Received: 4 October 2021 | Revised: 31 May 2022 | Accepted: 18 July 2022 DOI: 10.1111/nmo.14441 ORIGINAL ARTICLE Long-­lasting visceral hypersensitivity in a model of DSS-­induced colitis in rats Sergio López-­Estévez1,2 | Josep Manuel López-­Torrellardona1 | Marc Parera1 | Vicente Martínez1,2,3 1 Department of Cell Biology, Physiology and Immunology, Universitat Autònoma Abstract de Barcelona, Barcelona, Spain Background: Persistent visceral hypersensitivity is a key component of functional and 2 Neuroscience Institute, Universitat Autònoma de Barcelona, Barcelona, Spain inflammatory gastrointestinal diseases. Current animal models fail to fully reproduce 3 Centro de Investigación Biomédica the characteristics of visceral pain in humans, particularly as it relates to persistent en Red de Enfermedades Hepáticas y hypersensitivity. This work explores the validity of DSS-­induced colitis in rats as a Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain model to mimic chronic intestinal hypersensitivity. Methods: Exposure to DSS (5% for 7 days) was used to induce colitis in rats. Thereafter, Correspondence Vicente Martínez, Unit of Physiology, changes in viscerosensitivity (visceromotor responses to colorectal distension—­CRD), Veterinary School, Universitat Autònoma the presence of somatic referred pain (mechanosensitivity of the hind paws, von de Barcelona, 08193 –­ Bellaterra, Barcelona, Spain. Frey test) and the expression (qRT-­PCR) of sensory-­related markers (colon, lumbosa- Email: vicente.martinez@uab.es cral DRGs, and lumbosacral spinal cord) were assessed at different times during the 35 days period after colitis induction. Results: Following colitis, a sustained increase in visceromotor responses to CRD were observed, indicative of the presence of visceral hypersensitivity. Responses in animals without colitis remained stable over time. In colitic animals, somatic referred hypersensitivity was also detected. DSS-­induced colitis was associated to a differen- tial expression of sensory-­related markers (with both pro-­ and anti-­nociceptive action) in the colon, lumbosacral DRGs and lumbosacral spinal cord; indicating the presence of peripheral and central sensitization. Conclusions and Inferences: DSS-­induced colitis in rats is associated to the genera- tion of a long-­lasting state of visceral (colonic) hypersensitivity, despite clinical colitis resolution. This model reproduces the changes in intestinal sensitivity characteristics of inflammatory and functional gastrointestinal disorders in humans and can be used in the characterization of new pharmacological treatments against visceral pain. KEYWORDS colitis, hypersensitivity, intestinal inflammation, pain sensitization, visceral pain This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2022 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd. Neurogastroenterology & Motility. 2022;00:e14441.  wileyonlinelibrary.com/journal/nmo | 1 of 12 https://doi.org/10.1111/nmo.14441 2 of 12 | LÓPEZ-­E STÉVEZ et al. 1 | I NTRO D U C TI O N Highlights Visceral hypersensitivity is a common component of inflammatory • Rats with DSS-­induced colitis develop long-­lasting vis- and functional gastrointestinal (GI) disorders in humans. Indeed, per- ceral hypersensitivity, as assessed during colorectal sistent alterations in GI sensitivity with altered pain thresholds and distension. signs of hypersensitivity are considered as key findings in functional • Intestinal hypersensitivity is likely to result from the GI disorders, particularly in irritable bowel syndrome (IBS).1,2 Similar interaction between central and peripheral sensitizing long-­lasting alterations in sensitivity, although with some inconsist- mechanisms. encies, are also reported during the active and inactive phases of • This model represents a valid tool for the characteriza- inflammatory bowel disease (IBD).3,4 Despite the clinical importance tion of new therapeutic targets and the validation of of visceral pain, the development of effective treatments directed new drugs for the treatment of visceral pain of intestinal towards the specific control of this pain modality has been ham- origin. pered by: (i) the complex physiology of visceral pain; (ii) the differ- ences in the neural and immune substrata underlying visceral vs. somatic pain; and (iii) the difficulties in developing animal models with high translational validity, particularly as it relates to persistent/ more similar to Crohn's disease.31 Visceral pain responses during 5–­7 chronic hypersensitivity. DSS-­induced colitis have been studied mainly in mice. Although with Taking this into account, numerous animal models have been some contradictory data, several studies consistently indicate the developed for the assessment of visceral sensitivity in normal con- presence of intestinal (colonic) hypersensitivity during DSS-­induced ditions or in states of sensitization. A systematic review of these inflammation, either acute or chronic.32–­36 models is out of the scope of the present work and can be found With the objective of broadening the available models