TY - JOUR
AU - PhD, Robert J. Callister,
AB - Background Abdominal pain frequently accompanies inflammatory disorders of the gastrointestinal tract (GIT), and animal models of GIT inflammation have been developed to explore the role of the central nervous system (CNS) in this process. Here, we summarize the evidence from animal studies for CNS plasticity following GIT inflammation. Methods A systematic review was conducted to identify studies that: (1) used inflammation of GIT organs, (2) assessed pain or visceral hypersensitivity, and (3) presented evidence of CNS involvement. Two hundred and eight articles were identified, and 79 were eligible for analysis. Results Rats were most widely used (76%). Most studies used adult animals (42%) with a bias toward males (74%). Colitis was the most frequently used model (78%) and 2,4,6-trinitrobenzenesulfonic acid the preferred inflammatory agent (33%). Behavioral (58%), anatomical/molecular (44%), and physiological (24%) approaches were used alone or in combination to assess CNS involvement during or after GIT inflammation. Measurement times varied widely (<1 h–> 2 wk after inflammation). Blinded outcomes were used in 42% studies, randomization in 10%, and evidence of visceral inflammation in 54%. Only 3 studies fulfilled our criteria for high methodological quality, and no study reported sample size calculations. Conclusions The included studies provide strong evidence for CNS plasticity following GIT inflammation, specifically in the spinal cord dorsal horn. This evidence includes altered visceromotor responses and indices of referred pain, elevated neural activation and peptide content, and increased neuronal excitability. This evidence supports continued use of this approach for preclinical studies; however, there is substantial scope to improve study design. spinal cord, dorsal horn, colitis, pancreatitis, central sensitization Severe abdominal pain and visceral hypersensitivity are debilitating symptoms of inflammatory disorders of the gastrointestinal tract (GIT), such as inflammatory bowel disease (IBD) and pancreatitis.1,–4 GIT inflammation, however, does not seem to be the sole factor responsible for pain in patients with these conditions. This is based on the observation that 30% to 50% of IBD patients in clinical remission continue to experience abdominal pain,5,–7 which is often severe enough to warrant ongoing narcotic medication.8,–10 Similarly, patients with chronic pancreatitis frequently suffer abdominal pain despite successful endoscopic or surgical interventions.11,12 Recent articles have highlighted major issues in chronic pain management in these distinct conditions, including the lack of effective pharmacological intervention and decreased patient’s quality of life because of the development of depression and anxiety.7,13,–15 Interestingly, pancreatitis and other heptopancreatobiliary disorders are common extraintestinal manifestations of IBD.16,17 Therefore, there is a need to improve our understanding of the relationship between GIT inflammation and pain. The mechanisms responsible for the development of chronic abdominal pain as a consequence of inflammation in the GIT are still largely unknown. Clinically, there is evidence that altered central nervous system (CNS) function (plasticity) plays a role in the development of the chronic pain state that can accompany IBD and pancreatitis.13,18,–20 A large proportion of IBD patients in clinical remission not only continue to experience chronic abdominal pain without active inflammation in the gut5,–7 but also frequently experience “referred pain,” whereby poorly localized pain from the viscera is referred to other body locations.20,–24 This phenomenon is thought to occur when there is overlap between visceral and somatic sensory pathways within the CNS.25 Similarly, reorganization of CNS pathways has been documented in patients with chronic pancreatitis.4 Therefore, CNS dysfunction seems to play an important role in both the development and maintenance of chronic pain in inflammatory conditions of the GIT. In response to this need for a better understanding of the central mechanisms underlying pain associated with visceral inflammation, animal models have been developed to mimic IBD, pancreatitis, and other painful conditions of the GIT. These models generally involve chemically induced inflammation of a target organ and subsequent assessment of CNS properties/function during acute inflammation or when inflammation has resolved. In this review, we summarize the current preclinical evidence for altered CNS function in animal models of visceral pain as a consequence of GIT inflammation. This review highlights a number of areas where study design could be improved to enhance the integration of findings across studies and laboratories. Finally, we identify a number of areas where additional data are required to improve our understanding of the central mechanisms that underlie inflammatory-mediated GIT pain. Methods Literature Search A systematic literature search was conducted using Ovid SP databases (Embase and Medline). The details of our search strategy are provided in Table 1. Once the search was completed, duplicates