Digging deeper into pain: an ethological behavior assay correlating well-being in mice with human pain experience.

ABSTRACT: The pressing need for safer, more efficacious analgesics is felt worldwide. Preclinical tests in animal models of painful conditions represent one of the earliest checkpoints novel therapeutics must negotiate before consideration for human use. Traditionally, the pain status of laboratory animals has been inferred from evoked nociceptive assays that measure their responses to noxious stimuli. The disconnect between how pain is tested in laboratory animals and how it is experienced by humans may in part explain the shortcomings of current pain medications and highlights a need for refinement. Here, we survey human patients with chronic pain who assert that everyday aspects of life, such as cleaning and leaving the house, are affected by their ongoing level of pain. Accordingly, we test the impact of painful conditions on an ethological behavior of mice, digging. Stable digging behavior was observed over time in naive mice of both sexes. By contrast, deficits in digging were seen after acute knee inflammation. The analgesia conferred by meloxicam and gabapentin was compared in the monosodium iodoacetate knee osteoarthritis model, with meloxicam more effectively ameliorating digging deficits, in line with human patients finding meloxicam more effective. Finally, in a visceral pain model, the decrease in digging behavior correlated with the extent of disease. Ultimately, we make a case for adopting ethological assays, such as digging, in studies of pain in laboratory animals, which we believe to be more representative of the human experience of pain and thus valuable in assessing clinical potential of novel analgesics in animals.

A note on estimating absolute cytosolic Ca2+ concentration in sensory neurons using a single wavelength Ca2+ indicator.

Ca2+ imaging is frequently used in the investigation of sensory neuronal function and nociception. In vitro imaging of acutely dissociated sensory neurons using membrane-permeant fluorescent Ca2+ indicators remains the most common approach to study Ca2+ signalling in sensory neurons. Fluo4 is a popular choice of single-wavelength indicator due to its brightness, high affinity for Ca2+ and ease of use. However, unlike ratiometric indicators, the emission intensity from single-wavelength indicators can be affected by indicator concentration, optical path length, excitation intensity and detector efficiency. As such, without careful calibration, it can be difficult to draw inferences from differences in the magnitude of Ca2+ transients recorded using Fluo4. Here, we show that a method scarcely used in sensory neurophysiology - first proposed by Maravall and colleagues (2000) - can provide reliable estimates of absolute cytosolic Ca2+ concentration ([Ca2+]cyt) in acutely dissociated sensory neurons using Fluo4. This method is straightforward to implement; is applicable to any high-affinity single-wavelength Ca2+ indicator with a large dynamic range; and provides estimates of [Ca2+]cyt in line with other methods, including ratiometric imaging. Use of this method will improve the granularity of sensory neuron Ca2+ imaging data obtained with Fluo4.

Transcriptomic profiling reveals a pronociceptive role for angiotensin II in inflammatory bowel disease.

ABSTRACT: Visceral pain is a leading cause of morbidity in inflammatory bowel disease (IBD), contributing significantly to reduced quality of life. Currently available analgesics often lack efficacy or have intolerable side effects, driving the need for a more complete understanding of the mechanisms causing pain. Whole transcriptome gene expression analysis was performed by bulk RNA sequencing of colonic biopsies from patients with ulcerative colitis (UC) and Crohn's disease (CD) reporting abdominal pain and compared with noninflamed control biopsies. Potential pronociceptive mediators were identified based on gene upregulation in IBD biopsy tissue and cognate receptor expression in murine colonic sensory neurons. Pronociceptive activity of identified mediators was assessed in assays of sensory neuron and colonic afferent activity. RNA sequencing analysis highlighted a 7.6-fold increase in the expression of angiotensinogen transcripts, Agt , which encode the precursor to angiotensin II (Ang II), in samples from UC patients ( P = 3.2 × 10 -8 ). Consistent with the marked expression of the angiotensin AT 1 receptor in colonic sensory neurons, Ang II elicited an increase in intracellular Ca 2+ in capsaicin-sensitive, voltage-gated sodium channel subtype Na V 1.8-positive sensory neurons. Ang II also evoked action potential discharge in high-threshold colonic nociceptors. These effects were inhibited by the AT 1 receptor antagonist valsartan. Findings from our study identify AT 1 receptor-mediated colonic nociceptor activation as a novel pathway of visceral nociception in patients with UC. This work highlights the potential utility of angiotensin receptor blockers, such as valsartan, as treatments for pain in IBD.