to as- elsewhere [see as examples: Regmi and Shah (2020); Johnson et al. sess visceral pain, particularly as it related to the presence of (2020); West and McVey Neufeld (2021); Accarie and Vanuytsel chronic hypersensitivity, the present work evaluates the validity of (2020)].8–­11 Several animal models of intestinal hypersensitivity have DSS-­induced colitis in rats as a model to assess intestinal (colonic) been developed based on the evidence that inflammation seems to sensitivity, with special emphasis in the presence of long-­term sen- be a pathophysiological component of inflammatory and functional sitization. Moreover, we also assessed changes in the expression GI disorders, as mentioned above. Overall, postinflammatory mod- of sensory-­related markers at the periphery (colon and dorsal root els have been based on the local administration (intracolonic ene- ganglion) or the spinal cord level, as a way to gain insight into the mas) of different active compounds (such as acetic acid, capsaicin, mechanisms underlying inflammation-­associated changes in viscero- mustard oil, zymosan, trinitrobenzene sulfonic acid (TNBS)-­ or dini- sensitivity following DSS exposure. trobenzene sulfonic acid (DNBS))12–­16 or the experimental infection with biological agents (such as Trichinella spiralis, Nippostrongylus brasiliensis or Campylobacter species).17–­19 From the available mod- 2 | M ATE R I A L S A N D M E TH O DS els, a recent report recommends the use of the intracolonic TNBS postinflammatory model of colonic hypersensitivity as the principal 2.1 | Animals model for screening novel treatments for visceral pain originating within the GI tract.9 This recommendation was based on four char- Female adult Sprague–­D awley rats (Crl:OFA[SD]; 10–­12 week–­ acteristics of the model: simplicity, characterization in rodent spe- old at the beginning of the study; Charles River, France) were cies, reproducibility across laboratories and construct validity and used. Female rats were used because of the higher prevalence translational relevance. However, data available on the TNBS model of GI dysfunctions associated with visceral pain, such as IBS, in is limited and can be considered insufficient as to endorse its use women. 37,38 Given the inconsistent observations related to the over similar models. potential effect of the estrous cycle in sensitivity, the phase One of the most commonly used models of intestinal inflam- of the cycle was not taken into consideration in the present mation is based on the dextran sulfate sodium (DSS) exposure in studies. 39–­4 4 Animals were group-­h oused (3–­4 animals per cage) rodents. This model has strong similarities with the characteristic in standard cages and were maintained in conventional conditions histopathology of intestinal inflammation in humans and it is re- in an environmentally controlled room (20–­2 2°C, 12 h light: dark garded as a valid model of human intestinal inflammation. The DSS-­ cycle), with food and water ad libitum, except when receiving induced colitis model has been extensively validated in both rats20–­26 DSS. Animals were allowed to acclimatize to the animal facility for and mice.7,27–­29 Some reports suggest that the DSS model elicits an at least 1 week prior to the start of the studies. All experiments inflammatory response more similar to human IBD than the TNBS were performed in accordance with EU regulations and were ap- model.30 In fact, exposure to DSS elicits a colitic response that, ac- proved by the Ethical Committee of the Universitat Autònoma cording to its histopathological features and immunologic profile, de Barcelona (protocols 3958 and 3961) and the Generalitat de resembles human ulcerative colitis, while TNBS-­induced colitis is Catalunya (protocol 9915). LÓPEZ-­E STÉVEZ et al. | 3 of 12 2.2 | DSS-­induced colitis When calculating the magnitude of the balloon pressure signals, the first and last 4.5 s of each pulse were excluded, since these Rats received a solution of 5% DSS (45 kDa; TdB Consultancy AB, reflect artifacts produced by the barostat during the inflation and Upssala, Sweden) in their drinking water for 7 days to induce colitis. deflation process and do not originate from the animal. The total The same protocol has been previously used for the induction of co- response to a CRD protocol was calculated as the area under curve 20–­24 litis in rats. DSS solutions were prepared freshly on a daily basis (AUC) of the corresponding VMRs registered during the distension while control rats received normal tap water. Throughout the study, time, corrected for the basal activity. body weight, the general state, and the presence of clinical signs of inflammation were assessed individually on a daily basis. 