Possible implications of animal models for the assessment of visceral pain

Abstract Acute pain, provoked generally after the activation of peripheral nociceptors, is an adaptive sensory function that alerts the individual to avoid noxious stimuli. However, uncontrolled acute pain has a maladaptive role in sensory activity leading to development of a chronic pain state which persists even after the damage is resolved, or in some cases, in the absence of an initial local acute injury. Huge numbers of people suffer from visceral pain at least once during their life span, leading to substantial health care costs. Although studies reporting on the mechanism of visceral pain are accumulating, it is still not precisely understood. Therefore, this review aims to elucidate the mechanism of visceral pain through an evaluation of different animal models and their application to develop novel therapeutic approaches for treating visceral pain. To assess the nociceptive responses in viscera, several visceral pain models such as inflammatory, traction, stress and genetic models utilizing different methods of measurement have been devised. Among them, the inflammatory and traction models are widely used for studying the visceral pain mechanism of different disease conditions and post‐operative surgery in humans and animals. A hapten, 2,4,6‐trinitrobenzene sulfonic acid (TNBS), has been extensively used as an inflammatory agent to induce visceral pain. The traction model seems to cause a strong pain stimulation and autonomic reaction and could thus be the most appropriate model for studying the underlying visceral pain mechanism and for probing the therapeutic efficacies of various anesthetic and analgesics for the treatment of visceral pain and hyperalgesia.

Animal models have played a pivotal role in our understanding of the mechanisms underlying the pathophysiology of visceral pain. 2 Over the years, a number of studies have investigated the visceral pain provoked by traction of visceral organs in various species.
Boscan et al 6  TNBS-induced ileitis goats to study VH and reported that electroacupuncture attenuates VH through down-regulation of spinal PAR-2 and CGRP. The persistent pancreatitis pain induced by injection of dibutyltin dichloride in the tail vein, along with 10% alcohol in drinking water, has also been investigated in rats. 13 A number of studies have elucidated the association of stress with a lowered pain sensation thresholds and enhanced pain perception that parallels the altered brain circuit activity. 14 During stress, a neuroendocrine hormone, ie a corticosteroid, is secreted in response to activation of the hypothalamic-pituitary-adrenal axis that not only acts at mineralocorticoid and glucocorticoid receptors throughout the body, but also promotes enhanced sensitivity of neurons to both noxious and innocuous stimuli, which in turn promotes the development of chronic pain. 15 Zheng et al 16 reported that chronic stress induces visceral as well as somatosensory hyperalgesia in a chronic, intermittent stress rat model. The epigenetic modulation of gene expression could be responsible for the mechanism underlying the persistent effects of stress on visceral sensitivity. A growing line of evidence shows that changes in DNA-methylation and histone-acetylation patterns within the brain and spinal cord of rats result in alterations in nociceptive signaling via increased expression of pro-and anti-nociceptive gene expression. 17,18 Among the various animal models for studying visceral pain, the traction model seems most effective for studying the underlying mechanism of visceral pain and drug efficacy because it results in direct activation of the nociceptors and the signal that consequently releases histamine from the mesenteric mast cells is also stronger. 6,19,20 The abdomen is the site most prone to both acute and chronic painful syndromes resulting from visceral diseases, referred pain coming from adjacent structures, and/or systemic injuries. 21 The various diseases or disorders of abdominal and uro-genital organs that result in visceral pain include obstruction, intussusception, neoplasms, volvulus, extra-luminal compression, intra-abdominal abscess, celiac disease, typhlitis, intestinal or biliary colic, gall stones, incarcerated or strangulated hernia, intestinal and mesentery infarcts, ovarian cysts, tumors, endometriosis and urolithiasis. Besides these, surgeries of the gastrointestinal tract and pelvic organs also produce visceral pain in animals and human beings.
This review of visceral pain research outlines the mechanism of visceral pain provoked by inflammation, stress, genetic and repetitive stimulation of visceral organs in various animal models.