KV7 but not dual small and intermediate KCa channel openers inhibit the activation of colonic afferents by noxious stimuli.

In numerous subtypes of central and peripheral neurons, small and intermediate conductance Ca2+-activated K+ (SK and IK, respectively) channels are important regulators of neuronal excitability. Transcripts encoding SK channel subunits, as well as the closely related IK subunit, are coexpressed in the soma of colonic afferent neurons with receptors for the algogenic mediators ATP and bradykinin, P2X3 and B2, highlighting the potential utility of these channels as drug targets for the treatment of abdominal pain in gastrointestinal diseases such as irritable bowel syndrome. Despite this, pretreatment with the dual SK/IK channel opener SKA-31 had no effect on the colonic afferent response to ATP, bradykinin, or noxious ramp distention of the colon. Inhibition of SK or IK channels with apamin or TRAM-34, respectively, yielded no change in spontaneous baseline afferent activity, indicating these channels are not tonically active. In contrast to its lack of effect in electrophysiological experiments, comparable concentrations of SKA-31 abolished ongoing peristaltic activity in the colon ex vivo. Treatment with the KV7 channel opener retigabine blunted the colonic afferent response to all applied stimuli. Our data therefore highlight the potential utility of KV7, but not SK/IK, channel openers as analgesic agents for the treatment of abdominal pain.NEW & NOTEWORTHY Despite marked coexpression of small (Kcnn1, Kcnn2) and intermediate (Kcnn4) conductance calcium-activated potassium channel transcripts with P2X3 (P2rx3) or bradykinin B2 (Bdkrb2) receptors in colonic sensory neurons, pharmacological activation of these channels had no effect on the colonic afferent response to ATP, bradykinin or luminal distension of the colon. This is in contrast to the robust inhibitory effect of the KV7 channel opener, retigabine.

Beta 3-adrenoceptor agonism ameliorates early-life stress-induced visceral hypersensitivity in male rats.

Visceral hypersensitivity, a hallmark of disorders of the gut-brain axis, is associated with exposure to early-life stress (ELS). Activation of neuronal ß3-adrenoceptors (AR) has been shown to alter central and peripheral levels of tryptophan and reduce visceral hypersensitivity. In this study, we aimed to determine the potential of a ß3-AR agonist in reducing ELS-induced visceral hypersensitivity and possible underlying mechanisms. Here, ELS was induced using the maternal separation (MS) model, where Sprague Dawley rat pups were separated from their mother in early life (postnatal day 2-12). Visceral hypersensitivity was confirmed in adult offspring using colorectal distension (CRD). CL-316243, a ß3-AR agonist, was administered to determine anti-nociceptive effects against CRD. Distension-induced enteric neuronal activation as well as colonic secretomotor function were assessed. Tryptophan metabolism was determined both centrally and peripherally. For the first time, we showed that CL-316243 significantly ameliorated MS-induced visceral hypersensitivity. Furthermore, MS altered plasma tryptophan metabolism and colonic adrenergic tone, while CL-316243 reduced both central and peripheral levels of tryptophan and affected secretomotor activity in the presence of tetrodotoxin. This study supports the beneficial role of CL-316243 in reducing ELS-induced visceral hypersensitivity, and suggests that targeting the ß3-AR can significantly influence gut-brain axis activity through modulation of enteric neuronal activation, tryptophan metabolism, and colonic secretomotor activity which may synergistically contribute to offsetting the effects of ELS.

Sensitization of colonic nociceptors by IL-13 is dependent on JAK and p38 MAPK activity.

The effective management of visceral pain is a significant unmet clinical need for those affected by gastrointestinal diseases, such as inflammatory bowel disease (IBD). The rational design of novel analgesics requires a greater understanding of the mediators and mechanisms underpinning visceral pain. Interleukin-13 (IL-13) production by immune cells residing in the gut is elevated in IBD, and IL-13 appears to be important in the development of experimental colitis. Furthermore, receptors for IL-13 are expressed by neurons innervating the colon, though it is not known whether IL-13 plays any role in visceral nociception per se. To resolve this, we used Ca2+ imaging of cultured sensory neurons and ex vivo electrophysiological recording from the lumbar splanchnic nerve innervating the distal colon. Ca2+ imaging revealed the stimulation of small-diameter, capsaicin-sensitive sensory neurons by IL-13, indicating that IL-13 likely stimulates nociceptors. IL-13-evoked Ca2+ signals were attenuated by inhibition of Janus (JAK) and p38 kinases. In the lumbar splanchnic nerve, IL-13 did not elevate baseline firing, nor sensitize the response to capsaicin application, but did enhance the response to distention of the colon. In line with Ca2+ imaging experiments, IL-13-mediated sensitization of the afferent response to colon distention was blocked by inhibition of either JAK or p38 kinase signaling. Together, these data highlight a potential role for IL-13 in visceral nociception and implicate JAK and p38 kinases in pronociceptive signaling downstream of IL-13.