were removed, and abstracts of the remaining articles were examined. Retrieved records were screened for relevance before inclusion. Articles that did not fit the inclusion criteria were noted but not analyzed further. Table 1. Search Strategy for Ovid SP Databases Using the Advanced Search Function     View Large Table 1. Search Strategy for Ovid SP Databases Using the Advanced Search Function     View Large Inclusion Criteria Original research articles were included based on the following criteria: (1) the animal model involved inflammation, (2) inflammation occurred in at least in one of the following organs of the GIT: esophagus, stomach, liver, pancreas, gallbladder, appendix, and small or large intestine, (3) studies demonstrated the presence of pain, or visceral hypersensitivity, or examined factors that could contribute to pain or visceral hypersensitivity, and (4) the measures used were considered evidence of CNS involvement in visceral pain processing. All searches were limited to English-language journal articles published before July 12, 2012. Exclusion Criteria Review articles were excluded, although their reference lists were searched to identify additional studies that met the inclusion criteria. Other articles excluded were dissertations and conference proceedings, studies of cancer or neuropathic pain (i.e., where inflammation is not the sole cause of “pain”), and studies using the acetic acid writhing test, as this test is not organ specific. Study Selection Two investigators (Farrell and Callister) independently evaluated abstracts of identified articles according to the selection criteria, and potentially relevant articles were retrieved in full. In cases of initial disagreement on an article’s eligibility, inclusion was decided after discussion leading to consensus between investigators. Initial agreement between investigators on inclusion of articles was assessed using percentage agreement and the kappa statistic. Data Extraction The following data were extracted from the included studies: authors, year of publication, animal strain, animal age and sex, GIT section/organ that was inflammed, method of inflammation (including inflammatory agent, route of administration, and dose), time points at which measurements were made (h/d/wk postinflammation), techniques used to measure effects of inflammation on CNS function, and the outcomes of these measures. In studies with multiple interventions, only data from uninflamed (control) and inflammed groups were considered for analysis. Methodological Quality Assessment of Studies We developed a 10-point checklist to assess the bias of reporting and quality of study design (Fig. 2). These 10 points were developed to evaluate key aspects of study design, such as randomization of animals into experimental groups, and to assess how thoroughly study objectives were reported and measured. Studies were deemed to be of higher quality if they reported randomization of animals into experimental groups, blinded analysis, and objective evidence of visceral inflammation (via histology, Evan’s blue extravasation, and/or myeloperoxidase analysis). Results Selection of Studies The process followed for article selection is presented in Figure 1. A total of 208 articles were identified. Of these, 112 were excluded because they did not meet the inclusion criteria or contained aspects of the exclusion criteria, whereas a further 33 were excluded because they were reviews. A total of 79 articles were eligible for detailed analysis, including an additional 16 from review article reference lists. There was a high level of agreement on inclusion/exclusion26,27 between the 2 investigators who screened the retrieved articles (percent agreement = 0.92, kappa score = 0.82). Figure 1. View largeDownload slide Flow diagram for literature searching and screening. Two hundred eight articles were identified using Embase and Medline databases. Articles were included/excluded based on relevance, resulting in the inclusion of 63 articles. Reference lists of reviews were also scanned, leading to inclusion of a further 16 articles. A total of 79 articles were included for further analysis. Figure 1. View largeDownload slide Flow diagram for literature searching and screening. Two hundred eight articles were identified using Embase and Medline databases. Articles were included/excluded based on relevance, resulting in the inclusion of 63 articles. Reference lists of reviews were also scanned, leading to inclusion of a further 16 articles. A total of 79 articles were included for further analysis. Characteristics of Included Studies The characteristics of studies that identified and investigated CNS involvement in the processing of GIT inflammatory pain were examined to determine whether commonality existed in study design and outcomes. These study characteristics are shown in Tables 2 and 3. Rats were the most widely used species in models of GIT inflammation (76%), followed by mice (22%), cats (1%28), and rabbits (1%29). Visceral inflammation was induced in animals considered to be adults (by the authors) in 33 studies (42%) and in neonates in 1 study (1%30). Surprisingly, 45 studies (57%) reported