| NATURE OF VISCER AL PAIN
Visceral pain is the complex sensory experience arising from the viscera of the abdominal, thoracic, and pelvic cavities. We experience visceral pain because damaged or injured internal organs and tissues activate pain receptors. The pain may be accompanied by symptoms such as nausea, vomiting, changes in vital signs and emotional manifestations. There is no clear demarcation between the acute and chronic states of visceral pain.
Acute pain has an adaptive sensory function as it alerts the individual to avoid noxious stimuli, 20 but uncontrolled acute pain has a maladaptive role in the sensory activity leading to development of a chronic pain state. In general, acute pain persists for a short time after injuries or inflammation. However, Kruszka and Kruszka 22 reported that acute pelvic pain originating from the lower abdomen or pelvis may persist, alarmingly, for three months. Different pathological conditions such as ectopic pregnancy, hematoma of corpus luteum, ovarian cyst, ovarian tumor, ovarian torsion, acute salpingo-oophoritis, fibroids, uterine cancer, cervical cancer, ovulation, appendicitis, fracture of pelvis and hernia provoke an intense acute pelvic pain which is challenging to investigate because the many symptoms are subtle and non-specific. Some of these conditions may be life-threatening (ectopic pregnancy, appendicitis and ruptured ovarian cysts) or may threaten fertility (pelvic inflammatory diseases, ovarian torsion). 22 On the other hand, chronic visceral pain persists much longer, sometimes over a lifetime. [22][23][24][25] It is a symptom of several diseases and disorders but not, in and of itself, a disease process.
Visceral nociceptors do not have specialized receptors and neurotransmitters. Moreover, the neuroanatomy of visceral nociception -the neurotransmitters, receptors and ion channels that modulate visceral pain -is qualitatively and quantitatively different from that of the systems modulating somatic and neuropathic pain. 1 Therefore, various animal models have been devised to study the mechanism underlying diseases causing visceral pain. It has been supposed that pain mechanisms serve as a natural protective mechanism against noxious stimuli by changing the physiology and behavior to reduce or avoid further damage, and promote recovery. 20 A better understanding of the mechanism of peripheral and central sensitization can help to explain why prevention of sensitization is critical to recovery and will illuminate the multiple stages in the process that could be altered by different analgesic therapies. Moreover, the effective management of pain after surgery is a major welfare issue in human and animal practice. Management of acute postoperative pain remains suboptimal because nearly 80% of patients report subjectively moderate to extreme pain following surgery. 26 Despite the various studies aimed at unraveling the mechanism of visceral pain, it is still less well understood than that of somatic pain. The reason may primarily be the diverse nature of visceral pain, compounded by multiple factors such as sexual dimorphism, psychological stress, genetic traits, and the nature of predisposing disease. These multiple contributing factors create a huge challenge for the development of an ideal animal model that closely mimics the disease condition being investigated. Over the past few decades, substantial numbers of animal models of visceral pain have been generated to improve management of pain and our understanding of the underlying mechanisms of visceral pain.

| MECHANIS M OF VISCER AL PAIN
Noxious stimuli including inflammatory agents and mechanical stimuli activate the nociceptors connected to unmyelinated C-fibers and thinly myelinated Aδ-fibers via specific receptors or ion channels sensitive to mechanical stimuli or inflammatory agents. 27 Damaged tissue and inflammatory cells release different chemical mediators that activate or modify the stimulus response properties of nociceptors. The underlying mechanism of visceral pain and hyperalgesia is illustrated in Figure 1. Primary hyperalgesia, which occurs at the site of injury, is the consequence of increased input from nociceptors sensitized by the stimulus. These stimulated nociceptors connected to Aδ-and C-afferent fibers transmit the amplified sensory discharges to the CNS, which results in an increase in the pain originating from the primary hyperalgesic area, called secondary hyperalgesia. Pain pathways (P) become activated in the CNS which stimulates tactile receptors connected to Aβ-afferents leading to the activation of tactile pathways (T). 28 The consequence of amplification of the nociceptive input from the injured area is access to pain neurons (P) resulting into touch-evoked pain called allodynia. 29

| Peripheral sensitization
The physiology of pain in animals and humans involves the peripheral process of detecting a noxious stimulus (mechanical, thermal or chemical) and transmission of the impulses to the spinal cord. They are modulated and projected to the brain for central processing of the information, which determines the perception of the noxious stimulus. 28 Except for nociceptive pain, all other types of pain are considered as clinical or pathological, often involving tissue damage with inflammation or nerve damage. The Aδ-and C-fibers are the two types of sensory fibers conducting most of the nociceptive signals to the SDH, while the large Aβ-fibers transmit other sensory information (pressure, touch, and vibration) to the CNS. They do not transmit pain impulses, but work in the spinal cord to modulate painful stimuli. 28,29 Aδ-fibers are associated with intense and pricking pain, and rapidly conduct impulses (5-20 m/s) while C-fibers are associated with dull, burning pain and slowly conduct impulses (0.5-1 m/s). 20 Both types of fibers innervate skin and deep somatic/ visceral structures but differ in their ratios. The reported ratio of Aδ-to C-fibers is 1:1-2 in cutaneous nerves, and 1:8-10 in visceral nerves. Peripheral sensitization occurs when inflammation at the site of injury creates an increased response to a normally painful stimulus. These sensitized nociceptors evoke a stronger response to any given stimulus than in the normal state and their thresholds may be reduced such that even innocuous stimuli can activate them. 20 Additionally silent nociceptors, not activated in the normal state, act as noxious stimuli to cause hyperalgesia and allodynia. In addition to sensitization at the peripheral tissue and pain receptors, the CNS is also sensitized.