Sensitization of colonic nociceptors by TNFα is dependent on TNFR1 expression and p38 MAPK activity.

Visceral pain is a leading cause of morbidity in gastrointestinal diseases, which is exacerbated by the gut-related side-effects of many analgesics. New treatments are needed and further understanding of the mediators and mechanisms underpinning visceral nociception in disease states is required to facilitate this. The pro-inflammatory cytokine TNFα is linked to pain in both patients with inflammatory bowel disease and irritable bowel syndrome, and has been shown to sensitize colonic sensory neurons. Somatic, TNFα-triggered thermal and mechanical hypersensitivity is mediated by TRPV1 signalling and p38 MAPK activity respectively, downstream of TNFR1 receptor activation. We therefore hypothesized that TNFR1-evoked p38 MAPK activity may also be responsible for TNFα sensitization of colonic afferent responses to the TRPV1 agonist capsaicin, and noxious distension of the bowel. Using Ca2+ imaging of dorsal root ganglion sensory neurons, we observed TNFα-mediated increases in intracellular [Ca2+ ] and sensitization of capsaicin responses. The sensitizing effects of TNFα were dependent on TNFR1 expression and attenuated by p38 MAPK inhibition. Consistent with these findings, ex vivo colonic afferent fibre recordings demonstrated an enhanced response to noxious ramp distention of the bowel and bath application of capsaicin following TNFα pre-treatment. Responses were reversed by p38 MAPK inhibition and absent in tissue from TNFR1 knockout mice. Our findings demonstrate a contribution of TNFR1, p38 MAPK and TRPV1 to TNFα-induced sensitization of colonic afferents, highlighting the potential utility of these drug targets for the treatment of visceral pain in gastrointestinal disease. KEY POINTS: The pro-inflammatory cytokine TNFα is elevated in gastrointestinal disease and sensitizes colonic afferents via modulation of TRPA1 and NaV 1.8 activity. We further develop this understanding by demonstrating a role for p38 MAPK and TRPV1 in TNFα-mediated colonic afferent sensitization. Specifically, we show that: TNFα sensitizes sensory neurons and colonic afferents to the TRPV1 agonist capsaicin. TNFα-mediated sensitization of sensory neurons and colonic nociceptors is dependent on TNFR1 expression. TNFα sensitization of sensory neurons and colonic afferents to capsaicin and noxious ramp distension is abolished by inhibition of p38 MAPK. Collectively these data support the utility of targeting TNFα, TNFR1 and their downstream signalling via p38 MAPK for the treatment of visceral pain in gastrointestinal disease.

Pain Severity Correlates With Biopsy-Mediated Colonic Afferent Activation But Not Psychological Scores in Patients With IBS-D.

INTRODUCTION: Despite heterogeneity, an increased prevalence of psychological comorbidity and an altered pronociceptive gut microenvironment have repeatedly emerged as causative pathophysiology in patients with irritable bowel syndrome (IBS). Our aim was to study these phenomena by comparing gut-related symptoms, psychological scores, and biopsy samples generated from a detailed diarrhea-predominant IBS patient (IBS-D) cohort before their entry into a previously reported clinical trial. METHODS: Data were generated from 42 patients with IBS-D who completed a daily 2-week bowel symptom diary, the Hospital Anxiety and Depression score, and the Patient Health Questionnaire-12 Somatic Symptom score and underwent unprepared flexible sigmoidoscopy. Sigmoid mucosal biopsies were separately evaluated using immunohistochemistry and culture supernatants to determine cellularity, mediator levels, and ability to stimulate colonic afferent activity. RESULTS: Pain severity scores significantly correlated with the daily duration of pain (r = 0.67, P < 0.00001), urgency (r = 0.57, P < 0.0005), and bloating (r = 0.39, P < 0.05), but not with psychological symptom scores for anxiety, depression, or somatization. Furthermore, pain severity scores from individual patients with IBS-D were significantly correlated (r = 0.40, P < 0.008) with stimulation of colonic afferent activation mediated by their biopsy supernatant, but not with biopsy cell counts nor measured mediator levels. DISCUSSION: Peripheral pronociceptive changes in the bowel seem more important than psychological factors in determining pain severity within a tightly phenotyped cohort of patients with IBS-D. No individual mediator was identified as the cause of this pronociceptive change, suggesting that nerve targeting therapeutic approaches may be more successful than mediator-driven approaches for the treatment of pain in IBS-D.