animal weight but not age. There was a sex bias across studies, where 58 studies used males (74%) and females were used in 12 (15%). Eight studies (10%) used both sexes, and sex was not reported in 1 study (1%29). Table 2. Characteristics of Colon Studies                         View Large Table 2. Characteristics of Colon Studies                         View Large Table 3. Characteristics of Pancreas and Other Viscera Studies             View Large Table 3. Characteristics of Pancreas and Other Viscera Studies             View Large Colitis was the most frequently used model of visceral inflammation (78%), followed by pancreatitis (19%). By comparison, inflammation of the esophagus (1%28), stomach (1%31), and gallbladder (1%29) were rarely used. A variety of chemical agents have been used to induce inflammation (Tables 2 and 3). Trinitrobenzene sulfonic acid (TNBS) was most commonly used in models of colitis (33%) and cerulein (29%) in models of pancreatitis. Other inflammatory agents used included mustard oil, zymosan, turpentine, and acetic acid. Finally, the time points after the induction of inflammation at which outcome measures were made varied widely from <1 hour (22%), 1 to 24 hours (31%), 2 to 7 days (23%), 8 to 14 days (11%), and >2 weeks (13%). Therefore, there is great variance in both the characteristics of the models of gastrointestinal inflammation and the time points after the induction of inflammation at which measurements of CNS properties were made. Methodological Quality/Risk of Bias The methodological quality of included studies was assessed using our 10-point checklist, and these results are shown in Figure 2. Only 10% of studies reported randomization of animals into experimental groups.31,–38 Blinded outcome measures were undertaken in 42% of studies, and objective evidence of visceral inflammation (histology, myeloperoxidase assay, or Evan’s blue extravasation) was obtained in 54% of studies (Tables 2 and 3). Two studies did not report the dose of the inflammatory agent used: 1 of these modeled pancreatitis39 and the other cystic pain.29 No study reported sample size calculations. Figure 2. View largeDownload slide Methodological quality of included studies. A 10-point checklist was developed to assess study-design quality of the included articles. Dark gray bars indicate the proportion of articles that met each criterion; light gray bars indicate the proportion of studies that did not. Numbers of studies that meet each criterion are shown on each bar. Figure 2. View largeDownload slide Methodological quality of included studies. A 10-point checklist was developed to assess study-design quality of the included articles. Dark gray bars indicate the proportion of articles that met each criterion; light gray bars indicate the proportion of studies that did not. Numbers of studies that meet each criterion are shown on each bar. Only 3 studies (4%) were considered to be of high methodological quality according to our criteria33,34,37 (see Methods: Methodological Quality Assessment of Studies). Each of these 3 studies used models of colitis. Two studies used the chemical dextran sodium sulfate as an inflammatory agent and examined the long-term effects of visceral inflammation (49 d after dextran sodium sulfate withdrawal) on neural activation in the spinal cord and referred hypersensitivity.33,34 The immediate early gene product, c-Fos, was used as a marker of neural activation. The third study used the TNBS model of colitis and measured the visceromotor response (VMR) and referred bladder hypersensitivity 1 week after induction of inflammation.37 Evidence for Altered CNS Function as a Measure of Gastrointestinal Pain The experimental approaches used to assess altered CNS pain processing mechanisms in the identified studies can be divided into 3 major categories: behavioral, anatomical/molecular, and physiological. Behavioral Approaches In preclinical models of visceral inflammation, confirming that an animal is in pain or at least exhibits visceral hypersensitivity is essential before mechanisms that drive pain can be explored. This is often inferred on the basis of patterns of behavior. Of the included studies, 58% examined behavioral responses after visceral inflammation. These behaviors include the VMR and measures of referred pain via sensory threshold testing (e.g., von Frey hairs). Ness and Gebhart40 have published a detailed description of the VMR. The VMR is a reflex that acts via a spinal cord–brainstem–spinal cord loop and, therefore, implicates the CNS in the regulation of involuntary behavioral responses to pain.40,41 Only 29% of included studies assessed VMR after visceral inflammation, and 21 of 28 of these studies were undertaken in models of colitis,25,30,42,–60 where the VMR was elicited by colorectal distension (CRD). Each study reported an increase in the magnitude of responses to distension and/or a decrease in the distension pressure required to elicit a given response amplitude/intensity during or after visceral inflammation.25,36,42,–48,52,53,55,–63 Two studies used a model of gastric inflammation31 and recorded spontaneous abdominal contractions during pancreatitis as an index of