Motor, mood, and memory impairments persist during remission periods in chronic colitis and are influenced by neuroinflammation and sex

The FASEB Journal. 2024;38:e70133. | 1 of 17 https://doi.org/10.1096/fj.202400837R wileyonlinelibrary.com/journal/fsb2 Received: 12 April 2024 | Revised: 25 September 2024 | Accepted: 15 October 2024 DOI: 10.1096/fj.202400837R RESEARCH ARTICLE Motor, mood, and memory impairments persist during remission periods in chronic colitis and are influenced by neuroinflammation and sex GemaSotelo- Parrilla | AlejandroRuiz- Calero | PabloGarcía- Miranda | María L.Calonge | María D.Vázquez- Carretero | María J.Peral This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology. Gema Sotelo- Parrilla and Alejandro Ruiz- Calero should be considered joint first authors. Departamento de Fisiología, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain Correspondence Pablo García- Miranda and María D. Vázquez- Carretero, Departamento de Fisiología, Facultad de Farmacia, Universidad de Sevilla, C/Profesor García González, n° 2, 41012 Sevilla, Spain. Email: [email protected] and m[email protected] Funding information Ministerio de Ciencia e Innovación (MCIN), Grant/Award Number: PID2019- 105632RB- I00 Abstract Ulcerative colitis is a chronic pathology characterized by relapsing–remitting phases of intestinal inflammation. Additionally, some patients develop neuropsychiatric disorders, such as depression and anxiety, or cognitive deficits. We aimed to investigate whether the development of chronic colitis elicits memory, locomotion, and mood impairments. It further examined whether these impairments are influenced by the relapsing–remitting phases of the colitis or by sex. Here, we used a chronic colitis model in male and female rats, induced with sodium dextran sulfate, mirroring the phases of human ulcerative colitis. Our results revealed that the severity of colitis was slightly higher in males than females. Chronic colitis triggered motor and shortterm memory deficits and induced anxiety- and depressionlike behaviors that remained throughout the development of the disease. There are also sex differences under control or inflammatory conditions. Therefore, in both situations, females compared to males displayed: (i) slightly lower locomotion, (ii) increased anxietylike behaviors, (iii) similar depressionlike behaviors, and (iv) similar shortterm memory deficit. Additionally, under control conditions, the mRNA levels of IL- 1β, IL- 6, and TNF- α were higher in the female hippocampus. In both sexes, when chronic colitis was established, the neuroinflammation was evidenced by increased mRNA levels of these three cytokines in the hippocampus and in the motor and prefrontal cortices. Interestingly, this neuroinflammation was slightly greater in males. In summary, we show that the development of chronic colitis caused persistent behavioral abnormalities, highlighting sex differences, and that could be a consequence, at least in part, of the increase in IL- 1β, IL- 6, and TNF- α in the brain. KEYWORDS anxiety, depression, memory, motor activity, sex differences, ulcerative colitis Abbreviations: DAI, disease activity index; DSS, sodium dextran sulfate; IBD, inflammatory bowel diseases; IL-1β, interleukin-1 beta; IL-6, interleukin-6; PBS, phosphate buffer saline; PFA, paraformaldehyde; SEM, standard error of the mean; TNF-α, tumor necrosis factor-alpha; UC, ulcerative colitis 2 of 17 | SOTELOPARRILLA etal. 1 | INTRODUCTION Ulcerative colitis (UC) is one of two inflammatory bowel diseases (IBD) characterized by chronic inflammation of the intestine with relapsing–remitting phases, and severe symptoms including diarrhea, rectal bleeding, and weight loss.1 Its pathogenesis is complex and unclear, and factors such as age, sex, genetics, and immunity affect its evolution.1–3 In addition, neuropsychiatric disorders, such as depression and anxiety, are more prevalent in UC patients than in the general population.3,4 These patients declare reduced emotional wellbeing, cognitive dysfunctions, and mood disturbances.5 These symptoms are associated with poor quality of life of UC patients, especially during acute episodes.4,6 Mood alterations, such as depression or anxietylike behaviors, and memory deficits, were observed in UC animal models.7 However, in these models, results on the effects of colitis on locomotion are controversial. Several authors reported a reduced motor activity,8–12 whereas others did not find effects.13–19 Most studies on behaviors used an acute model of colitis and performed the tests at a time point of the treatment despite being human UC a chronic progressive condition with differences between the active and resolution phases.4,20 Sex differences have also been found in the frequency and clinical course of mood disorders in UC patients, with women with UC being more likely than men to develop anxiety and depression.3,4 However, the results of sex differences were not clear when an animal model of experimental colitis was used. In these models, for example, Painsipp