2.4 | Evaluation of referred mechanical somatic hyperalgesia: von Frey test 2.3 | Assessment of colonic sensitivity: colorectal distension To evaluate referred mechanical sensitivity, animals were located into compartment enclosures in a test chamber with a framed metal mesh For the assessment of colonic sensitivity, the colorectal distension floor, through which von Frey monofilaments were applied for pain as- (CRD) method was used, following, with minor modifications, pre- sessment (bending force range from 1 to 26 g; North Coast Medical, viously published procedures.45 Before starting experiments, rats Inc.; Gilroy, California, USA). Animals were habituated to the testing were gradually habituated (45–­75 min per day, at 15 min steps) to environment for a 30–­40 min period before starting the procedure. Bollmann cages to reduce motion artifacts and minimize the ef- Somatic referred pain was quantified by measuring the withdrawal fects of stress-­related responses. On the day of the experiment, rats response to punctate mechanical stimulation of the hind paws, as were anesthetized with isoflurane (Isoflo; Esteve, Barcelona, Spain). previously described.7 For each animal, the mean measure of the right A plastic balloon (made in-­house; length: 2.5 cm; diameter: 1.5 cm) and left hid paw was taken as the measure of sensitivity. The up-­down with connecting catheter was inserted in the distal colon (2 cm from method was used to assess pain thresholds, which represent the me- the base of the balloon to the anus). The catheter was fixed with chanical threshold that elicits 50% of the maximal response.49 The per- tape to the tail of the animal. Animals were then placed in Bollmann centage of DSS-­induced algesic effect was calculated as: % = [(PWB cages and allowed to recover from anesthesia for at least 20–­30 min -­ PWI) / PWB] * 100, where PWB and PWI are the basal paw threshold before starting the CRD protocol. (g) and paw threshold on the day of interest, respectively. The balloons were connected to pressure transducers (P-­602, CFM-­ k33, 100 mmHg, Bronkhorst HI-­TEC, Veenendal, The Netherlands) to control intraballoon pressure during the CRD procedure. A customized 2.5 | Experimental protocols barostat system was used to manage balloon inflation and to mea- sure pressure changes during CRD. The pressure was monitored and Animals were weighted and randomly distributed in two ex- kept constant by a custom computer software (Pharmlab 5.0 online, perimental groups (control and colitis; experimental Day −1). AstraZeneca R&D, Mölndal, Sweden) running on a standard computer. Thereafter they received 5% DSS during 7 consecutive days to in- The same system served also for data acquisition. duce colitis (experimental Days 0–­7 ). The control group received For CRD, a protocol consisting of repetitive phasic distensions normal tap water during the same period. After finishing the in- at 80 mmHg, with a pulse duration of 30 s at 5 min intervals was duction of colitis, a resting period of 9 days (experimental Days 7–­ used. This protocol served to induce acute mechanical hypersen- 16) was allowed and thereafter somatic (von Frey test) and visceral sitivity and has been extensively used to assess colonic mecha- sensitivity was assessed at regular intervals throughout the fol- nosensitvity.45–­4 8 Rapid pressure changes in the balloon, which lowing 20 days period (experimental Days 16–­35), with a 3–­4 days reflect contractions of the abdominal muscles, served to assess between consecutive tests. Basal somatic and visceral sensitiv- visceromotor responses (VMRs). The analogue input channels ity were assessed at the start of the DSS exposure (experimental were sampled with individual sampling rates, and digital filtering Days −1 and 0 for somatic and visceral sensitivity, respectively). In was performed on the signals. The balloon pressure signals were addition, at experimental Days 0 (basal), 21, and 35, some animals sampled at 50 Hz. A highpass filter at 1 Hz was used to separate were euthanized for the assessment of inflammation (see below). the contraction-­induced pressure changes from the slow varying Figure 1 shows a schematic representation of the experimental pressure generated by the barostat. A customized software (CDR protocols. Analytics v2.0, J4 Style) was used to quantify the magnitude of the balloon pressure signals. The average rectified value of the balloon pressure signals was calculated for 30 s before the pulse 2.6 | Samples collection (i.e., baseline activity) and for the duration of the pulse. This mea- sure allows the contractions of the abdominal muscles to be dis- At experimental Days 0, 21, and 35, control and DSS-­treated rats tinguished from the relaxations or compensations of the barostat. were deeply anesthetized with isoflurane (Isoflo; Esteve, Barcelona, 4 of 12 | LÓPEZ-­E STÉVEZ et al. F I G U R E 1 Schematic representation of the experimental protocols followed in the study Spain) and euthanatized by exsanguination through intracardiac lamina propria), and presence of inflammatory infiltrate (0: normal; puncture. Thereafter, a medial laparotomy was performed, the ceco-­ 1: mild localized infiltrate; 2: mild generalized infiltrate; 3: severe colonic region localized and the colon dissected. Afterward, a tissue generalized infiltrate). Histopathological scoring was performed in a sample from the proximal colon was collected and frozen immedi- blinded manner (coded slides) by two independent researchers. ately in liquid nitrogen. In addition, dorsal root ganglia (DRGs) cor- responding to the lumbosacral region and the lumbosacral region of the spinal cord (L3-­S2) were also collected and frozen immediately. 