pain.64 Pain originating in viscera is often “referred” to other body regions, such as skin or even other viscera.21 Such referred pain is thought to occur because of “overlap” between visceral and somatic sensory pathways within the CNS.25,62,65 Referred pain after visceral inflammation was measured in 23% of the included studies. Models of colitis were used in 10% studies, and most (7%) measured paw and/or abdominal hypersensitivity via von Frey or heat withdrawal latency. The remainder examined referred hypersensitivity of the bladder66 or urethral sphincter hypersensitivity during bladder distension.67 The noncolitis studies (13%) assessed referred hypersensitivity, via von Frey or heat beam application to the abdomen, in models of pancreatitis. Von Frey testing has consistently shown that animals “withdraw” their limbs at lower thresholds during inflammation and after recovery from both colitis and pancreatitis.32,–34,61,–63,68,–79 In contrast, responses to plantar heat are more varied, with some studies reporting a decrease in paw withdrawal latency (i.e., increased sensitivity),36,62,80 no change,61 or even an increase in latency (decreased sensitivity).81 Similarly, assessments of viscerovisceral cross talk in models of colitis have shown reduced micturition latency37,62,66 and increased urethral sphincter responses to electrical stimulation.67 Both are indicative of bladder overreactivity. Only 6% of studies used a combination of VMR and referred pain testing to assess CNS involvement after inflammation.36,37,61,–63 The data from these studies did not differ from single-assessment approaches and are included in the above description. Anatomical/Molecular Approaches It is clear that visceral inflammation can result in long-term abdominal pain and referred hyperalgesia in both experimental and clinical settings.20,62,63,73,82,83 However the precise mechanisms underlying these changes are poorly understood. It has been suggested that remodeling of CNS nociceptive networks is largely responsible,84,–86 although, until recently, this has been relatively unexplored in models of visceral inflammation. Immunohistochemistry (IHC) has been used to investigate the properties of spinal cord and brainstem neurons during and after visceral inflammation.29,33,–35,45,46,48,63,64,68,75,77,79,80,86,–97 This approach is based on the premise that structural remodeling of neural networks in the CNS underlies the exaggerated reflex behaviors observed after visceral inflammation.84,–86 Similarly, molecular analyses have been used to investigate altered expression of key proteins and signaling molecules in the CNS after visceral inflammation. Markers of neural activation Markers of neural activation in the spinal cord and brainstem during visceral inflammation are commonly used to evaluate CNS involvement in pain processing. Two markers are widely used: c-Fos, and phosphorylated ERK (pERK).98 c-Fos or pERK98 IHC were used in 23% studies to assess CNS involvement in nociceptive processing after visceral inflammation. Most used c-Fos (20%), and expression of either marker was consistently increased.29,33,–35,45,63,64,68,80,86,–88,90,92,–94,96,97 Neural activation was maximal in spinal cord segments T13–L1 and L6–S2 in models of colitis,33,–35,45,48,63,86,87,93,94,96,97 T3–L1 in pancreatitis,64,68,80,88,90,92 and T6 for gallbladder inflammation.29 Labeling was observed in laminas I to VII and/or X, reflecting the diffuse termination of visceral afferents in the spinal cord compared to their cutaneous counterparts.29,33,–35,45,63,64,68,80,86,–88,90,92,–94,96,97 c-Fos labeling was also elevated in brainstem regions, such as the periaqueductal gray, dorsal raphe nucleus, pontine parabrachial nucleus, locus coeruleus, and the nucleus of the solitary tract after colonic93 and pancreatic80 inflammation. These nuclei are well-known nociceptive centers, which provide descending inhibition to the spinal cord.99 Gene or protein expression of c-Fos and pERK in spinal cord homogenates were measured in 6% studies after visceral inflammation.29,49,–51,69 Three studies49,–51 observed increased c-Fos mRNA in models of colitis, whereas 2 studies29,69 reported increased pERK mRNA and protein levels after colitis69 or gallbladder inflammation.29 Notably, 1 study showed that increased nuclear expression of pERK was accompanied by the development of referred pain after colitis.69 c-Fos and pERK IHC were combined with neuroanatomical tracing in 2 studies to identify the specific neural populations/pathways activated by visceral inflammation.86,97 Colonic central afferent terminals were retrogradely labeled and identified along with pERK in the dorsal horn after colitis and CRD. The density of adjacent labeled terminals and pERK-positive neurons in the dorsal horn increased and labeling expanded from superficial to deep dorsal horn layers after inflammation.86 Similarly, spinal neurons projecting to the parabrachial nucleus were retrogradely labeled with fluorogold before colitis and CRD. Fluorogold and c-Fos double labeling was then examined in spinal cord segments known to receive colonic inputs. Notably, double-labeled neurons were more prevalent (increased by 15%) in