relative levels of alternatively spliced Nrcam variants are critical for neuropathic pain genesis. Targeting Nrcam alternative splicing via the antisense oligonucleotides may be a new potential avenue in neuropathic pain management.

Functional dyspepsia (FD) is a common functional gastrointestinal disorder. Gastric hypersensitivity (GHS) is a hallmark of this disorder but the cellular mechanisms remain largely unknown. Stressors during gestational period could have effects on the offspring's tissue structure and function, which may predispose to gastrointestinal diseases. The aim of this study was to test whether prenatal maternal stress (PMS) induces GHS and to investigate role of acid-sensing ion channel (ASIC)/ nuclear factor-κB (NF-κB) signaling by examining Asic1 methylation status in adult offspring rats. GHS in response to gastric distention was examined by electromyography recordings. Changes in neuronal excitability were determined by whole-cell patch-clamp recording techniques. Demethylation of CpG islands of Asic1 was determined by methylation-specific PCR and bisulfite sequencing assay. PMS produced GHS in adult offspring rats. Treatment with Amiloride, an inhibitor of ASICs, significantly attenuated GHS and reversed hyperexcitability of gastric-specific dorsal root ganglion (DRG) neurons labeled by the dye DiI. Expression of ASIC1 and NF-κBp65 were markedly enhanced in T7-T10 DRGs. Furthermore, PMS led to a significant demethylation of CpG islands in the Asic1 promoter. A chromatin immunoprecipitation assay showed that PMS also enhanced the ability of NF-κBp65 to bind the promoter of Asic1 gene. Blockade of NF-κB using lentiviral-p65shRNA reversed upregulation of ASIC1 expression, GHS and the hyperexcitability of DRG neurons. These data suggest that upregulation of ASIC1 expression is attributed to Asic1 promoter DNA demethylation and NF-κB activation, and that the enhanced interaction of the Asic1 and NF-κBp65 contributes to GHS induced by PMS.

Burn injuries and associated complications present a major public health challenge. Many burn patients develop clinically intractable complications, including pain and other sensory disorders. Recent evidence has shown that dendritic spine neuropathology in spinal cord sensory and motor neurons accompanies central nervous system (CNS) or peripheral nervous system (PNS) trauma and disease. However, no research has investigated similar dendritic spine neuropathologies following a cutaneous thermal burn injury. In this retrospective investigation, we analyzed dendritic spine morphology and localization in alpha-motor neurons innervating a burn-injured area of the body (hind paw). To identify a molecular regulator of these dendritic spine changes, we further profiled motor neuron dendritic spines in adult mice treated with romidepsin, a clinically approved Pak1-inhibitor, or vehicle control at two postburn time points: Day 6 immediately after treatment, or Day 10 following drug withdrawal. In control treated mice, we observed an overall increase in dendritic spine density, including structurally mature spines with mushroom-shaped morphology. Pak1-inhibitor treatment reduced injury-induced changes to similar levels observed in animals without burn injury. The effectiveness of the Pak1-inhibitor was durable, since normalized dendritic spine profiles remained as long as 4 days despite drug withdrawal. This study is the first report of evidence demonstrating that a second-degree burn injury significantly affects motor neuron structure within the spinal cord. Furthermore, our results support the opportunity to study dendritic spine dysgenesis as a novel avenue to clarify the complexities of neurological disease following traumatic injury.

The discovery and development of new and potentially nonaddictive pain therapeutics requires rapid, yet clinically relevant assays of nociception in preclinical models. A reliable and scalable automated scoring system for nocifensive behavior of mice in the formalin assay would dramatically lower the time and labor costs associated with experiments and reduce experimental variability. Here, we present a method that exploits machine learning techniques for video recordings that consists of three components: key point detection, per frame feature extraction using these key points, and classification of behavior using the GentleBoost algorithm. This approach to automation is flexible as different model classifiers or key points can be used with only small losses in accuracy. The adopted system identified the behavior of licking/biting of the hind paw with an accuracy that was comparable to a human observer (98% agreement) over 111 different short videos (total 284 min) at a resolution of 1 s. To test the system over longer experimental conditions, the responses of two inbred strains, C57BL/6NJ and C57BL/6J, were recorded over 90 min post formalin challenge. The automated system easily scored over 80 h of video and revealed strain differences in both response timing and amplitude. This machine learning scoring system provides the required accuracy, consistency, and ease of use that could make the formalin assay a feasible choice for large-scale genetic studies.