Sensitization of knee-innervating sensory neurons by tumor necrosis factor-α-activated fibroblast-like synoviocytes: an in vitro, coculture model of inflammatory pain.

ABSTRACT: Pain is a principal contributor to the global burden of arthritis with peripheral sensitization being a major cause of arthritis-related pain. Within the knee joint, distal endings of dorsal root ganglion neurons (knee neurons) interact with fibroblast-like synoviocytes (FLS) and the inflammatory mediators they secrete, which are thought to promote peripheral sensitization. Correspondingly, RNA sequencing has demonstrated detectable levels of proinflammatory genes in FLS derived from arthritis patients. This study confirms that stimulation with tumor necrosis factor (TNF-α) results in expression of proinflammatory genes in mouse and human FLS (derived from osteoarthritis and rheumatoid arthritis patients), as well as increased secretion of cytokines from mouse TNF-α-stimulated FLS (TNF-FLS). Electrophysiological recordings from retrograde labelled knee neurons cocultured with TNF-FLS, or supernatant derived from TNF-FLS, revealed a depolarized resting membrane potential, increased spontaneous action potential firing, and enhanced TRPV1 function, all consistent with a role for FLS in mediating the sensitization of pain-sensing nerves in arthritis. Therefore, data from this study demonstrate the ability of FLS activated by TNF-α to promote neuronal sensitization, results that highlight the importance of both nonneuronal and neuronal cells to the development of pain in arthritis.

Galanin suppresses visceral afferent responses to noxious mechanical and inflammatory stimuli.

Galanin is a neuropeptide expressed by sensory neurones innervating the gastrointestinal (GI) tract. Galanin displays inhibitory effects on vagal afferent signaling within the upper GI tract, and the goal of this study was to determine the actions of galanin on colonic spinal afferent function. Specifically, we sought to evaluate the effect of galanin on lumbar splanchnic nerve (LSN) mechanosensitivity to noxious distending pressures and the development of hypersensitivity in the presence of inflammatory stimuli and colitis. Using ex vivo electrophysiological recordings we show that galanin produces a dose-dependent suppression of colonic LSN responses to mechanical stimuli and prevents the development of hypersensitivity to acutely administered inflammatory mediators. Using galanin receptor (GalR) agonists, we show that GalR1 activation, but not GalR2/3 activation, suppresses mechanosensitivity. The effect of galanin on colonic afferent activity was not observed in tissue from mice with dextran sodium sulfate-induced colitis. We conclude that galanin has a marked suppressive effect on colonic mechanosensitivity at noxious distending pressures and prevents the acute development of mechanical hypersensitivity to inflammatory mediators, an effect not seen in the inflamed colon. These actions highlight a potential role for galanin in the regulation of mechanical nociception in the bowel and the therapeutic potential of targeting galaninergic signaling to treat visceral hypersensitivity.

Human osteoarthritic synovial fluid increases excitability of mouse dorsal root ganglion sensory neurons: an in-vitro translational model to study arthritic pain.