etal.13 found that the female mice suffer more depressionlike behaviors and the males more anxiety, other authors, even though using female and male rodents in their studies, did not make a sex differentiation on behavior,21,22 and others did not find differences between sexes.9,16,18 Regarding the motor behaviors, there are scarcely any reports examining whether, in the colitis models, the sex affects these behaviors. Consequently, these limited studies indicated a comparable reduction in locomotion across both sexes, with no discernible effect in either sex.9,13,16,18,22 Gut–brain axis could be the route that leads to these impairments in behaviors as has been suggested.7,20,23 The peripheral inflammation caused by chronic colitis can elicit a sustained neuroinflammation over time, which is considered an important inducer for the development of neurodegenerative diseases.24 This inflammatory environment in the brain is possibly produced by the activated microglia and astrocytes that release proinflammatory cytokines, such as interleukin- 1 beta (IL- 1β), interleukin- 6 (IL- 6) and tumor necrosis factoralpha (TNF- α).25 These proinflammatory cytokines may modify neuronal functions resulting in behavioral abnormalities. Although neuroinflammation has been observed in various brain regions of animals with colitis and is considered a plausible responsible for behavioral impairments,7,26 there is limited direct evidence supporting this link. Previous studies, including one of ours, have indirectly associated increased expression of proinflammatory cytokines with behavioral alterations.16,22,27 We previously reported that acute colitis in male rats triggered neuroinflammation and increased excitability of the motor cortex and impaired motor activity and coordination.27 In this work, we used a DSS- based model of chronic colitis in rats, considered a wellestablished animal model for studying UC. Moreover, we can take advantage of investigating the relapsing–remitting phases of UC and more specifically the changes with the activity of the disease, since in humans it would require an exhaustive followup of the patient who goes through these periods.28 We asked whether, in male and female rats, in these phases throughout the development of chronic colitis: (i) motor activity, mood, and memory are altered, (ii) neuroinflammation occurs in the motor cortex, hippocampus, and/or prefrontal cortex, and, (iii) these effects are different depending on the sex of the animal. So far, no studies have dealt with the evolution of such motor activity, mood, and memory impairments that occur throughout the development of chronic colitis considering relapsing–remitting phases and sex differences. 2 | MATERIALS AND METHODS 2.1 | Animals Male and female Wistar rats (45 dayold) were used. This study included 16–30 adult rats per experimental group, evenly divided into 8–15 males and 8–15 females. They were humanely handled, and efforts were made to minimize the number of animals used and their suffering. The procedures concerning the protection of experimental animals were following the guidelines of the European Union Council (Directive 2010/63/ UE) and the Spanish Royal Decree (BOE 34/11370, 2013). The research protocol was approved by the Animal Ethics Committee of the University of Seville and Junta de Andalucía (Number of approval project: CEEAUS2021- 14, 21/03/2022/051). The rats were obtained from the “Centro de experimentación animal de la Universidad de Sevilla”. Animals were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) plus xylazine (10 mg/kg) and sacrificed. 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 3 of 17 SOTELOPARRILLA etal. 2.2 | Biological preparations Once the rats were sacrificed, the colons were removed and washed with icecold saline solution and their weights and lengths measured. Segments of the distal colon were either fixed by overnight incubation with phosphate buffer saline (PBS; in mM, 137 NaCl, 2.7 KCl,10 Na2HPO4, and 1.8 KH2PO4; pH 7.4) containing 4% paraformaldehyde (PFA) for histological analysis or frozen in liquid nitrogen and stored at −80°C until its use in RNA extraction to perform PCR- measurements. The hippocampus, motor, and prefrontal cortices were also removed, frozen, and stored at −80°C until their use for PCR measurements. 