thoracolumbar versus lumbosacral spinal cord. This suggests thoracolumbar segments play an important role in the processing of visceral inflammation.97 Markers of pain-associated neuropeptides The neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the SP receptor (neurokinin [NK1]), have long been associated with the induction and maintenance of various chronic pain states73,100,–105 and transmission of nociceptive signals to higher centers along the pain neuroaxis.106,107 The expression of SP and CGRP were examined in 6% of studies using IHC.35,77,79,86,93 Increases in the density and intensity of SP and CGRP labeling in the spinal cord dorsal horn have been documented in models of colitis35,86,93 and pancreatitis.77,79 Furthermore, increases in SP and CGRP coexisted with increased pain behaviors in 4% studies,77,79,93 suggesting a role for these neuropeptides in the development of long-term and referred hyperalgesia. Similarly, spinal cord expression of the NK1 receptor was examined in 3 studies89,91,95 in models of colitis. This work showed that both NK1 receptor-expressing neuron incidence and NK1 receptor internalization increased. Such internalization is considered an indicator of neuronal activation.89,91 Similarly, de novo expression of the NK1 receptor in dorsal column projection neurons was observed after colitis.95 Spinal cord mRNA or protein expression of neuropeptides and inflammatory markers were examined in 5%49,51,108,109 studies using colitis. Increased mRNA and protein expression of SP108,109 and NK1 and NK2 receptors49,51 have been documented after colitis. Similarly, CGRP expression was examined in 4 studies. Increases,49,108 decreases,109 or no change51 in expression were reported. Spinal cord mRNA expression of inflammatory markers, constitutive and inducible nitric oxide synthases (NOSs), interleukin-1β, tumor necrosis factor-α, and cyclooxygenase 1 and 2 were increased in 2 studies49,51 after the resolution of colitis. Two studies also showed a correlation between increases in neuropeptides and inflammatory markers and enhanced behavioral responses to CRD.49,51 Finally, 1 study109 examined SP and CGRP expression in the bladder after colonic inflammation. Increased SP and CGRP mRNA and protein were reported in the bladder for up to 30 days after the induction of colitis,109 and this was associated with neurogenic inflammation of the bladder. Other factors contributing to CNS processing of visceral pain Additional anatomical changes within the CNS after visceral inflammation have been explored in a further 5 studies (6%).46,48,75,86,110 Two of these examined spinal cord expression of neuronal NOS and NADPH to investigate the role of the NO cascade after colitis.46,48 Increased neuronal NOS and NADPH expression was shown in the spinal cord and was accompanied by an enhanced VMR. This exaggerated response was attenuated using intrathecal NOS inhibitors.46 One study showed that pancreatitis caused microglial activation within the dorsal horn. This was correlated with the development of referred pain and reversed by the microglia inhibitor minocycline.75 By using Evan’s blue plasma extravasation, 1 study showed (anatomically) that acute inflammation of the colon could induce cross-organ inflammation.110 Colitis resulted in plasma extravasation in the bladder and uterine horns. These cross-organ effects were eliminated after cutting the hypogastric nerve. This intervention did not prevent colon extravasation after bladder inflammation, suggesting central mechanisms regulate cross-organ sensitization.110 Notably, estrus stage appeared to modulate these effects. One study examined the role of phosphorylation in visceral pain using Western blot analysis after colitis.38 PKC-γ and -ε were activated via membrane translocation in lumbosacral spinal cord segments, and this activation correlated with spontaneous pain behaviors.38 Importantly, PKC has been shown to activate ERK and increase dorsal horn neuron excitability via modulation of A-type potassium channels.111 Finally, 1 study showed that protein expression of GluR6, a subunit of the excitatory glutamate receptor, was increased in the sacral spinal cord after colitis and correlated with increased spontaneous pain behaviors. Suppression of GluR6 in the spinal cord using antisense oligodeoxynucleotides attenuated these pain behaviors.112 Physiological Evidence Extracellular recording Behavioral and anatomical/molecular evidence from animal studies have shown altered CNS properties after visceral inflammation. However, these approaches provide little functional information in regard to changes in cellular physiology. Sixteen studies (18%) examined in vivo responses of CNS neurons during and after visceral inflammation using extracellular recording techniques.28,30,38,55,56,63,113,–122 This approach allows action potential (AP) discharge to be assessed before and after inflammation. In most of these studies (11/16), recordings were made from spinal cord neurons. Acute or previous colitis increased background or spontaneous activity in recorded neurons in 13% studies.30,38,56,63,114,115,118,–121 Similarly, 16% studies reported increased AP discharge after both