Critical evaluation of animal models of visceral pain for therapeutics development: A focus on irritable bowel syndrome.

The treatment of chronic visceral pain represents a substantial therapeutic need. In patients with IBS, abdominal pain is the major symptom that compels patients to seek medical attention. Unfortunately, there are few effective therapies to treat patients with chronic visceral pain and those that are available have undesirable side-effects. In this position paper, we have brought together ideas from members of multiple disciplines and societies in an attempt to provide a consensus on the rodent models that should be employed in the future development of new treatment approaches for visceral pain, particularly that associated with IBS. An important key message is that there are no animal models of IBS, but rather models that have translational relevance with high face and construct validity, which are available. These models should be used in the development of novel approaches to treat IBS and to further enhance our understanding of the mechanisms underlying visceral pain.

Pain, whether it be somatic, neuropathic or visceral, results in significant disability for every section of the community. Visceral pain may arise from any of the thoracic, pelvic, or abdominal organs. In direct contrast to somatic pain, visceral pain is often variable in its experience, poorly localized, and characterized by hypersensitivity to stimuli such as chemical or mechanical distension. Visceral pain is common and is a leading worldwide cause of healthcare utilization 1 . Unexplained abdominal pain is the sixth and tenth most common cause of hospital admission in women and men respectively 2 . In the United States, gastrointestinal (GI) disorders associated with chronic abdominal pain result in excess of 12 million office consultations per annum 3 . In the United Kingdom, it has been estimated that non-specific abdominal pain costs the economy in excess of £100 million per annum 4 . Despite its prevalence, visceral pain is often clinically challenging to delineate its etiology based on symptoms and clinical evaluation alone. Visceral pain is a presenting feature of both organic origin and disorders of gut brain interaction, although there is frequently overlap between the two 5 , 6 . Organic disorders, where histologically definable pathology is associated with visceral pain includes cancer, infectious processes, nephrolithiasis, ischemic disorders, gastro-esophageal reflux disease, peptic ulceration, cholecystitis, pancreatitis, inflammatory bowel disease and viscus perforation. However, chronic visceral pain also frequently occurs in the absence of distinct pathology such as in the highly prevalent “nociplastic” conditions of irritable bowel syndrome (IBS) and functional dyspepsia. Chronic visceral pain can result in changes within the central nervous system which may lead to behavioral symptoms such as anxiety and depression 7 . Chronic visceral pain adversely impacts on mood, physical activity, sleep, quality of life and drives healthcare usage 8 . Notwithstanding the sizeable numbers of patients with chronic and often incapacitating visceral pain, current clinical management options are limited, and short- and long-term outcomes are suboptimal 9 . There are few effective analgesic options to effectively manage chronic visceral pain and those that are available, such as opioids are limited by adverse side-effect profiles such as drowsiness, altered bowel habit and nausea 10 . Moreover, chronic opioid use can paradoxically lead to a hyperalgesic state and worsening of symptoms 11 . Given this unmet clinical need, the development of novel analgesic therapeutics, with defined targets derived from preclinical studies, is urgently needed. Whilst various animal models have played an important role our understanding of visceral pain, our understanding is far from complete 12 , 13 . This position paper will contextualize chronic visceral pain using the example of IBS and will focus on the pathophysiology of visceral pain while providing a critical review of current animal models. We will also highlight and prioritize those models and assays with the highest face/construct validity and delineate the perceived gaps. Chronic weekly visceral pain, associated with a change in frequency or form of stool are the central defining features of IBS 14 . Although the development and maintenance of chronic visceral pain in IBS incompletely understood, it can be conceptualized as due to aberrations occurring at potentially multiple levels of the bidirectional circuit of communication from the gut to the brain, the gut brain axis 15 . As such, visceral pain may arise due to a peripheral augmentation of the primary afferent signal, or may be due to central sensitization that occurs due to alterations in descending modulation or by central facilitation at spinal/supraspinal levels 16 . In patients with IBS, pain can be experienced anywhere in the abdomen, although typically it is felt in the left and right iliac fossae and is often related to defecation 17 . The quality of visceral pain is frequently described by patients as aching, dull, cramping, sickening, deep, or squeezing. Visceral pain is poorly localized and frequently is associated with autonomic epiphonema such as pallor, sweating, changes