To develop non-opioid therapies for postoperative incisional pain, we must understand its underlying molecular mechanisms. In this study, we assessed global gene expression changes in dorsal root ganglia neurons in a model of incisional pain to identify pertinent molecular pathways. Male, Sprague-Dawley rats underwent infiltration of 1% capsaicin or vehicle into the plantar hind paw (n = 6-9/group) 30 min before plantar incision. Twenty-four hours after incision or sham (control) surgery, lumbar L4-L6 dorsal root ganglias were collected from rats pretreated with vehicle or capsaicin. RNA was isolated and sequenced by next generation sequencing. The genes were then annotated to functional networks using a knowledge-based database, Ingenuity Pathway Analysis. In rats pretreated with vehicle, plantar incision caused robust hyperalgesia, up-regulated 36 genes and downregulated 90 genes in dorsal root ganglias one day after plantar incision. Capsaicin pretreatment attenuated pain behaviors, caused localized denervation of the dermis and epidermis, and prevented the incision-induced changes in 99 of 126 genes. The pathway analyses showed altered gene networks related to increased pro-inflammatory and decreased anti-inflammatory responses in dorsal root ganglias. Insulin-like growth factor signaling was identified as one of the major gene networks involved in the development of incisional pain. Expression of insulin-like growth factor -2 and IGFBP6 in dorsal root ganglia were independently validated with quantitative real-time polymerase chain reaction. We discovered a distinct subset of dorsal root ganglia genes and three key signaling pathways that are altered 24 h after plantar incision but are unchanged when incision was made after capsaicin infiltration in the skin. Further exploration of molecular mechanisms of incisional pain may yield novel therapeutic targets.

Understanding the mechanisms that drive transition from acute to chronic pain is essential to identify new therapeutic targets. The importance of endogenous resolution pathways acting as a "brake" to prevent development of chronic pain has been largely ignored. We examined the role of interleukin-10 (IL-10) in resolution of neuropathic pain induced by cisplatin. In search of an underlying mechanism, we studied the effect of cisplatin and IL-10 on spontaneous activity (SA) in dorsal root ganglia neurons. Cisplatin (2 mg/kg daily for 3 days) induced mechanical hypersensitivity that resolved within 3 weeks. In both sexes, resolution of mechanical hypersensitivity was delayed in Il10 mice, in WT mice treated intrathecally with neutralizing anti-IL-10 antibody, and in mice with cell-targeted deletion of IL-10R1 on advillin-positive sensory neurons. Electrophysiologically, small- to medium-sized dorsal root ganglia neurons from cisplatin-treated mice displayed an increase in the incidence of SA. Cisplatin treatment also depolarized the resting membrane potential, and decreased action potential voltage threshold and rheobase, while increasing ongoing activity at -45 mV and the amplitude of depolarizing spontaneous fluctuations. In vitro addition of IL-10 (10 ng/mL) reversed the effect of cisplatin on SA and on the depolarizing spontaneous fluctuation amplitudes, but unexpectedly had little effect on the other electrophysiological parameters affected by cisplatin. Collectively, our findings challenge the prevailing concept that IL-10 resolves pain solely by dampening neuroinflammation and demonstrate in a model of chemotherapy-induced neuropathic pain that endogenous IL-10 prevents transition to chronic pain by binding to IL-10 receptors on sensory neurons to regulate their activity.

High frequency spontaneous activity in injured primary afferents has been proposed as a pathological mechanism of neuropathic pain following nerve injury. Although spinal infusion of glial cell line-derived neurotrophic factor (GDNF) reduces the activity of injured myelinated A-fiber neurons after 5th lumbar (L5) spinal nerve ligation (SNL) in rats, the implicated molecular mechanism remains undetermined. The fast-inactivating transient A-type potassium current (IA) is an important determinant of neuronal excitability, and five voltage-gated potassium channel (Kv) alpha-subunits, Kv1.4, Kv3.4, Kv4.1, Kv4.2, and Kv4.3, display IA in heterologous expression systems. Here, we examined the effect of spinal GDNF infusion on IA and the expression of these five Kv mRNAs in injured A-fiber neurons using the in vitro patch clamp technique and in situ hybridization histochemistry. GDNF infusion reversed axotomy-induced reduction of the rheobase, elongation of first spike duration, and depolarization of the resting membrane potential. L5 SNL significantly reduced the current density of IA and GDNF treatment reversed the reduction. Among the examined Kv mRNAs, only the change in Kv4.1-expression was parallel with the change in IA after SNL and GDNF treatment. These findings suggest that GDNF should reduce the hyperexcitability of injured A-fiber primary afferents by IA recurrence. Among the five IA-related Kv channels, Kv4.1 should be a key channel, which account for this IA recurrence.