CRD and pelvic nerve stimulation and a decreased activation threshold for AP generation after colitis30,56,63,113,–117,119,120,122 and esophageal inflammation.28 Other studies have examined the effect of visceral inflammation on the temporal characteristics of AP discharge. Two populations of dorsal horn neurons have been described (abrupt and sustained firing) according to the way they respond to CRD.123,124 Two studies reported decreased activity in abrupt dorsal horn lumbosacral neurons after colitis,56,63 and another 2 reported increased activity in abrupt thoracolumbar neurons.63,122 Similarly, 2 studies reported increased activity of sustained neurons in the lumbosacral spinal cord.56,122 In contrast, 1 study reported a decreased proportion of neurons inhibited by CRD in the thoracolumbar spinal cord.122 This supports the notion that spinal cord processing after inflammation is somehow enhanced in thoracolumbar versus lumbosacral spinal cord as documented in anatomical studies.97 Referred pain The development of referred hypersensitivity in both somatic and visceral structures has been frequently observed in animal models of visceral inflammation and in patients.20,22,–25 Electrophysiological assessment of viscerosomatic convergence in the spinal dorsal horn was conducted in 3 studies. After colitis, more neurons exhibited colonic–somatic convergence: that is, they responded to both CRD and somatic pinch/brush. In addition, the sensitivity and size of peripheral somatic receptive fields increased.30,117,119 Another study demonstrated viscerovisceral convergence in dorsal horn neurons using bladder distension after colitis.121 AP discharge in neurons excited by bladder distension increased, whereas the duration of AP inhibition in neurons normally inhibited by bladder distension decreased.121 These changes were observed in both convergent and nonconvergent colon–bladder neurons, suggesting widespread sensitization of dorsal horn neurons.121 Cross-sensitization of bladder afferents, presumably by dorsal horn viscerovisceral convergence during colitis, was demonstrated in 2 studies (3%) using single-unit pelvic nerve recordings.66,125 Bladder afferents showed increased spontaneous activity during colitis. This subsided after inflammation had resolved, but the responses of pelvic nerve afferent fibers to bladder distension and intravesical capsaicin increased during and after resolution of colitis. A proportion of fibers were also sensitive to intravesical bradykinin and SP.66,125 Activation of affective brain centers during visceral inflammation The activation of nociceptive brainstem sites and cortical structures was recently explored using functional magnetic resonance imaging (fMRI) in a model of pancreatitis.39 The study reported increased fMRI signaling in the periaqueductal gray, dorsal raphe nucleus, rostral ventromedial medulla, and the lateral thalamus 1 week after the induction of inflammation. Increased fMRI signaling was also observed in limbic or “affective” brain regions, such as the amygdala, parietal cortex, and midcingulate/retrosplenial cortices.39 In most cases, these increases were attenuated by morphine, implicating pain directly in this subcortical and cortical activation. Discussion This systematic review demonstrates that considerable behavioral, anatomical, and physiological evidence exists for CNS plasticity, particularly in the dorsal horn of the spinal cord, following visceral inflammation. This suggests that improving our understanding of how visceral inflammation alters processing in the CNS may uncover new therapeutic strategies to manage patients with inflammatory pain from GIT organs.126 In this review, we have identified aspects of study design that would improve the quality of future studies. Importantly there is a need for additional studies that are relevant to explaining patients’ inflammation-based GIT pain and that explore mechanisms underlying plasticity in the spinal cord during and after visceral inflammation. Quality of Preclinical Studies Only 3 studies were deemed high quality33,34,37 according to our criteria (reported randomization of animals into experimental groups, blinded analysis, and objective evidence of visceral inflammation). Failure to randomize into control and treatment groups was a major limitation in many studies, and less than half the studies used blinded outcome assessments. Sample size calculations and the appropriate statistical powering were not reported in any of the studies assessed. Although many of the studies may have been appropriately powered for the main outcome variables/endpoints they reported, there is much less certainty regarding secondary variables. Future studies should report sample size calculations for primary outcome variables, report the actual P values for all measures, and explore the use of effect size calculations to provide more objective indications of the magnitude of reported differences. These recommendations for improving study design are in agreement with recent articles that have highlighted significant flaws in the experimental design of animal studies.127,128 Lack of randomization, blinding, poor statistical power, and