OBJECTIVES: Knee OA is a leading global cause of morbidity. This study investigates the effects of knee SF from patients with OA on the activity of dorsal root ganglion sensory neurons that innervate the knee (knee neurons) as a novel translational model of disease-mediated nociception in human OA. METHODS: Dissociated cultures of mouse knee neurons were incubated overnight or acutely stimulated with OA-SF (n = 4) and fluid from healthy donors (n = 3, Ctrl-SF). Electrophysiology and Ca2+-imaging determined changes in electrical excitability and transient receptor potential channel function, respectively. RESULTS: Incubation with OA-SF induced knee neuron hyperexcitability compared to Ctrl-SF: the resting membrane potential significantly increased (F(2, 92) = 5.6, P = 0.005, ANOVA) and the action potential threshold decreased (F(2, 92) = 8.8, P = 0.0003, ANOVA); TRPV1 (F(2, 445) = 3.7, P = 0.02) and TRPM8 (F(2, 174) = 11.1, P < 0.0001, ANOVA) channel activity also increased. Acute application of Ctrl-SF and OA-SF increased intracellular Ca2+ concentration via intra- and extracellular Ca2+ sources. CONCLUSION: Human OA-SF acutely activated knee neurons and induced hyperexcitability indicating that mediators present in OA-SF stimulate sensory nerve activity and thereby give rise to knee pain. Taken together, this study provides proof-of-concept for a new method to study the ability of mediators present in joints of patients with arthritis to stimulate nociceptor activity and hence identify clinically relevant drug targets for treating knee pain.

Functional and anatomical deficits in visceral nociception with age: a mechanism of silent appendicitis in the elderly?

The ability to sense visceral pain during appendicitis is diminished with age leading to delay in seeking health care and poorer clinical outcomes. To understand the mechanistic basis of this phenomenon, we examined visceral nociception in aged mouse and human tissue. Inflamed and noninflamed appendixes were collected from consenting patients undergoing surgery for the treatment of appendicitis or bowel cancer. Supernatants were generated by incubating samples in buffer and used to stimulate multiunit activity in intestinal preparations, or single-unit activity from teased fibres in colonic preparations, of young and old mice. Changes in afferent innervation with age were determined by measuring the density of calcitonin gene-related peptide-positive afferent fibres and by counting dorsal root ganglia back-labelled by injection of tracer dye into the wall of the colon. Finally, the effect of age on nociceptor function was studied in mouse and human colon. Afferent responses to appendicitis supernatants were greatly impaired in old mice. Further investigation revealed this was due to a marked reduction in the afferent innervation of the bowel and a substantial impairment in the ability of the remaining afferent fibres to transduce noxious stimuli. Translational studies in human tissue demonstrated a significant reduction in the multiunit but not the single-unit colonic mesenteric nerve response to capsaicin with age, indicative of a loss of nociceptor innervation. Our data demonstrate that anatomical and functional deficits in nociception occur with age, underpinning the atypical or silent presentation of appendicitis in the elderly.

Adenosine triphosphate is co-secreted with glucagon-like peptide-1 to modulate intestinal enterocytes and afferent neurons.

Enteroendocrine cells are specialised sensory cells located in the intestinal epithelium and generate signals in response to food ingestion. Whilst traditionally considered hormone-producing cells, there is evidence that they also initiate activity in the afferent vagus nerve and thereby signal directly to the brainstem. We investigate whether enteroendocrine L-cells, well known for their production of the incretin hormone glucagon-like peptide-1 (GLP-1), also release other neuro-transmitters/modulators. We demonstrate regulated ATP release by ATP measurements in cell supernatants and by using sniffer patches that generate electrical currents upon ATP exposure. Employing purinergic receptor antagonists, we demonstrate that evoked ATP release from L-cells triggers electrical responses in neighbouring enterocytes through P2Y2 and nodose ganglion neurones in co-cultures through P2X2/3-receptors. We conclude that L-cells co-secrete ATP together with GLP-1 and PYY, and that ATP acts as an additional signal triggering vagal activation and potentially synergising with the actions of locally elevated peptide hormone concentrations.

Single-cell RNAseq reveals seven classes of colonic sensory neuron.

OBJECTIVE: Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons. DESIGN: Unbiased single-cell RNA-sequencing was performed on retrogradely traced mouse colonic sensory neurons isolated from both thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia associated with lumbar splanchnic and pelvic spinal pathways, respectively. Identified neuronal subtypes were validated by single-cell qRT-PCR, immunohistochemistry (IHC) and Ca2+-imaging. RESULTS: Transcriptomic profiling and unsupervised clustering of 314 colonic sensory neurons revealed seven neuronal subtypes. Of these, five neuronal subtypes accounted for 99% of TL neurons, with LS neurons almost exclusively populating the remaining two subtypes. We identify and classify neurons based on novel subtype-specific marker genes using single-cell qRT-PCR and IHC to validate subtypes derived from RNA-sequencing. Lastly, functional Ca2+-imaging was conducted on colonic sensory neurons to demonstrate subtype-selective differential agonist activation. CONCLUSIONS: We identify seven subtypes of colonic sensory neurons using unbiased single-cell RNA-sequencing and confirm translation of patterning to protein expression, describing sensory diversity encompassing all modalities of colonic neuronal sensitivity. These results provide a pathway to molecular interrogation of colonic sensory innervation in health and disease, together with identifying novel targets for drug development.