in body temperature or tachycardia 18 . Visceral pain is frequently accompanied by affective complications such as anxiety and emotional distress. A further characteristic of pain in IBS is that it is associated with cross-organ sensitization of other viscera. For example, dysmenorrhea of uterine origin may result in colonic allodynia where pain is evoked by normally non-noxious physiological stimuli such as feces and gaseous distension 19 . Chronic visceral pain in IBS can result in limitations in physical functional ranging from mild to severe. Consequentially, IBS may exert a major impact deleterious on day-to- day activities, relationships and work 20 . In the United States, IBS total costs are estimated to be in the order of $30 billion per annum 21 . In patients with IBS, the clinical examination may demonstrate abdominal tenderness on palpation, but such patients may also have superficial or deep allodynia or hyperalgesia. Patients with IBS frequently demonstrate increased sensitivity to mechanical distension of the GI tract, so called visceral hypersensitivity. Increasing visceral hypersensitivity is associated with increasing severity of IBS symptoms irrespective of comorbid anxiety or depression 22 . The duration and severity of pain in IBS is independently associated with healthcare seeking 23 . The treatment of pain in IBS is multifaceted, complex and often must be individualized in the context of a multidisciplinary environment 24 . Long-term follow-up demonstrates that up to 20% of IBS patients symptoms worsen over time, although in 30–50% symptoms remain stable 25 . Predictive factors of worsening symptoms include pain, previous surgery, somatic symptoms and co-morbid anxiety and depression. As discussed above, the causes of chronic visceral pain in patients with IBS are incompletely understood due to the multifactorial etiology of the disorder. Pathological sensitization of primary afferents in the gut can occur due to multiple mechanisms that are not mutually exclusive. In the gut, crosstalk between microbiota (commensal or pathogenic), immune cells, nerves, and potentially glia can enhance nociceptive signaling and activation of ‘silent’ nociceptors. In the spinal cord, enhanced pain signals can sensitize the dorsal horn neurons and there can be a loss of endogenous descending inhibitory signaling from the brain. Within the brain, chronic stress can remodel corticolimbic circuits that promote central sensitization and dis-inhibition (can inhibit descending inhibition to the dorsal horn of the spinal cord). While there are as of yet no definitive biomarkers for visceral pain, preclinical studies in animal models of visceral hypersensitivity have identified multiple potential factors that could represent therapeutic targets. Compounds that have received Food and Drug Administration (FDA) approval to treat IBS target guanylate cyclase-c receptors (linaclotide), serotonin receptors (alosetron, tegaserod), chloride channels (lubiprostone), opioid receptors (eluxadoline), and the microbiota (rifaximin) 26 . Animal models have provided evidence that growth factors (BDNF, GDNF, NGF), soluble neurotransmitters (NO, H 2 S), voltage gated ion-channels (Ca V , K V , Na V , HCN, TRPA1, TRPC4, TRPM8, TRPV1), ligand gated ion-channels (ASIC, P2X), GABA-ergic signaling, glutamatergic signaling, steroid hormones (estrogen, glucocorticoid, mineralocorticoid, progesterone), G protein-coupled receptors (α/β-adrenoceptors, CB, CRH, histamine, NK, NOP, P2Y), protease activated receptors (PAR), toll-like receptors, and cytokine receptors can also modify visceral sensitivity. Further, rodent models have shown that intracellular kinase pathways, mast cell stabilizers, microglia, and epigenetic modifications of DNA or histones also may affect visceral sensitivity. Additional studies of potential pathophysiological processes mediating visceral hypersensitivity have also implicated changes in tight junctions leading to changes permeability of the intestinal epithelium. Thus, there are multiple potential mechanisms within the gut, the spinal cord, and the brain that can lead to the chronic visceral pain experienced by IBS patients. Rodent models are most commonly employed to assess the pathophysiology of nociception and the development of new treatment approaches for patients with visceral pain. In 2019 a workshop entitled Critical Evaluation of Animal Pain Models for Therapeutics Development was funded by the National Institute of Neurological Disorders and Stroke (NINDS) who brought to Washington DC a team of experts in the pain models field. The participants were charged with providing a short list of models that could be used to screen and develop novel therapeutics that are devoid of abuse potential to treat pain. Although there exist over 500 different “models” of visceral pain in the published literature, only a few have found utility in more than one laboratory and even fewer have demonstrated both face value (looks like the disorder in humans) and predictive value in association with therapeutics. The visceral pain subgroup (headed by authors BGVM and TJN) focused on visceral pain that is either [i] unexplained pathologically, yet evident functionally or [ii] caused by persistent inflammation. Although models designed to mimic inflammatory bowel disease, IBS, bladder pain