inappropriate statistical analysis has been identified as the major factors contributing to bias and false positive results.127,128 Other Aspects of Study Design Rats have been the main species used in preclinical studies to date; however, the use of mice has increased over the past decade. Animal age and/or age range should be more accurately reported because it is known that the properties of spinal neurons involved in pain processing change with age.129 These oversights potentially introduce uncertainty about the effects of intervention-induced plasticity versus changes associated with developmental mechanisms or interactions between the 2. There is also more scope for studies that investigate the effects of early-life events30,130,131 on long-term nociceptive processing and the effects of repeated inflammatory exposure on pain vulnerability. This is particularly relevant because of the increasing incidence of IBD in childhood and the potential effects of disease on their developing nervous system.132 Male animals were used in almost three-quarters of studies. There is clear evidence in the clinical literature that nociceptive processing differs between the sexes, at least for somatic pain.133,–136 The data for visceral pain disorders are, however, less clear. For example, there are no sex-related differences in baseline rectal discomfort or noxious rectrosigmoid distension in IBS patients137,138; however, ovarian hormone levels influence abdominal pain in both IBD and IBS patients.139,140 Unfortunately, the preclinical literature has not shed much light on the effect of sex on central visceral pain processing. Only 10% studies used both sexes. One reported that estrus stage enhanced CNS-mediated cross-organ inflammation.110 These data are consistent with studies showing ovarian hormones can affect responses to visceral stimulation, such as CRD.141,142 Importantly, 1 of these studies demonstrated that changes were mediated by spinal estrogen receptors.141 Therefore, future studies on CNS visceral pain-processing mechanisms should be mindful of sex-related confounds, compare males/females, and report estrus stage. A wide variety of noxious agents and doses have been used to induce visceral inflammation. There is a clear need for studies that compare the effects of different agents so that the impact of specific inflammatory agents are known and can be subsequently manipulated. Similarly, there is a need to undertake studies that explore the effect of the dose of inflammatory agent. Doses are often selected to ensure an effect and to reduce variability in results. This approach may be experimentally satisfying, however, to better approximate the clinical situation variation in the induction of pain is required to better understand the manner in which pain develops in humans with differing degrees of GIT dysfunction. Perhaps one of the most clinically relevant and troubling findings for the translation of preclinical work is when CNS outcomes are measured after visceral inflammation. This varied greatly, from as little as 20 minutes to >2 weeks. This is important as chronic inflammatory conditions such as IBD or functional gastrointestinal disorders involve periods of remission and relapse,143,144 and IBD patients often continue to experience abdominal pain during periods of remission.5,6,144 Thus, there is a pressing need for a time series analysis using preclinical models. A minority of studies using models of colitis (20% of studies) measured CNS outcomes after inflammation had resolved.30,33,34,43,49,51,59,60,62,63,86,109 Thus, the long-term effects of inflammation on CNS plasticity are relatively unexplored. In addition, studies that measured long-term CNS changes only inflamed the colon once. It is, therefore, unclear whether the time courses used in the preclinical studies accurately reflect inflammatory bowel conditions. To improve clinical relevance, future studies should consider inflaming the colon more than once, to mimic the natural cyclical progression of IBD relapse and remission.145 Preclinical models of pancreatitis have, however, assessed CNS outcomes at time points that closely mirror disease progression. Patients with acute pancreatitis are generally hospitalized for <10 days,146 and 73% of the included studies measured CNS outcomes within 2 weeks of inflammation. Chronic pancreatitis is, however, a progressive disease often caused by alcohol abuse.147,–150 One study attempted to model the long-term induction of chronic pancreatitis using alcohol and a high fat diet for 10 weeks.80 Thus, preclinical models of pancreatitis provide an example where knowledge of the clinical pathology can be used to provide more relevant data in terms of mechanisms underlying altered CNS function after visceral inflammation. Evidence for CNS Plasticity Anatomical and molecular studies have consistently demonstrated changes in the spinal cord following visceral inflammation. Most assessed immediate early gene expression (c-Fos and pERK). Others used SP and CGRP expression because they are found in 70% to 90% of visceral afferents.151 Importantly, both c-Fos and pERK expression is attenuated with analgesics,152,153 suggesting these