5-oxoETE triggers nociception in constipation-predominant irritable bowel syndrome through MAS-related G protein-coupled receptor D.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that is characterized by chronic abdominal pain concurrent with altered bowel habit. Polyunsaturated fatty acid (PUFA) metabolites are increased in abundance in IBS and are implicated in the alteration of sensation to mechanical stimuli, which is defined as visceral hypersensitivity. We sought to quantify PUFA metabolites in patients with IBS and evaluate their role in pain. Quantification of PUFA metabolites by mass spectrometry in colonic biopsies showed an increased abundance of 5-oxoeicosatetraenoic acid (5-oxoETE) only in biopsies taken from patients with IBS with predominant constipation (IBS-C). Local administration of 5-oxoETE to mice induced somatic and visceral hypersensitivity to mechanical stimuli without causing tissue inflammation. We found that 5-oxoETE directly acted on both human and mouse sensory neurons as shown by lumbar splanchnic nerve recordings and Ca2+ imaging of dorsal root ganglion (DRG) neurons. We showed that 5-oxoETE selectively stimulated nonpeptidergic, isolectin B4 (IB4)-positive DRG neurons through a phospholipase C (PLC)- and pertussis toxin-dependent mechanism, suggesting that the effect was mediated by a G protein-coupled receptor (GPCR). The MAS-related GPCR D (Mrgprd) was found in mouse colonic DRG afferents and was identified as being implicated in the noxious effects of 5-oxoETE. Together, these data suggest that 5-oxoETE, a potential biomarker of IBS-C, induces somatic and visceral hyperalgesia without inflammation in an Mrgprd-dependent manner. Thus, 5-oxoETE may play a pivotal role in the abdominal pain associated with IBS-C.

The extracellular matrix glycoprotein tenascin-X regulates peripheral sensory and motor neurones.

KEY POINTS: Tenascin-X (TNX) is an extracellular matrix glycoprotein with anti-adhesive properties in skin and joints. Here we report the novel finding that TNX is expressed in human and mouse gut tissue where it is exclusive to specific subpopulations of neurones. Our studies with TNX-deficient mice show impaired defecation and neural control of distal colonic motility that can be rescued with a 5-HT4 receptor agonist. However, colonic secretion is unchanged. They are also susceptible to internal rectal intussusception. Colonic afferent sensitivity is increased in TNX-deficient mice. Correspondingly, there is increased density of and sensitivity of putative nociceptive fibres in TNX-deficient mucosa. A group of TNX-deficient patients report symptoms highly consistent with those in the mouse model. These findings suggest TNX plays entirely different roles in gut to non-visceral tissues - firstly a role in enteric motor neurones and secondly a role influencing nociceptive sensory neurones Studying further the mechanisms by which TNX influences neuronal function will lead to new targets for future treatment. ABSTRACT: The extracellular matrix (ECM) is not only an integral structural molecule, but is also critical for a wide range of cellular functions. The glycoprotein tenascin-X (TNX) predominates in the ECM of tissues like skin and regulates tissue structure through anti-adhesive interactions with collagen. Monogenic TNX deficiency causes painful joint hypermobility and skin hyperelasticity, symptoms characteristic of hypermobility Ehlers Danlos syndrome (hEDS). hEDS patients also report consistently increased visceral pain and gastrointestinal (GI) dysfunction. We investigated whether there is a direct link between TNX deficiency and GI pain or motor dysfunction. We set out first to learn where TNX is expressed in human and mouse, then determine how GI function, specifically in the colon, is disordered in TNX-deficient mice and humans of either sex. In human and mouse tissue, TNX was predominantly associated with cholinergic colonic enteric neurones, which are involved in motor control. TNX was absent from extrinsic nociceptive peptidergic neurones. TNX-deficient mice had internal rectal prolapse and a loss of distal colonic contractility which could be rescued by prokinetic drug treatment. TNX-deficient patients reported increased sensory and motor GI symptoms including abdominal pain and constipation compared to controls. Despite absence of TNX from nociceptive colonic neurones, neuronal sprouting and hyper-responsiveness to colonic distension was observed in the TNX-deficient mice. We conclude that ECM molecules are not merely support structures but an integral part of the microenvironment particularly for specific populations of colonic motor neurones where TNX exerts functional influences.