2.9 | Gene expression: quantitative reverse Frozen samples were stored at −80°C until analysis. Moreover, tis- transcription-­PCR sue samples (about 3 cm) of the middle colon were collected and fixed overnight in 4% paraformaldehyde. Afterward, tissues were Total RNA was extracted from frozen tissue samples using TRI paraffin embedded and 5 μm-­thick sections obtained. reagent with Ribopure Kit (Ambion/Applied biosystems, Foster City, California, USA). RNA was purified by precipitation with lithium chloride. 50 Thereafter, a two-­s tep quantitative real-­time 2.7 | Clinical and macroscopic assessment of PCR (RT-­qPCR) was performed. RNA samples were converted inflammation into cDNA with a High-­C apacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-­time quantitative PCR (iTaq Universal™ Clinical assessment of inflammation included daily monitoring of SYBR® Green Supermix, Bio-­R ad) was incubated on the Bio-­R ad body weight, appearance of feces, and general health condition, as CFX384 Touch Real-­T ime PCR Detection System (Bio-­R ad). All previously described.7,28 A score (0–­8) was assigned for health con- samples were analyzed in triplicate. For each sample, the cycle dition (including hunch posture, piloerection, fecal consistency, and thresholds were obtained and data were analyzed using the com- anal inflammation); where 0 indicates normal activity/fur/fecal con- parative Ct method (2−ΔΔCt), non-­inflammed vehicle-­t reated ani- tent/no anal inflammation, 1 indicates abnormal gait/bristly fur/wet mals served as the calibrator. 51 PrimePCR™ SYBR® Green Assay anus/loose fecal content and 2 indicates prostrated animal/dirty fur/ (Bio-­R ad) for interferon γ (IFN-­γ ; qRnoCID0006848), interleukin watery or bloody anus/diarrhea. 1β (IL-­1β; qRnoCID0004680), and interleukin 10 (IL-­10; qRno- At necropsy, the macroscopic appearance of the colon (inflamma- CID0005930) were used for inflammatory response. Cannabinoid tory score) was scored based on: consistency of fecal content (score receptor type 1 (CB1; qRnoCED0008430) and type 2 (CB2; qRno- 0–­3) and presence of visible fecal blood (score 0–­3). Additionally, the CED0008595), μ-­opioid receptor (MOR; qRnoCED0003071), tran- extent of oedema (0–­3), stiffness (0–­2), thickness (0–­3), and pres- sient receptor potential vanilloid 1 (TRPV1; qRnoCID0003147), ence of ulcerations (0–­1) were also assessed; equivalent to a maxi- and 3 (TRPV3; qRnoCID0007727), nerve growth factor (NGF; mum total score of 15. qRnoCID0003911), receptor activity modifying protein 1 (Ramp1; qRnoCED0001653), and σ1 receptor (σ1R; qRnoCED0005919) were used for the assessment of neuronal/sensory activity. Actin 2.8 | Histopathological studies β (Actb; qRnoCID0056984) and hypoxanthine-­guanine phospho- ribosyltransferase 1 (Hprt1; qRnoCED0057020) were used as en- Hematoxylin–­eosin-­stained sections were obtained from 4% dogenous reference genes. paraformaldehyde-­fixed colon specimens, following standard proce- dures, for their histological examination. A histopathological score (ranging from 0, normal, to 12, maximal alterations) was assigned to 2.10 | Statistical analysis each animal. Parameters scored included: structure of the luminal epithelium (0: normal; 1: mild alterations; 2: local destruction; 3: gen- Data are expressed as mean ± SEM. For RT-­qPCR data, a robust analy- eralized destruction), state of the lower part of the crypts (0: normal; sis (one iteration) was used to obtain mean ± SEM. Data were analyzed 1: mild alterations; 2: local destruction; 3: generalized destruction), by one-­ or two-­way ANOVA, with or without repeated measures, as occurrence of edema (0: normal; 1: mild local edema in submucosa appropriate, followed, when necessary, by a Bonferroni's multiple and/or lamina propria; 2: moderate diffuse edema in submucosa and/ comparisons test. Data were considered statistically significant when or lamina propria; 3: severe generalized edema in submucosa and/or p < 0.05. All statistical analyses were performed using SPSS program LÓPEZ-­E STÉVEZ et al. | 5 of 12 (version 17 for Windows, IBM, Madrid, Spain) or GraphPad Prism 7 (GraphPad Software, La Jolla, California, USA). 3 | R E S U LT S In non-­inflamed animals, none of the parameters assessed showed time-­related changes. Therefore, control data obtained at experi- mental Days 0, 21, and 35 have been pooled as a single control group. 