| Central sensitization
Animal studies involving direct electrophysiological recordings from spinal neurons suggest that central sensitization may also be an important mechanism in generating VH and pain. Central axons of firstorder neurons synapse onto second-order neurons in the SDH. They terminate predominantly in laminae-I, II, and V of the SDH on projection neurons and local interneurons. 33 Laminae-I and -II receive direct primary afferent inputs from Aδ-and C-fibers. 28  Aδ-, C-and Aβ-fibers. 28,34,39 Consequently, WDR neurons can play a role in the segmental suppression of pain. 40 Primary (first-order) neurons project from here to second-order neurons in supraspinal centers in the ascending pathway of the dorsal horn of spinal cord which convey stimuli transduced by nociceptors to the brain-stem, thalamus, limbic structure, and finally to neo-cortex. 34

| ME A SUREMENT OF VISCER AL PAIN IN L ABOR ATORY ANIMAL S
Quantitative measurement of pain severity, especially in non-verbal animals that are used as disease models, can be difficult to obtain, but several developments in behavioral neuroscience are making the measurement of pain more consistent, automated and accurate. 41 Some of the objective methods of visceral pain measurement used in different visceral pain models and experimental conditions in rodents have been summarized in Table 1.

| Dynamic weight bearing
Dynamic weight bearing (DWB) is a non-subjective and non-reflexive method for the evaluation of inflammatory-driven abdominal pain in a mouse model. 42 Some researchers have used the DWB test as a tool for measuring non-evoked inflammatory hyperalgesia in a mouse model, 43 and it has also been shown to be an effective, objective and predictable test for studying both the pathophysiological mechanisms involved in arthritic nociception in mice and for evaluating novel analgesic drugs against arthritis. 44

| Grimace scale and burrowing
The grimace scale, originally developed to assess the pain in nonspeaking humans, was recently validated for pain assessment in several laboratory, farm and exotic animals (mouse, rat, rabbit, horse, cat, cattle, sheep, pig, ferret and seal). 45 The rat grimace scale (RGS) rates pain from 0 (no pain) to 2 (severe pain) based on changes in four facial expressions, ie orbital tightening, nose/cheek flattening, ear changes and whisker changes. 46 Langford et al 47 did not observe grimacing in acute (<10 minutes) and chronic (>1 day) contexts, but subsequent researchers have reported seeing changes in facial expression lasting only a few seconds to minutes or extending to several months post injury. 45 The feline grimace scale, however, includes five action units -ear position, orbital tightening, muzzle tension, whisker change and head position. The scores using this scale were reported higher during naturally occurring acute pain than in control cats. 48 A recent study in an acute and chronic colitis model of rat showed that RGS increased during both acute and chronic phases but burrowing only decreased during the acute phase and suggested using RGS as an pain scale and welfare improvement tool. 49 Rater training is mandatory for performing grimace scale research. At a minimum, observers need some years of species-specific working experience to score the pain efficiently using the grimace scale. Burrowing, a spontaneous and self-motivated behavior, can be used as a measure of spontaneous or stimulus-evoked nociception in mice and rats. 50 Burrowing behavior is displayed by all rodents and represents another very easy way to quantify the quality of life, as one simply needs to measure the weight of the burrowing material (eg gravel) at the beginning and end of any desired time period. 45 This behavior has been shown to be disrupted by different visceral nociceptive processes such as laparotomy, 51 nerve injury 52 and colitis. 53

| Telemetry
During animal experimentation, even when a person is simply present in a room, a mouse may hide the signs of pain, making monitoring of low to moderate pain very difficult. 56 To overcome such problems, telemetry has been used, which allows monitoring without the presence of the investigator in the vicinity of the animal. Telemetry has been established to record physiological parameters such as heart rate, core body temperature, or blood pressure in mice. 57 Nijsen et al 58 used it as an appropriate tool for measuring the visceromotor and cardiovascular responses to averse, noxious duodenal distension continuously and simultaneously in the rats kept in a home cage, without additional handling-related or restraint-induced stress.