2.3 | Induction and assessment of experimental chronic colitis Rats with similar body weight values were randomized into untreated or water groups (control) and sodium dextran sulfate (DSS)- treated groups. The control rats' groups drank normal water. Chronic colitis was induced by administering three cycles of 4% DSS (MW 40 kDa; PanReac AppliChem, Spain) for 7 days in drinking water.28 Between DSS cycles, rats received normal tap water for 7 days, except for the third DSS cycle where they only received water for 2 days (Figure1A). This treatment mimics the relapsing–remitting periods of inflammation as those of ulcerative colitis in humans. Throughout the DSS treatment, von Frey filaments were used to assess nociceptive responses by applying the stimulus in the abdominal area in both DSS- treated and untreated female and male rats. Induction and progression of the colon inflammation were evaluated by determining clinical manifestations using the disease activity index (DAI) and colon parameters, such as weight/length ratio, histological score, and mRNA relative expression of colonic proinflammatory cytokines, as previously described.29 For DAI evaluation, every 2–3 days, throughout the DSS treatment, the animals were monitored for body weight gain, stool consistency, and the presence of blood in feces. For histological scoring, at the end of each DSS or water period and in parallel with control groups, rats were sacrificed, and paraffinembedded sections of the removed distal colon were stained with hematoxylin/eosin. Colon injury was graded by evaluating the destruction of epithelium, the dilatation of crypts, the loss of goblet cells, the inflammatory cell infiltrate, edema, and crypt abscesses. For DAI or FIGURE 1 Evaluation of colitis in DSS- treated female and male rats. (A) Experimental design. Either female or male rats received three cycles of DSS in their drinking water for 7 days, with a 7- day recovery period between each cycle. Controls received normal tap water throughout the experiment. Behavioral tasks were conducted on days: 0 (pretest), 9, 16, 23, 30, and 37 (red). Rats were euthanized and assessed for colon histological alterations at all time points indicated. (B) The disease activity index (DAI) was assessed every 2–3 days. (C) Colon weight/length ratio. Data are means ± SEM (n = 8–15 animals per group). ANOVA followed by the Tukey post hoc test: ap < .05 males vs females and bp < .001 DSS vs water. Female Male WaterDSS 0.00 0.04 0.08 0.12 b b a 0.16 Colon weight / length ratio (g/cm) 0714 21 28 35 42 0 1 2 3 4 5 6 Days DAI b b b b b b b b b b b b b b b b b b b b b b a,b a,b bb bb (A) (B) (C) 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 of 17 | SOTELOPARRILLA etal. histological assessment, each of the items was scored on a scale of 0 to 3 and added together to obtain a cumulative score. 2.4 | Relative quantification of realtime PCR Realtime PCR was carried out as previously described.29 Briefly, total RNA was extracted from the distal colon, hippocampus, prefrontal cortex, and primary motor cortex of control and DSS- treated rats using RNeasy® kit (Qiagen). cDNA was synthesized from 1 μg of total RNA using QuantiTect® reverse transcription kit (Qiagen) as described by the manufacturer. The primers used are given in TableS1. Realtime PCR was performed with NZYSupreme qPCR Green Master Mix® (MB41903; Nzytec). β- Actin served as a reference gene and was used for sample normalization. The comparative Ct method was applied to determine the mRNA relative expression and the lowest value measured of each gene was set at 1. Results are also presented as fold change for each cytokine. Fold changes are calculated by dividing the relative expression of each gene in rats with DSS treatment by that in rats with normal water (control). All these determinations were performed in samples taken on day 37 of DSS treatment. 2.5 | Behavioral tests The behavioral tests conducted in this study were the open field test and the novel object recognition test. They were first administered on day 0 (pretest) to all animals before randomizing them in water (control) and DSS groups. Then, as indicated in Figure1A, the tests were performed on days: 9, 16, 23, 30, and 37 of DSS treatment, which is 1–2 days after each period, in the case of DSS to avoid coinciding with the sudden changes in the peaks of inflammation. Control groups are evaluated in parallel. 2.5.1 | Open field test This test was used to assess the motor activity and the state of mind of rats, either untreated or DSS treated. It was conducted in a normally illuminated room, where the animals were acclimated for 30 min. The open field was a white plastic opaque cage measuring 50 cm long × 35 cm wide × 35 cm high. The cage floor was divided into 15 squares and placed in a soundfree room. A video camera was mounted above the open field to record the movements of each animal. The rat was placed in the center of the cage and left to explore freely for 5 min, and all its movements were recorded. The cage was cleaned with 70% ethanol and dried after each rat session to remove olfactory cues. The following parameters were manually counted: percentage of time spent in the center area, percentage of immobility time, number of crossings, number of rearings, and number of groomings. Furthermore, DeepLabCut software was used to measure automatically: the mean speed and distance traveled in the whole and in the center of the cage, the number of transitions from the periphery to the center of the cage, total time spent in the corners, and immobility time spent in the corners, following the methodology previously described30–33 with some modifications. 