3.1 | Colitis development and follow-­up Rats not exposed to DSS showed a linear body weight increase throughout the experimental time, with no clinical signs of inflam- mation. In contrast, animals receiving DSS showed changes in body weight and presented clinical signs, consistent with the development of colitis, in a time-­related manner (Figure 2). Body weight showed a decline from experimental Day 2 (p < 0.05 vs. body weight at Day 0; p < 0.05 vs. body weight in animals without colitis at the correspond- ing days), with a peak reduction of 7.0 ± 2.9% by Day 17. Thereafter, body weight increased slowly but without recovering the growth ratio observed in control conditions (Figure 2A). In the same animals, F I G U R E 2 Clinical characterization of DSS-­induced colitis in clinical signs of colitis, such as piloerection, loose feces/watery diar- rats: time-­course changes in body weight (% change from Day 0, rhea, and fecal blood were observed, with a peak between experi- taken as 100%) and clinical signs. The gray square denotes the mental Days 7 and 10 (p < 0.05 vs. control group; Figure 2B), likely period of DSS exposure. Data are mean ± SEM (n = 5–­16) reflecting individual variability in the response to DSS and the devel- opment of inflammation. Thereafter, clinical signs subsided and were using this protocol. CRD evoked a significant increase in VMRs vs. absent from experimental Day 21 onward. basal activity (first distension: 0.21 ± 0.04; p < 0.05 vs. basal activ- At experimental Days 21 and 35, the colon of animals exposed to ity). Moreover, VMRs showed a distension-­related increase along DSS showed some macroscopic signs of inflammation (Figure 3A–­C) the CRD protocol (first distension: 0.21 ± 0.04, p < 0.05 vs. 12th and histopathological alterations, particularly as it relates to the pres- distension: 0.39 ± 0.5), with an overall 201 ± 27% increase in the ence of inflammatory infiltrate through the colonic wall (Figure 3D-­E). response observed from the first to the last distension (Figure 5A). In all cases, expression (RT-­qPCR) of IFN-­γ, IL-­1β, and IL-­10 was Animals not exposed to DSS maintained this response to phasic CRD detectable and quantifiable in colon, lumbosacral DRGs, and lum- throughout the experimental time with only small, non-­significant, bosacral spinal cord. In animals with colitis, time-­related changes fluctuations, as assessed in experimental Days 17, 21, 24, 28, 31, and in the expression of colonic proinflammatory cytokines, IFN-­γ and 35 (p > 0.05 two-­way ANOVA with time and inflammation as fac- IL-­1β, with an up-­regulation by experimental Day 21 (IL-­1β: p < 0.05 tors; Figure 5). Animals exposed to DSS showed increased VMRs to vs. control; Figure 4A) and a normalization in the expression by Day phasic CRD from experimental Day 17 when compared with healthy 35 were observed. Similar changes, although delayed in time, were animals. Increased responses to CRD were maintained on time until detected in DRGs and spinal cord. In this case, up-­regulation of pro-­ the end of the experimental time (p < 0.05 vs. control; Figure 5C). inflammatory cytokines increased with time, reaching statistically As an example, at experimental Day 35, in colitic animals VMRs to significant values at experimental Day 35 (Figure 4B,C). phasic CRD increased from the first (0.40 ± 0.06) to the last disten- The gene expression of the anti-­inflammatory cytokine IL-­10 was sion (0.88 ± 0.12; p < 0.05 vs. VMR during the first distension), with only modified at the lumbosacral spinal cord at experimental Day 35, an overall 255 ± 33% increase from the first to the last distension showing a significant up-­regulation (p < 0.05 vs. control; Figure 4). (Figure 5). 3.2 | Effects of DSS-­induced colitis on colonic 3.3 | Effects of DSS-­induced colitis on referred sensitivity somatic mechanical sensitivity In control conditions (experimental Day 0), repetitive phasic CRD at In colitic rats, small fluctuations in referred mechanical sensitiv- 80 mmHg elicited VMRs similar to those described elsewhere when ity, manifested as a reduction in the force needed to evoke a paw 6 of 12 | LÓPEZ-­E STÉVEZ et al. F I G U R E 3 Macro-­ and micro-­scopic assessment of the colon at the time of necropsy in healthy controls and animals with DSS-­induced colitis at experimental Days 21 (d21) and 35 (d35). A: macroscopic assessment of the colon (maximum possible scoring: 15; see methods for details); B: colon length; C: colonic relative weight; D: colonic histopathological scores (maximum possible scoring: 12; see methods for details). Data are mean ± SEM of 4–­5 animals per group. *: p < 0.05 vs. control group (in panel D, used for the complete histopathological score). E: Representative microphotographs showing hematoxylin–­ eosin-­stained colonic sections from control and DSS-­treated animals at experimental Days 21 (d21) and 35 (d35) F I G U R E 4 Effects of DSS-­induced colitis on the expression of pro-­ (IFN-­γ and IL-­1β) and anti-­inflammatory cytokines (IL-­10) in colon (A), lumbosacral DRGs (B), and lumbosacral spinal cord (C) in control conditions (denoted at experimental Day 0, d0) and at experimental Days 21 (d21) and 35 (d35). Data are mean ± SEM, n = 4–­5 per group. *: p < 0.05 vs. control group withdrawal reflex, was observed from experimental Day 16 (vs. sen- 3.4 | Effects of DSS-­induced colitis on sensory-­ sitivity measures determined at experimental Day −1, taken as basal related markers sensitivity). However, significance was only achieved at experimen- tal Days 16 and 27 (Figure 6) and no