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Chronic abdominal pain in the absence of ongoing disease is the hallmark of disorders of gut-brain interaction (DGBIs), including irritable bowel syndrome (IBS). While the etiology of DGBIs remains poorly understood, there is evidence that both genetic and environmental factors play a role. In this study, we report the identification and validation of arginine-vasopressin receptor 1A (Avpr1a) as a novel candidate gene for visceral hypersensitivity (VH), a primary peripheral mechanism underlying abdominal pain in DGBI/IBS. Comparing 2 C57BL/6 (BL/6) substrains (C57BL/6NTac and C57BL/6J) revealed differential susceptibility to the development of chronic VH following intrarectal zymosan instillation, a validated preclinical model for postinflammatory IBS. Using whole-genome sequencing, we identified a single-nucleotide polymorphism differentiating the 2 strains in the 5' intergenic region upstream of Avpr1a, encoding the protein Avpr1a. We used behavioral, histological, and molecular approaches to identify distal colon-specific gene expression and neuronal hyperresponsiveness covarying with Avpr1a genotype and VH susceptibility. While the 2 BL/6 substrains did not differ across other gastrointestinal phenotypes (eg, fecal water retention), VH-susceptible BL/6NTac mice had higher colonic Avpr1a mRNA and protein expression. These results parallel findings that patients' colonic Avpr1a mRNA expression corresponded to higher pain ratings. Moreover, neurons of the enteric nervous system were hyperresponsive to the Avpr1a agonist arginine-vasopressin, suggesting a role for enteric neurons in the pathology underlying VH. Taken together, these findings implicate differential regulation of Avpr1a as a novel mechanism of VH susceptibility as well as a potential therapeutic target specific to VH. PERSPECTIVE: This article presents evidence of Avpr1a as a novel candidate gene for VH in a mouse model of IBS. Avpr1a genotype and/or tissue-specific expression represents a potential biomarker for chronic abdominal pain susceptibility.
Assuntos
Dor Crônica , Camundongos Endogâmicos C57BL , Receptores de Vasopressinas , Dor Visceral , Animais , Dor Visceral/genética , Receptores de Vasopressinas/genética , Camundongos , Masculino , Dor Crônica/genética , Sistema Nervoso Entérico/metabolismo , Neurônios/metabolismo , Modelos Animais de Doenças , Colo , Polimorfismo de Nucleotídeo Único , Síndrome do Intestino Irritável/genética , Hiperalgesia/genéticaRESUMO
Background and aims: Spinal cord injury (SCI) affects roughly 300,000 Americans with 17,000 new cases added annually. In addition to paralysis, 60% of people with SCI develop neurogenic bowel (NB), a syndrome characterized by slow colonic transit, constipation, and chronic abdominal pain. The knowledge gap surrounding NB mechanisms after SCI means that interventions are primarily symptom-focused and largely ineffective. The goal of the present studies was to identify mechanism(s) that initiate and maintain NB after SCI as a critical first step in the development of evidence-based, novel therapeutic treatment options. Methods: Following spinal contusion injury at T9, we observed alterations in bowel structure and function reflecting key clinical features of NB. We then leveraged tissue-specific whole transcriptome analyses (RNAseq) and fecal 16S rRNA amplicon sequencing in combination with histological, molecular, and functional (Ca2+ imaging) approaches to identify potential mechanism(s) underlying the generation of the NB phenotype. Results: In agreement with prior reports focused on SCI-induced changes in the skin, we observed a rapid and persistent increase in expression of calcitonin gene-related peptide (CGRP) expression in the colon. This is suggestive of a neurogenic inflammation-like process engaged by antidromic activity of below-level primary afferents following SCI. CGRP has been shown to disrupt colon homeostasis and negatively affect peristalsis and colon function. As predicted, contusion SCI resulted in increased colonic transit time, expansion of lymphatic nodules, colonic structural and genomic damage, and disruption of the inner, sterile intestinal mucus layer corresponding to increased CGRP expression in the colon. Gut microbiome colonization significantly shifted over 28 days leading to the increase in Anaeroplasma, a pathogenic, gram-negative microbe. Moreover, colon specific vagal afferents and enteric neurons were hyperresponsive after SCI to different agonists including fecal supernatants. Conclusions: Our data suggest that SCI results in overexpression of colonic CGRP which could alter colon structure and function. Neurogenic inflammatory-like processes and gut microbiome dysbiosis can also sensitize vagal afferents, providing a mechanism for visceral pain despite the loss of normal sensation post-SCI. These data may shed light on novel therapeutic interventions targeting this process to prevent NB development in patients.