2.5.2 | Novel object recognition test This test was used to assess shortterm memory and is based on the rat's attraction to novelty and the unknown. Before experimentation, rats were acclimated to the room for 30 min in the home cage. The task was conducted in the same opaque white plastic cage used in the openfield test after allowing the rats to explore freely for 5 min. The task consisted of three phases: training, resting, and testing. The movements performed by the rats during the training and the testing phases were recorded on video. During the training phase, the rat was placed in the center of the cage and allowed to freely explore for 10 min two identical objects placed on the opposite sides of the cage. The total time that the animal explores the two identical objects in the training phase (expressed as a percentage of the total time) was also measured. Subsequently, during the resting phase, the rat was placed in the home cage for 1 h. For the testing phase, one of the objects was replaced with a novel one, and then the rat was placed back in the cage and allowed to freely explore for 10 min. The total time that each rat investigated each object (nose facing the object within a maximum distance of 2 cm) was analyzed and used to calculate the discrimination index. This index assesses the preference for the novel object and was calculated by dividing the total time exploring the new object by the total time exploring both objects and multiplying by 100. A value of 50% or more indicates that the rat prefers the novel object which, in turn, points out that the rat remembers and recognizes the familiar one. All objects were appropriately cleaned using 70% ethanol to remove odor cues between trials. Objects designated as familiar or novel were randomized among behavioral trials. To ensure that all the objects used were equally attractive to the rats, a pretest for saliency was performed with a different group of rats with no previous experience 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 5 of 17 SOTELOPARRILLA etal. with the objects. The saliency test was conducted using the same objects that would be used later for the novel object recognition test and the value obtained for the discrimination index was 50 ± 2 (mean ± SEM), n = 30, indicating that the rats did not prefer any of the objects over another. 2.6 | Statistical analysis Data are presented as the mean ± the standard error of the mean (SEM). Statistical analyses were performed separately for male rats (n = 8–15) and female rats (n = 8–15) to investigate sexspecific effects. In addition, combined analyses were performed with data pooled from both sexes (n = 16–30). The n was reported in the figure legends. Comparisons between the experimental groups were evaluated with the ANOVA followed by Tukey's post hoc test using the GraphPad Prism software version 8.0. The differences were set to be significant at p < .05. The Pearson correlations were performed using the R 4.3.2 version (RStudio Team, 2023, Boston, MA, USA), and the graphics were built using the ggplot2 package version 3. 5.0.34 3 | RESULTS 3.1 | The development of DSS- induced colitis presents sex differences once the chronic colitis is established We start the study by evaluating clinical manifestations and histology of the colon to assess DSS- induced colitis severity along with the development of the disease in male and female rats. For that we use the disease activity index (DAI) and score the colon injury using the parameters previously described in Methods section. The DSS model used here (Figure1A) resembles the development of ulcerative colitis, since it provides an alternation between relapsing–remitting phases similar to those currently occurring in humans. All rats exposed to the DSS cycles developed these characteristic phases of chronic colitis. Figure1B shows increased DAI values in both male and female rats compared to control rats, peaking on days 7, 21 and 35, whereas remission values are observed on days 16 and 30. The DAI was higher in males than in females only at the end of DSS- treatment when chronic colitis is established. Figure1C reveals that the colon weight/length ratio on day 37 was higher in DSS- treated rats indicating colonic inflammation in those animals. The results also showed that this relationship was greater in males compared to females, again indicating higher severity in males at this point of treatment. Histological analysis performed along DSS treatment (Figure2A–C) shows that colon injury, modified with the pace of the development of colitis, was significantly higher on day 9 followed by days 23 and 37 and lower on days 16 and 30. The score indicating the loss of crypt architecture and the infiltration of inflammatory cells in the mucosa and submucosa (Figure2C) throughout the development of colitis was similar in females and males, and although not statistically significant the damage was slightly greater in males on day 37. Moreover, we examined the mRNA relative abundance of colon proinflammatory cytokines IL- 1β, IL- 6, and TNF- α on day 37 of DSS treatment, when chronic colitis is established. Figure3A shows that the mRNA abundance of the three cytokines increased in the colon of DSS- treated rats with data from females and males combined. Beside these changes, we found sex differences since IL- 1β and TNF- α increased in the colon of both sexes but IL- 6 only in DSS- treated males, compared to untreated rats (Figure3B). In addition, the observed increase in IL- 1β and IL- 6, due to DSS treatment, was higher in males as compared to females, but that of TNF- α was similar in both sexes. We did not find sex differences in the mRNA abundances of the colonic proinflammatory cytokines under control conditions (Figure3B). These results indicate that while chronic colitis develops similarly in both sexes males experienced slightly more severe chronic colitis. 