correlation with visceromo- mRNA for the sensory-­related markers evaluated was detected in tor responses during CRD was found (see Figure S1). No changes all samples, with the exception of Ramp1 which was not detected in somatic sensitivity were observed in control conditions (data not in two spinal cord samples. In control rats, expression levels re- shown). mained stable over time. As it relates to the colon, in animals with LÓPEZ-­E STÉVEZ et al. | 7 of 12 F I G U R E 5 Effect of DSS-­induced colitis on VMR responses to phasic repetitive CRD. A: VMRs to repetitive phasic CRD in healthy and DSS-­induced colitic rats. VMRs to CRD were assessed in the same animals, either healthy or colitic, before the induction of colitis, experimental Days 0 (d0), and 35 days after DSS exposure (d35). B: Cumulative responses to phasic CRD (AUC) of the experimental groups included in A. C: Time-­course changes in the cumulative responses to phasic CRD (AUC) throughout the experimental time. Data represents % change from the VMRs determined in basal conditions at experimental Day 0, taken as 100%. Data are mean ± SEM (n = 7–­16). *: p < 0.05 vs. control group (two-­way ANOVA with time and inflammation as factors) Within the lumbosacral spinal cord, an overall up-­regulation of anti-­nociceptive-­related markers (p < 0.05 for CB1, CB2, and MOR vs. control; Figures 7 and 8) and a down-­regulation of pro-­nociceptive-­ related markers (p < 0.05 for TRPV1 and 3 and Ramp1 vs. control; Figures 7 and 8) was detected following DSS-­induced colitis. Spinal σ1Rs were up-­regulated following DSS exposure (p < 0.05 vs. con- trols, regardless the time considered; Figures 7 and 8). 4 | DISCUSSION Robust models for the study of visceral pain and the characteriza- tion of potential therapeutic targets are still a need. Taking into ac- count the clinical characteristics of visceral pain of intestinal origin F I G U R E 6 Time course changes in mechanical referred somatic these models should, ideally, involve states of long-­lasting hyper- sensitivity in animals with DSS-­induced colitis as assessed in the hind paws [relative to experimental Day −1 (d-­1), taken as basal sensitivity. In the present report, we present evidence supporting sensitivity, 0% change] at experimental Days 16 (d16), 20 (d20), the potential value of DDS-­induced colitis in rats as a way to induce 27 (d27) and 34 (d34) (see Figure 1 for details of the experimental long-­lasting colonic hypersensitivity, thus providing an animal model protocol). Data are mean ± SEM of n = 8 animals per group. *: to investigate visceral pain mechanisms, to characterize new thera- p < 0.05 vs. basal sensitivity (experimental Day −1) peutic targets and for the validation/screening of new drugs. It is well recognized that inflammation and pain are associated DSS-­induced colitis, only the expression of TRPV1 and Ramp1, on phenomena in multiple organic systems, including somatic and vis- experimental Day 35, was affected, with a significant up-­regulation ceral structures. Within the GI tract, inflammatory and functional (both p < 0.05 vs. control; Figures 7 and 8). On the contrary, colonic bowel disorders are characterized by the coexistence of inflamma- expression of σ1Rs was significantly down-­regulated (p < 0.05 vs. tion, of variable intensity, and visceral pain, with chronic states of control, for experimental Days 21 and 35; Figures 7 and 8). hypersensitivity as a distinctive feature.1,2 Taking this into account, In lumbosacral DRGs, only σ1R expression was affected, with animal models to study visceral pain arising from the gut are largely a dual response including a down-­regulation at early times (exper- based on the induction of inflammation or irritation (with the con- imental Day 21) (p < 0.05 vs control) followed by an up-­regulation at sequent immune activation) of the GI tract.8,9,11,52 In this context, experimental Day 35 (p < 0.05 vs. control; Figures 7 and 8). DSS-­induced colitis is a well-­validated and accepted model of human 8 of 12 | LÓPEZ-­E STÉVEZ et al. F I G U R E 7 Heat map showing the relative expression (mRNA) of sensory-­ related markers in colon, lumbosacral DRGs, and lumbosacral spinal cord in control conditions (healthy animals) and after DSS-­induced colitis [experimental Days 21 (d21) and 35 (d35)]. The cross (X) denotes the two spinal cord samples in which no expression of Ramp1 was detected. See also Figure 8 colitis (in particular ulcerative colitis).7,27–­29 Compared with other in- suggest an inflammation-­associated remodeling of central (spinal) flammatory models, such as TNBS/DNBS-­induced inflammation, the mechanisms that might be related to alterations in pain sensitivity, as DSS model induces a less aggressive inflammatory response that, previously suggested for other models of gastrointestinal inflamma- according to some authors, resembles in a more accurate manner the tion, such as during T. spiralis-­induced enteritis.53 Altogether, these 30 inflammation observed in humans. In the present conditions, ex- observations support the presence of changes in visceral sensitivity posure to DSS led to the induction of a state of colitis, as determined after clinical DSS-­induced colitis resolution. by the presence of clinical signs consistent with those previously In animals without colitis, phasic CRD elicited VMRs indicative reported for this model. 