Human visceral nociception: findings from translational studies in human tissue.

Peripheral sensitization of nociceptors during disease has long been recognized as a leading cause of inflammatory pain. However, a growing body of data generated over the last decade has led to the increased understanding that peripheral sensitization is also an important mechanism driving abdominal pain in highly prevalent functional bowel disorders, in particular, irritable bowel syndrome (IBS). As such, the development of drugs that target pain-sensing nerves innervating the bowel has the potential to be a successful analgesic strategy for the treatment of abdominal pain in both organic and functional gastrointestinal diseases. Despite the success of recent peripherally restricted approaches for the treatment of IBS, not all drugs that have shown efficacy in animal models of visceral pain have reduced pain end points in clinical trials of IBS patients, suggesting innate differences in the mechanisms of pain processing between rodents and humans and, in particular, how we model disease states. To address this gap in our understanding of peripheral nociception from the viscera and the body in general, several groups have developed experimental systems to study nociception in isolated human tissue and neurons, the findings of which we discuss in this review. Studies of human tissue identify a repertoire of human primary afferent subtypes comparable to rodent models including a nociceptor population, the targeting of which will shape future analgesic development efforts. Detailed mechanistic studies in human sensory neurons combined with unbiased RNA-sequencing approaches have revealed fundamental differences in not only receptor/channel expression but also peripheral pain pathways.

Ex vivo study of human visceral nociceptors.

OBJECTIVE: The development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human tissue that could aid future target identification and drug evaluation. DESIGN: Electrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV4) modulation on mechanical responses. RESULTS: Two distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (-20.8±6.9%, n=6, p<0.05), a treatment for IBS, or application of HC067047 (-34.9±10.0%, n=7, p<0.05), a TRPV4 antagonist, highlighting the utility of the preparation to examine the mechanistic action of existing drugs or novel analgesics. Repeated application of bradykinin or ATP produced consistent afferent responses following desensitisation to the first application, demonstrating their utility as test stimuli to evaluate analgesic activity. CONCLUSIONS: Functionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.

Peripheral KV7 channels regulate visceral sensory function in mouse and human colon.

Background Chronic visceral pain is a defining symptom of many gastrointestinal disorders. The KV7 family (KV7.1-KV7.5) of voltage-gated potassium channels mediates the M current that regulates excitability in peripheral sensory nociceptors and central pain pathways. Here, we use a combination of immunohistochemistry, gut-nerve electrophysiological recordings in both mouse and human tissues, and single-cell qualitative real-time polymerase chain reaction of gut-projecting sensory neurons, to investigate the contribution of peripheral KV7 channels to visceral nociception. Results Immunohistochemical staining of mouse colon revealed labelling of KV7 subtypes (KV7.3 and KV7.5) with CGRP around intrinsic enteric neurons of the myenteric plexuses and within extrinsic sensory fibres along mesenteric blood vessels. Treatment with the KV7 opener retigabine almost completely abolished visceral afferent firing evoked by the algogen bradykinin, in agreement with significant co-expression of mRNA transcripts by single-cell qualitative real-time polymerase chain reaction for KCNQ subtypes and the B2 bradykinin receptor in retrogradely labelled extrinsic sensory neurons from the colon. Retigabine also attenuated responses to mechanical stimulation of the bowel following noxious distension (0-80 mmHg) in a concentration-dependent manner, whereas the KV7 blocker XE991 potentiated such responses. In human bowel tissues, KV7.3 and KV7.5 were expressed in neuronal varicosities co-labelled with synaptophysin and CGRP, and retigabine inhibited bradykinin-induced afferent activation in afferent recordings from human colon. Conclusions We show that KV7 channels contribute to the sensitivity of visceral sensory neurons to noxious chemical and mechanical stimuli in both mouse and human gut tissues. As such, peripherally restricted KV7 openers may represent a viable therapeutic modality for the treatment of gastrointestinal pathologies.