syndrome, pancreatitis, kidney stones and endometriosis were discussed, for purposes of the goals of the conference, the visceral pain subgroup settled on rodent models of colonic pain since those models had the greatest literature support for their use. It was immediately apparent that there are no rodent models that recapitulate the entire complex symptomatology of IBS. However, since the hallmark feature of IBS is visceral pain, a key requirement for the face value of the models is that they exhibit visceral hypersensitivity and express pain-like behaviors in response to luminal distention. Multiple secondary endpoints were also be considered (e.g., somatic hypersensitivity, GI transit and anxiety-like behavior). The visceral pain subgroup discussed multiple rodent models of visceral hypersensitivity induced following neonatal or adult stressors (acute or chronic, homotypic or heterotypic), enemas of irritants (dilute acetic acid, zymogen, deoxycholic acid), inflammation (or post-inflammatory), manipulation of central signaling, or from knockdown or knockout of genes. The reader is referred to a number of excellent and recent reviews describing in detail rodent models of visceral hypersensitivity 12 , 27 , 28 . The visceral pain subgroup considered that one model in particular was worthy of being considered for screening novel therapeutics: the intracolonic trinitrobenzene sulfonic acid (TNBS) pretreatment model. This recommendation is in contrast to recommendations of the 2016 National Institutes of Health (NIH) Functional Bowel Disorder workgroup whose goal was to identify animal models that could be employed for basic discovery into the etiology of IBS 15 , while the NINDs subgroup was tasked with identifying the best model for discovery of new therapeutics. Characterized in both male and female rats (as well as mice), the post-inflammatory TNBS model has shown reliability and reproducibility across multiple laboratories with high construct validity and translational relevance (i.e., it has been used to predict outcomes in human clinical trials), is relatively simple, and can be used for high throughput studies, and is applicable to a large segment of patients that develop IBS following an episode of enteritis 29 – 33 . For several weeks following intracolonic TNBS pretreatment, while active inflammation of the colon is present, visceromotor responses (VMRs; described more below) to colorectal distension (CRD) are enhanced. However, after a recovery period of 4–6 weeks, overt signs of inflammation disappear but hypersensitivity to CRD persists. There are statistically significant baseline differentials between pre- and post-TNBS VMRs at all CRD pressures ranging from 20 (non-noxious) to 60 (noxious) mmHg intensities and this preclinical model has already predicted clinical reversal of the pain state by active drugs 34 , 35 . Notably, the anti-hyperalgesic pharmacology associated with the early phases of the TNBS models (when persistent inflammation is still present) appear to differ from the anti-hyperalgesic pharmacology observed after colonic histology has returned to a normative state (that phase of the model most closely modelling IBS). The model has also been useful for reverse translational studies to investigate the mechanisms of colonic hypersensitivity 36 . Moreover, due to the female predominance of IBS, it was a consensus of the visceral pain subgroup that it is important to use female animals in any preclinical investigation of IBS. This issue is especially important since many IBS clinical trials enroll predominantly women and are underpowered to examine sex-related differences in the efficacy of a novel compound. We have found that reproducibility and rigor are improved by maintaining housing conditions that are stress-free, acclimating animals to the animal facility (7 days is recommended) and the laboratory (7 days is recommended). Performance of the experiments at a consistent time of day reduces diurnal variability. Although VMRs can be evoked in both anesthetized and unanesthetized states, we have found that performing experiments in their home cage gives more consistent results since it avoids a novel environmental stressor. Consistency in the age and rodent strain/source also limit variability. Also evident from the discussion by the NINDS task force was that in the development of novel therapeutics to treat chronic visceral pain, the efficacy of the compounds should be assessed in more than one model with etiologies that resemble specific groups of patients (e.g., for IBS, measures following adult stress or after exposure to early life adversity). That is, the TNBS pretreatment model is primarily useful for initial screening of analgesics for chronic visceral pain, but more disease-specific models should be tested prior to clinical trials in order to limit false predictions of efficacy that would be likely with use of a single animal model ( Box 1 ). Stress and negative emotions profoundly increase visceral pain in the absence of observable colonic injury. Multiple animal models have been developed that exhibit visceral hypersensitivity in adulthood in response to acute or chronic stressors. Although these models, including restraint stress (physical stress) and water avoidance (psychological stress) induce visceral hypersensitivity, they have low construct