3.2 | Motor activity is reduced throughout the development of chronic colitis and partially affected by sex Motor activity analysis of the animals was carried out using the open field test. The results are summarized in Figure4. First, we observed that under control conditions, none of the parameters evaluated in either females or males showed differences when comparing day zero with other time points, indicating that motor behavior is not affected, whether analyzing data from both sexes combined or separated by sex (Figure4A,B). The development of chronic colitis in DSS- treated rats (combined data) led to a general diminution of locomotion, displaying a lower number of rearings and crossings, indicating a reduction in vertical and horizontal motor activity respectively, and a decreased traveled distance and average speed (Figure4A). These motor deficits were evident from day 9 and continued throughout the development of chronic colitis, there was no correspondence with the remitting–relapsing phases of 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 of 17 | SOTELOPARRILLA etal. the pathology observed in the DAI (Figure1B) and in the colon histology (Figure2). Furthermore, the differences between each time point and day zero were significant in all parameters indicating that DSS- treated rats do not return to normal locomotion at any time during the development of colitis. These colitisinduced motor deficits were also observed in both males and females when data were analyzed separately by sex (Figure4B). Sex differences were found in the average speed on day 9 of DSS treatment showing that females moved slower than males. In addition, there was a sex effect on the traveled distance in both the water and the DSS groups. Female rats traveled shorter distances compared to males under control conditions (water group) at all the time points measured, and under inflammatory conditions on days 9, 23, 30 and 37 of DSS treatment. We also calculated the differences in traveled distance between males and females under both control and DSS conditions (Table1). Comparison of these values indicated that the effect of colitis on that parameter was similar in both sexes, and therefore the observed differences between females and males were inherent to sex. Representative images of the movement tracks from the rats on day 37 (Figure4C) show this sex effect on the traveled distance. Taken together, motor deficits persisted during chronic colitis in both sexes and the female rats traveled shorter distances under both inflammatory and control conditions. FIGURE 2 Histological analysis of colon from DSS- treated and untreated female and male rats. Representative hematoxylin–eosinstained colon sections from water (A) and DSS (B) groups. The area indicated by the blue square is enlarged next to each image. Scale bar represents 400 μm in the lowmagnification images and 100 μm in magnified images. (C) Score of colon damage. Data are means ± SEM (n = 8–15 animals per group). ANOVA followed by the Tukey post hoc test: bp < .05 DSS vs water and cp < .05, ccp < .001 each time point of treatment vs day 9 of DSS treatment. DSSday: FEMALES MALES Histological score g 916 23 37 30 Water Water DSS DSS Female Male (A) (B) (C) 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 7 of 17 SOTELOPARRILLA etal. 3.3 | Anxietylike behaviors are induced throughout the development of chronic colitis and differ by sex Next, we wondered whether the rats' moodrelated behaviors throughout the development of chronic colitis were modified and whether these alterations, if any, were different due to sex. For this purpose, we used the openfield test and analyzed the parameters linked to anxiety. As in motor activity, in none of the evaluated parameters there were differences between day zero compared to other time points of water (control) groups. We found anxietylike behaviors induced by the development of chronic colitis at all the time points measured (combined data). DSS- induced colitis led to a reduction in the following parameters: (i) the time spent in the center of the cage, (ii) the number of transitions to the central area, and (iii) the locomotion in the center (traveled distance and average speed); and an increase in both, the total time spent and the time immobile in the corners (Figure5A,C). These modifications