markers are reliable indicators of elevated neural activation after visceral insults. Notably, pERK expression correlates strongly with pain behaviors, as targeting of pERK with MEK inhibitors reduces both spinal cord expression of pERK and cutaneous hypersensitivity29,69,110,141 after visceral inflammation. Similarly, elevated SP and CGRP expression correlated with increased pain behaviors in 3 studies,77,79,93 suggesting these central changes may contribute to the development of persistent and referred hyperalgesia. SP and CGRP have also been implicated in neurogenic inflammation of the bladder after resolution of colitis.109 Release of neuropeptides in the bladder may be explained by cross-organ sensitization because of convergence of colonic and bladder afferents onto neurons in the spinal cord dorsal horn. After colonic nociceptive signals converge with bladder afferents in the spinal cord, excitation can be relayed to the bladder by a phenomenon known as the dorsal root reflex, where an action potential develops in the spinal cord and is propagated to peripheral structures.154 This would result in peripheral release of SP and CGRP in the bladder and produce pain. Together, these anatomical studies provide strong evidence for CNS plasticity following visceral inflammation. Three major behavioral changes were observed during or after recovery from visceral inflammation: increased VMR; referred hypersensitivity to somatic/skin areas; and sensitization of other viscera. These changes likely represent central sensitization155 because of the convergence of visceral and somatic afferents in the spinal cord dorsal horn.25,65,156 Withdrawal responses to mechanical stimuli (i.e., von Frey hairs) following visceral inflammation consistently show hypersensitivity. However, responses to plantar heat vary, with some studies reporting increased sensitivity,36,62,80 decreased sensitivity,81 or no change.61 The mechanisms underlying the varied responses to mechanical and thermal stimuli are not known. In 1 study,61 where both mechanical and thermal responses were assessed, referred mechanical hyperalgesia was observed, but responses to heat were unchanged after colonic inflammation. This suggests referred hypersensitivity may be stimulus (or modality) specific, and it would be interesting to determine whether visceral and mechanosensitive somatic afferents preferentially converge on dorsal horn neurons.61 Extracellular recording has clearly demonstrated that visceral inflammation can alter the excitability, AP discharge patterns, and level of viscerosomatic convergence in spinal neurons. Although these functional data support the notion of central sensitization in spinal circuits after a visceral insult, the identity of the neurons involved, and the detailed mechanisms responsible for their altered output are unknown. Moreover, extracellular recording tends to introduce bias towards tonically active neurons,113 or those that respond to a stimulus (somatic brush/pinch or CRD) with sustained excitatory responses.116,117,120 In addition, only 2 major firing patterns, abrupt and sustained, have been identified in the dorsal horn123,124 using extracellular recording. This contrasts with the diversity of AP discharge patterns observed in animal studies using both in vitro and in vivo intracellular recording.157,158 Thus, our knowledge of the spinal neuron populations involved in central plasticity is limited. Finally, if new CNS-acting therapies are to be developed for visceral pain, we must understand what actually drives altered neuronal excitability. It has long been recognized that neuron output (in the form of AP discharge) depends on the combined action of excitatory or inhibitory synaptic inputs and intrinsic properties. Importantly, changes in either synaptic inputs or intrinsic properties can alter neuron output.159 Such changes in the intrinsic properties of sensory neurons (DRGs) have been demonstrated after TNBS-induced colitis using whole-cell patch clamp electrophysiology.160,161 At present, the detailed synaptic and intrinsic properties of spinal neurons that receive inputs from the GIT are uncharacterized, under either normal or inflammatory conditions. Future studies aimed at filling this knowledge gap are now required to determine the neurochemical phenotype, morphology, connectivity, and intrinsic and synaptic properties of spinal neurons involved in the central plasticity that clearly accompanies inflammation of the GIT. In summary, chronic pain management in patients with inflammatory diseases of the GIT is lacking. The virtual absence of pharmacological agents that can successfully relieve pain has highlighted the need for further research into the causes of chronic pain. 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TI - A Systematic Review of the Evidence for Central Nervous System Plasticity in Animal Models of Inflammatory-mediated Gastrointestinal Pain
JF - Inflammatory Bowel Diseases
DO - 10.1097/01.MIB.0000437499.52922.b1
DA - 2014-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/a-systematic-review-of-the-evidence-for-central-nervous-system-BJDphRyrgU
SP - 176
EP - 195
VL - 20
IS - 1
DP - DeepDyve
ER -