validity in that rodent and human stressors are quite different. Furthermore, both restraint and water avoidance stress, when repeated on a daily basis (usually 1-hr/day for 7–10 days), can lead to adaptation in certain strains of rat (e.g., Sprague Dawley strain) 37 . To address this, heterotypic stress paradigms have been introduced in which animals are randomly exposed to variable stressors (cold, restraint, water avoidance) to produce a persistent stress response along with an increase in colonic sensitivity 38 – 41 . Many IBS patients also report a history of early childhood adversity, including neglect, physical / verbal abuse and poverty. Animal models of early life stress have been developed including maternal separation (to model neglect), limited nesting (to model impoverished care) and odor attachment learning (to model attachment of an abusive caregiver). These early life pretreatments produce visceral hypersensitivity later in life and so have enhanced our understanding of the mechanisms underlying visceral pain, but they have low construct validity and their use as a first line screening model for future therapeutic development is not advisable since the paradigms employed are labor intensive and expensive due to the high cost of maintaining subjects until adulthood. To learn more about stress-induced visceral hypersensitivity and the use of specific rodent models, the reader is referred to a recent review on rodent models of stress-induced visceral hypersensitivity 28 . Commonly used endpoints to assess visceral sensitivity in experimental models are pseudoaffective responses, such as reflex changes in blood pressure or heart rate, or the aforementioned VMR to graded pressures of isobaric balloon distention in the distal GI tract. The same stimulus has been shown to produce pain in humans 42 , 43 . The technique of recording a VMR to CRD was originally described in 1988 44 and since then pseudoaffective responses to noxious stimuli have proven reliable in both rats and mice and are inhibited by known analgesics (e.g., opioids). Unfortunately, pseudoaffective responses are non-specific and affected by anesthesia. The most commonly method to measure VMRs in both rats and mice uses electromyography (EMG) electrodes surgically implanted onto the external oblique muscle. They are used to record the vigor and/or number of abdominal muscle contractions in response to graded CRD ranging from non-noxious to noxious intensities. Since the VMR to CRD can be performed in conscious rodents, it is possible to assess visceral sensitivity in freely moving animals to decrease the stress of the procedure. However, in our experience in mice, recording EMG activity requires some form of restraint to improve the electrical signal to noise ratio. In rats, an alternative to using surgically implanted EMG electrodes, is the direct observation of abdominal muscle contractions by an experienced investigator although this introduces a subjective component and may require observer blinding to group. Other laboratories have employed manometric techniques in freely moving animals to record pressures changes in the colorectal balloon as a measurement of visceral sensitivity 45 , 46 . Although this technique assesses visceral sensitivity and colonic motility simultaneously it has not been widely used by investigators in the visceral pain field possibly due to producing results such as stress-induced analgesia 45 , 47 that is seemingly contradictory to studies in rodents (and humans) using other endpoints such as the EMG demonstrating that stress induces visceral hyperalgesia 48 – 55 . While we do not know why manometry and EMG measurement of colonic sensitivity can produce different results in similar animal models, these studies highlight the need for carefully selected experimental controls that include factors such as surgical manipulation or chronic housing when using models of colonic hypersensitivity. Furthermore, some investigators have measured neuronal early-immediate gene expression (c-Fos and /or pERK) in the spinal cord in response to CRD. Although many non-nociceptive stimuli (stroking, hair brushing) produce a non-specific increase in c-Fos expression, pERK in the spinal cord is only activated in response to nociceptive stimuli 56 and offers an exciting alternative to complex electrophysiological recordings of colonic afferent nerve activity. As a minimum endpoint for screening efficacy, the NINDS task force recommended a statistically significant decrease in colonic hypersensitivity to less than 50% of the increase in responsiveness due to TNBS pretreatment. Linaclotide and 5-Hydroxytryptamine receptor 4 (5HT 4 ) agonists were deemed appropriate as active comparators for the late phase (no active inflammation, IBS-like) measures in TNBS pretreated rats. During active colonic inflammation, morphine was deemed an appropriate active comparator. For both phases, vehicle was deemed appropriate as an inactive control. Considering that chronic visceral pain is a major driving predictor of healthcare seeking and symptom chronicity, the development of novel therapeutic agents, based on validated animal models and assays, is central to improving outcomes in the patients with IBS. Multiple challenges exist in developing and using animal models to further understand the pathophysiology and