21–­24 As previously characterized in mice, of visceral pain, with the presence of acute mechanical sensitization; in rats DSS exposure leads to an acute state of colitis that partially similar to that previously described in similar experimental condi- 21,26 resolves over time generating a state of chronic, low-­degree, tions using the same distension paradigm.46 In animals with DSS-­ inflammation, which was evident up to 4 weeks after the induction induced colitis, similar responses to phasic CRD were observed. process. During this state, although no clinical signs were noticed However, overall responses to CRD were increased when compared and a gain of body weight was observed, histopathological and mo- to those obtained in non colitic animals, thus suggesting the pres- lecular alterations (moderate up-­regulation of pro-­inflammatory ence of inflammation-­associated hypersensitivity. Moreover, this cytokines) consistent with the presence of mild inflammation were response persisted over time, as assessed in repeated occasions still detected. These long-­term changes are reminiscent of the quies- between days 17 and 35 after the induction of inflammation. This cent phases observed in IBD patients when the flares of the disease suggests that the hyperalgesic responses associated to inflamma- resolve and of the low degree of inflammation/immune activation tion were long-­lasting, despite the resolution of colitis from a clin- described in IBS patients.1,2 Therefore, the model might bear utility ical point of view (see above). Indeed, we have observed a similar reproducing the functional/structural/molecular alterations of IBS hyperalgesic state in animals with DSS-­induced colitis up to 49 days or latent IBD. after the induction of inflammation (data not shown). Changes in Interestingly, evidence of long-­lasting immune activation outside VMRs during DSS-­induced colitis were, in magnitude, similar to the intestine, specifically in neural substrata important in sensory those described in other models of inflammation-­associated sen- processing from the colon, that is, lumbosacral DRGs and spinal sitization, such as the TNBS-­induced colitis model,13,14 but without cord was also observed. Moreover, expression changes of immune-­ the aggressiveness that characterizes the exposure to TNBS, as related mediators at these locations increased over time, thus further previously commented. Altogether, these observations suggest that supporting the presence of long-­lasting molecular alteration, even DSS-­induced colitis results in a reproducible long-­term state of co- outside the original site of inflammation, with potential significance lonic mechanical hyperalgesia, despite the clinical colitis resolution, in the processing of afferent sensory signals. These changes might thus representing a feasible animal model to study this condition. LÓPEZ-­E STÉVEZ et al. | 9 of 12 F I G U R E 8 Relative expression (mRNA) of sensory-­related markers in colon, lumbosacral DRGs, and lumbosacral spinal cord in control conditions and after DSS-­induced colitis [experimental Days 21 (d21) and 35 (d35)]. Data are mean ± SEM of 4–­5 animals per group (as shown individually in Figure 7). *: p < 0.05 vs. control 10 of 12 | LÓPEZ-­E STÉVEZ et al. Visceral pain sensitization, either in humans or in relevant ani- we assessed the expression of a set of inflammatory mediators pre- mal models, has been associated to peripheral and central sensiti- viously related to pain and sensitization mechanisms, but we cannot zation mechanisms implying changes in sensory pathways leading discard that other innate cytokines might be regulating these mech- to an increase in pain transmission and, therefore, to exacerbated anisms. Alternatively, a non-­inflammatory regulation of nociceptors pain manifestations (hypersensitivity). 54–­56 Inflammatory media- might also participate in the sensory changes observed here. tors, such as cytokines, released during inflammation have been It is noteworthy that in animals exposed to DSS the pro-­ directly implicated in peripheral sensitization. During DSS-­induced nociceptive σ1R followed a reverse pattern of expression. It was colitis, there is a significant local (colon) up-­regulation of pro-­ down-­regulated at the periphery (colon) and up-­regulated at cen- inflammatory cytokines (present observations).7,26,28 This might tral levels (lumbosacral spinal cord). Activation of σ1Rs in the spi- contribute to the increased responses to CRD observed in animals nal cord produce mechanical hypersensitivity and increased pain with colitis. Moreover, an up-­regulation in pro-­inflammatory cyto- responses in different animal models.64,65 Therefore, the changes kines was also observed at central sites related with the process- observed might support a direct role for σ1Rs on the development of ing of sensory signals arising from the colon (lumbosacral DRGs inflammation-­associated central sensitization. and lumbosacral region of the spinal cord). 