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Spinal cord injury (SCI) is a complex neurophysiological disorder, which can result in many long-term complications including changes in mobility, bowel and bladder function, cardiovascular function, and metabolism. In addition, most individuals with SCI experience some form of chronic pain, with one-third of these individuals rating their pain as severe and unrelenting. SCI-induced chronic pain is considered to be "high impact" and broadly affects a number of outcome measures, including daily activity, physical and cognitive function, mood, sleep, and overall quality of life. The majority of SCI pain patients suffer from pain that emanates from regions located below the level of injury. This pain is often rated as the most severe and the underlying mechanisms involve injury-induced plasticity along the entire neuraxis and within the peripheral nervous system. Unfortunately, current therapies for SCI-induced chronic pain lack universal efficacy. Pharmacological treatments, such as opioids, anticonvulsants, and antidepressants, have been shown to have limited success in promoting pain relief. In addition, these treatments are accompanied by many adverse events and safety issues that compound existing functional deficits in the spinally injured, such as gastrointestinal motility and respiration. Non-pharmacological treatments are safer alternatives that can be specifically tailored to the individual and used in tandem with pharmacological therapies if needed. This review describes existing non-pharmacological therapies that have been used to treat SCI-induced pain in both preclinical models and clinical populations. These include physical (i.e., exercise, acupuncture, and hyper- or hypothermia treatments), psychological (i.e., meditation and cognitive behavioral therapy), and dietary interventions (i.e., ketogenic and anti-inflammatory diet). Findings on the effectiveness of these interventions in reducing SCI-induced pain and improving quality of life are discussed. Overall, although studies suggest non-pharmacological treatments could be beneficial in reducing SCI-induced chronic pain, further research is needed. Additionally, because chronic pain, including SCI pain, is complex and has both emotional and physiological components, treatment should be multidisciplinary in nature and ideally tailored specifically to the patient.
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Thermal nociception involves the transmission of temperature-related noxious information from the periphery to the CNS and is a heritable trait that could predict transition to persistent pain. Rodent forward genetics complement human studies by controlling genetic complexity and environmental factors, analysis of end point tissue, and validation of variants on appropriate genetic backgrounds. Reduced complexity crosses between nearly identical inbred substrains with robust trait differences can greatly facilitate unbiased discovery of novel genes and variants. We found BALB/cByJ mice showed enhanced sensitivity on the 53.5°C hot plate and mechanical stimulation in the von Frey test compared to BALB/cJ mice and replicated decreased gross brain weight in BALB/cByJ versus BALB/cJ. We then identified a quantitative trait locus (QTL) on chromosome 13 for hot plate sensitivity (LOD = 10.7; p < 0.001; peak = 56 Mb) and a QTL for brain weight on chromosome 5 (LOD = 8.7; p < 0.001). Expression QTL mapping of brain tissues identified H2afy (56.07 Mb) as the top transcript with the strongest association at the hot plate locus (FDR = 0.0002) and spliceome analysis identified differential exon usage within H2afy associated with the same locus. Whole brain proteomics further supported decreased H2AFY expression could underlie enhanced hot plate sensitivity, and identified ACADS as a candidate for reduced brain weight. To summarize, a BALB/c reduced complexity cross combined with multiple-omics approaches facilitated identification of candidate genes underlying thermal nociception and brain weight. These substrains provide a powerful, reciprocal platform for future validation of candidate variants.
Assuntos
Nociceptividade , Locos de Características Quantitativas , Animais , Encéfalo , Mapeamento Cromossômico , Camundongos , Camundongos Endogâmicos BALB C , Locos de Características Quantitativas/genéticaRESUMO
Objectives: Functional Abdominal Pain (FAP) and Irritable Bowel Syndrome (IBS) are common recurrent abdominal pain diagnoses with the hallmark, lack of inflammation. To identify a biological signature for IBS/FAP in the colon, this study used genetic profiling to uncover gene expression changes associated with IBS/FAP and abdominal pain. Methods: Patients (8 to 17 years) newly diagnosed with IBS or FAP were enrolled in the study. At diagnostic colonoscopy, three rectal biopsies were collected, and gene expression analysis was performed using a Qiagen PCR Array. Relative fold difference in gene expression for 84 pain-associated genes was calculated using the 2-ΔΔ Cq method compared with pain-free controls. Factors affecting pain burden (Pain Burden Interview; PBI) were analyzed, including age, sex, rectal inflammation, and gene expression. Data were analyzed using multiple stepwise linear regression and 2-tailed t tests (P ≤ 0.05). Results: Of the 22 total patients in the study, 19 were diagnosed with either IBS-Constipation (frequency of 5.26%), IBS-Diarrhea (47.37%), IBS-Mixed (10.53%), or FAP (36.84%). IBS/FAP patients reported significantly higher pain burden at the time of diagnosis compared to pain-free controls (p < 0.001), as well as significantly higher abdominal pain (p = 0.01). Of the 84 genes, expression of GRIN1 (p = 0.02), MAPK3 (p = 0.04), P2X4 (p = 0.04), and PTGES3 (p = 0.02) were all significantly associated with PBI score. Discussion: Abdominal pain associated with IBS/FAP in pediatric patients may be linked to the expression of GRIN1, MAPK3, P2X4, and PTGES3, pointing to potential novel therapeutic targets for management of recurring abdominal pain.
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Drosophila melanogaster larvae detect noxious thermal and mechanical stimuli in their environment using polymodal nociceptor neurons whose dendrites tile the larval body wall. Activation of these nociceptors by potentially tissue-damaging stimuli elicits a stereotyped escape locomotion response. The cellular and molecular mechanisms that regulate nociceptor function are increasingly well understood, but gaps remain in our knowledge of the broad mechanisms that control nociceptor sensitivity. In this study, we use cell-specific knockdown and overexpression to show that nociceptor sensitivity to noxious thermal and mechanical stimuli is correlated with levels of Gαq and phospholipase Cß signaling. Genetic manipulation of these signaling mechanisms does not result in changes in nociceptor morphology, suggesting that changes in nociceptor function do not arise from changes in nociceptor development, but instead from changes in nociceptor activity. These results demonstrate roles for Gαq and phospholipase Cß signaling in facilitating the basal sensitivity of the larval nociceptors to noxious thermal and mechanical stimuli and suggest future studies to investigate how these signaling mechanisms may participate in neuromodulation of sensory function.