treatment of visceral pain in IBS. The most significant challenges include [i] a complex clinical phenotype with symptoms that overlap with other disorders (bladder pain syndrome, chronic pelvic pain, anxiety, depression), [ii] symptom heterogeneity (pain with constipation, pain with diarrhea and pain with both diarrhea and constipation), [iii] lack of diagnostic biomarkers and [iv] a pathophysiology of IBS that is multifactorial and incompletely understood. IBS is also a disorder with a female-predominance and symptoms can be triggered by colonic inflammation and stress, whilst early life stress serves as a risk factor. Taken together these challenges make IBS a demanding condition to treat. We consider that additional basic and clinical research is essential to further develop our understanding of visceral pain in IBS. Examples of gaps in our knowledge include a lack of understanding of the communication between the brain-gut-microbiome axis, as well as the cellular mechanisms responsible for central and enteric nervous system plasticity following exposure to stress and inflammatory insults. Using the latest biological and neuroimaging tools, animal studies are poised to enhance our basic knowledge that will hopefully lead to the identification of validated biomarkers that will produce better treatment approaches for IBS patients with severe abdominal pain. A significant limitation of the experimental models of visceral hypersensitivity is that the pain behavior must be evoked and is not spontaneous. Even when hypersensitivity to CRD is known to exist, there is no evidence of spontaneous behavior. In particular, models of early-life stress that have been shown to cause epigenetic remodeling within the brain and spinal cord that persists for the life of the animal (and perhaps to subsequent generations) and yet do not have a measurable spontaneous pain phenotype. One question is whether a spontaneous behavior indicative of pain in rodents exists and is not being measured (a technical limitation) or if there truly is no spontaneous pain (a limitation of the model). Several assays have been developed to attempt to measure spontaneous pain in rodents with neuropathic or inflammatory pain, such as the grimace scale, conditioned place preference/aversion, and monitoring spontaneous home cage activity 57 . While the grimace scale is starting to be applied to studies of visceral pain in colitis models with active inflammation, this type of visceral pain does not model hypersensitivity in IBS patients, and measurements after resolution of the inflammation still require colonic distension to evoke a response. Conditioned place preference/aversion has been used for assessing spontaneous pain in models of neuropathy and to test efficacy of analgesics, but this technique has yet to be applied to studies of visceral hypersensitivity. Similarly, measuring spontaneous home cage activity has only found subtle changes in behavior in mice with a neuropathic hind limb. Thus, detecting spontaneous visceral pain in rodents is still a nascent technique that has not proven useful to evaluate novel analgesics. Hence, at present, using colonic distension to evoke nociceptive visceral responses appears to be the best primary technique to assess efficacy of novel analgesics since the paradigm mimics the quantitative measurements of visceral hypersensitivity in humans. Other models exist which use the acute administration of irritant to evoke “spontaneous” behavioral responses (e.g. writhing) but the inescapable, high intensity nature of the stimulus along with the poor construct validity of the method, limits their use as they do not appear to have any additional value (are still evoked) and have significant ethical concerns. While all currently approved drugs for IBS have been shown to be efficacious in animal models of visceral hypersensitivity, not all drugs that are effective in animal models have proven to be clinically effective. Given the large number of targets that have shown efficacy in animal models of visceral hypersensitivity, the mystery is why there continues to be so few therapeutic options for IBS patients. While specifics about animal models were discussed previously, one potential reason for the apparent efficacy of many diverse compounds is the reporting bias in scientific literature, such that ‘positive’ results are published while “negative” results are not 58 . Many of the animal studies used only a single model of visceral hypersensitivity, and frequently those studies evaluated only a single sex. As noted before these factors can limit the potential to demonstrate efficacy in clinical trials with diverse patient populations and often a female predominance. When working with animal models it is essential to adhere to the highest level of experimental design and scientific rigor to produce robust and reproducible data. There are obviously important measures that can be taken to prevent bias such as blinding, sample size calculations, randomization of experimental groups, the inclusion of male and female animals, and appropriate statistics. For purposes of reproducibility, publications should also always provide important information about the strain, sex and age of the animals, their housing conditions, and diet to ensure experimental reliability within and beyond a single laboratory.