57,58 Therefore, it is One of the distinctive characteristics of visceral pain is its capac- feasible to assume that a similar cytokine-­mediated sensitization ity to generate referred somatic pain, revealed as sensitivity changes process might take place at central (spinal) relevant areas, as pre- (hypersensitivity) that can be detected in the paws, tail, or even the viously shown in other models of intestinal inflammation. 56 As a abdominal wall.5–­7 To explore this possibility, we also assessed the limitation of this study, it is worthy to consider that we have only presence of referred somatic pain in animals with visceral hypersen- evaluated expression changes at the mRNA, but not at the protein sitivity. For this, we evaluated time-­related mechanical sensitivity level, which might not necessarily follow the same pattern. Hence, (withdrawal responses) of the hind paws in the same animals with we cannot discard that sustained increased plasma levels of pro-­ DSS-­induced colitis used to assess viscerosensitivity. In the present inflammatory cytokines after clinical colitis resolution might con- conditions, moderate pro-­algesic responses to the mechanical prob- tribute to the sensitization process. Altogether, these data suggest ing of the hind paws were detected, without correlation with the that a combination of peripheral and central (spinal) sensitization visceromotor responses elicited by CRD, thus indicating a relatively might occur during DDS-­induced colitis as the underlying mech- poor association between DSS-­induced colitis and the induction of anism explaining the mechanical hypersensitivity observed in this referred somatic hyperalgesia. model. Interestingly, this reproduces the sensitization mechanisms In summary, we present evidence supporting the feasibility of postulated in humans with visceral (intestinal) hypersensitivity, DSS-­induced colitis in rats as an experimental model of long-­lasting such as during IBS, which is supposed to include central and pe- visceral hypersensitivity, reminiscent of the hypersensitive state 4,59 ripheral alterations in pain processing. observed in humans with functional and inflammatory GI disorders. To further understand the mechanisms mediating colitis-­ The changes in viscerosensitivity observed are likely to result from associated visceral hypersensitivity, the peripheral (colon) and the interaction between central (spinal cord) and peripheral (colon) central (spinal cord) expression of different sensory-­related mark- sensitizing mechanisms implicating a site-­specific modulation of ers, mediating both pro-­ and anti-­nociceptive responses, was also both pro-­ and anti-­nociceptive pathways and resulting in a persistent assessed. Interestingly, differential tissue-­ (colon vs. spinal cord) state of sensitization. The overall characteristics of the model make and sensory-­related (pro-­nociceptive vs. anti-­nociceptive) mod- feasible its use in the characterization of new therapeutic targets ulatory responses were detected during inflammation. Within the and the validation of new drugs for the treatment of visceral pain colon, an up-­regulation of the pro-­nociceptive markers TRPV1 and originating within the GI tract. In any case, these results warrant Ramp1 was detected, thus suggesting a facilitation of nociceptive further validation studies as to ensure the translational relevance responses consistent with the state of hyperalgesia observed in the of the model. same animals. This agrees with the described role of TRPV1 and its modulation during inflammation in other animal models as well as in AU T H O R C O N T R I B U T I O N S humans. 22,60–­62 On the contrary, the up-­regulation of Ramp1 could VM, Conceptualization, Methodology, Investigation, Resources, lead to an increase of CGRP-­mediated pro-­nociceptive responses Writing-­review & editing, Supervision. SL-­E, Methodology, at peripheral sites.63 Within the lumbosacral spinal cord, an overall Investigation, Formal analysis, Writing-­original draft, Writing-­review down-­regulation of pro-­nociceptive markers and an up-­regulation & editing. JML-­T, Methodology. MP, Methodology. of anti-­nociceptive markers was observed. These responses might seem incongruent with the presence of hypersensitivity and central C O N FL I C T O F I N T E R E S T spinal sensitization (as discussed above), but, as a whole, they can The authors declare no competing interests. be interpreted as the expression of compensatory mechanisms as an attempt to normalize nociceptive mechanisms and thus avoiding ORCID excessive/aberrant pain responses in states of sensitization. Here, Vicente Martínez https://orcid.org/0000-0001-5193-715X LÓPEZ-­E STÉVEZ et al. | 11 of 12 23. Hayashi Y, Aoyagi K, Morita I, Yamamoto C, Sakisaka S. Oral ad- REFERENCES ministration of mesalazine protects against mucosal injury and per- 1. 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