| Manometry
Manometry measures the pressures and the pattern of muscle contractions, particularly in the hollow visceral organs. Esophageal manometry is used to diagnose the conditions due to abnormalities in the contractions and strength of the esophageal muscle or in the sphincter at the lower end of the esophagus that results in pain, heartburn, and dysphagia. It is also used to study the visceral pain induced during CRD in irritable bowel syndrome (IBS) and the efficacy of different analgesics. 62 The applicability of noninvasive, surgery-free manometry of intracolonic pressure for assessing VMRs to CRD in mice has been reported. 64 The effects of transcutaneous electrical nerve stimulation (TENS) on esophageal motility and pain sensitivity were assessed in graded intraesophageal balloon distended human patients using computerized esophageal manometry and it was reported that TENS attenuates noncardiac chest pain of esophageal origin. 65

| Pain biomarkers
Proposed biomarkers of pain include body weight decreases and autonomic nervous system manifestations such as pupil dilation, blood pressure, blood flow, respiration, heart rate, skin conductance, fecal corticosterone, plasma norepinephrine, and heart rate variability. 45 Prostaglandin E2 (PGE2) is the predominant eicosanoid released after surgical trauma and has been associated with inflammation, pain, and fever, which result from the action of PGE2 on peripheral sensory neurons and on central sites within the spinal cord and brain. 66 PGE2 increases thermal and mechanical hypersensitivity, 67 exerting its actions by acting on a group of G-protein-coupled receptors (GPCRs). c-Fos and pERK can be used as markers for neuronal activation following noxious stimulation and tissue injury. 68

| Electrophysiology
The study of the electrical activity of nociceptors using exquisite

| Inflammatory pain models
The objective of this model is to provoke a painful condition that mimics the natural inflammation induced by chronic visceral pain.
Inflammatory pain is a big health concern causing suffering to millions in both humans and animals, especially during chronic inflammatory bowel diseases. The inflammatory pain model has helped scientists to understand the underlying mechanism of inflammatory pain and evaluate potential treatments.  TNBS diluted in ethanol is used as a hapten to disrupt the mucosal barrier of hollow visceral organs. 75 It has been regarded as a model for the study of ulcerative colitis and CD. TNBS/ethanol solution was injected into the ileal lumen of guinea pigs to elucidate the pathophysiology of ileal disease and the morphological and functional changes in neurons projecting to the ileal mucosa in the early stage after inflammatory damage. 76 Like TNBS, zymosan, an insoluble carbohydrate from the yeast cell wall, has also been used to induce colitis for studying the association of short-term sensitization of mechanoreceptors with long-term hypersensitivity to colon distention in the mice. 8 However, the severity of inflammation is milder compared to that caused by TNBS and generally does not produce mucosal ulceration.
An experimental model for the study of visceral pain associated with urinary bladder inflammation is developed by intravesicular injection of 0.2% acetic acid, 77 zymosan 9 or acrolein. 78 Initially, the intraperitoneal injection of cyclophosphamide (an antitumor agent) was employed to produce bladder inflammation-associated visceral pain, 79 but although the cyclophosphamide produces selective cystitis via its metabolite, acrolein, in the bladder, it has a severe systemic toxic effect that can complicate the evaluation of bladder pain.

| Traction pain models
Traction is the act of drawing or pulling the tissues or organ containing nociceptors from its normal position. It is important to note that the visceral organs are highly sensitive to traction, distension, ischemia and inflammation, which ultimately evoke visceral pain.   responses of peripheral pain regulatory pathways in a rat model of chronic, intermittent stress and reported that chronic stress induces both somatosensory and visceral hyperalgesia due to differential changes in endovanilloid and endocannabinoid pathways, and sodium channels in dorsal root ganglions innervating the lower extremities and pelvic viscera. Asano et al 84 reported that oral administration of aminophylline supressed the VH provoked in maternally separated rats, acetic acid-induced colitis rats and wrap restraint stress rats. The stressors/gene knockout, purpose of study and key results of different stress and genetic models for studying the underlying mechanism of visceral pain have been summarized chronologically in Table 4. Knockout models provide the opportunity to investigate the role of a specific gene in the regulation of colonic visceral sensitivity. Over the years, several studies have employed knockout models to unravel the gene-specific underlying mechanism of VH. Earlier studies 84,85 reported that knockout of corticotropin-releasing factor-1 receptor attenuates the VH in response to different CRD pressures in mice. It is well known that the voltage-gated sodium channel subtype Na V 1.7 is a prerequisite for sensing acute and inflammatory somatic pain in mice and humans. However, its involvement in pain originating from the viscera is still poorly understood. Hockely et al 86 investigated the role of Na V 1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor-specific Na V 1.7 knockout mouse and reported that Na V 1.7 knockout did not lead to any differences in pain-related responses and referred hyperalgesia to noxious mechanical and chemical stimuli in nerve-gut preparations in mouse.