were maintained along DSS treatment, without changes associated with the remitting–relapsing phases of the pathology and persisted when the DAI almost returned to normal in the remission periods. Comparisons of each time point with day zero were significant in all parameters revealing that the rats with colitis do not return to normal behavior at any time during the development of chronic colitis. These colitisinduced anxietylike behaviors were also observed in both males and females when data were analyzed separately by sex (Figure5B,C). Sex differences related to the parameters measured at the center of the cage were also found, such as the time spent, the number of transitions, and the traveled distance (Figure5B). Therefore, females exhibited lower values, including those from day zero, than those of males under control and inflammatory conditions in most of the time points measured. Differences between sexes in the parameters related to corners were also observed. However, in this case, most sexrelated effects were observed in the still time in the corners instead of in the total time spent over there (Figure5C). Thereby both control and DSS- treated females remained more time immobile at the corners than males at all the time points measured. We then calculated the differences between males and females under both control and DSS conditions for those parameters where the sexes differed in the water group (Table1). Comparison of these values indicated that the effect of colitis on those parameters was similar in both sexes, and therefore, the observed differences between females and males were inherent to sex. Representative activity tracking from the rats on day 37 shows these effects described so far (Figure4C). To sum up this section, the development of chronic colitis triggered anxietylike behaviors, with females exhibiting a more anxious profile than males under both control and inflammatory conditions. 3.4 | Depressionrelated behaviors are induced throughout the development of chronic colitis but without sex differences Next, we evaluated the effect of DSS- induced chronic colitis on two parameters related to depressivelike behaviors using again the openfield test: the number of groomings and the immobility time, understood as freezing time or the time that rats are completely quiet. First, the results showed that in the control groups none of the measured parameters exhibited differences between day zero and the other time points (Figure6A,B). DSS treatment increased immobility time and affected selfcare behavior, FIGURE 3 mRNA relative expression of proinflammatory cytokines in the colon of DSS- treated and untreated rats on day 37 of the treatment (chronic colitis). mRNA levels were expressed as relative abundance in each experimental group. The analysis was performed using (A) the combined measures from both sexes together or (B) separately. Data are means ± SEM (n = 16–30 animals per group in colitis effectrelated analysis; n = 8–15 animals per group in sex effectrelated analysis). ANOVA followed by the Tukey post hoc test: ap < .05 female vs male; bp < .001 DSS vs water. βα FemalesMales WaterDSS β α (A) (B) 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 of 17 | SOTELOPARRILLA etal. as reflected in a reduced number of groomings, whether the data were combined or analyzed separately by sex (Figure6A,B). These behavioral abnormalities began on day 9 of DSS treatment and were maintained throughout the development of chronic colitis. Again, compared to each time point with day zero, differences were significant in both parameters, revealing that at any time the rats with colitis return to normal behavior. No differences between sexes have been found under control or inflammatory conditions. These results indicate that the development of chronic colitis promotes depressionrelated behaviors equally in both sexes. 3.5 | Shortterm memory is impaired throughout the development of chronic colitis but without sex differences To also evaluate whether chronic colitis alters cognitive functions, we assessed shortterm memory using the novel object recognition test in DSS- treated and untreated rats, and in both females and males. First, we analyzed the total time that rats spent exploring the two identical objects and then, after changing one of the objects for a new one, we determined the discrimination index. Data are shown combined (Figure7A) and separated by sex (Figure7B). The results revealed that the chronic colitis did not modify the FIGURE 4 Motor activity in DSS- treated and untreated female and male rats. The parameters related to motor activity, measured using the open field test, were analyzed either (A) for both sexes combined or (B) separately. (C) Representative images of the movement tracks of the rats on day 37 (chronic colitis). Each purple spot of the image represents 1 s. Data are means ± SEM (n = 16–30 animals per group in colitis effectrelated analysis; n = 8–15 animals per group in sex effectrelated analysis). ANOVA followed by the Tukey post hoc test: ap < .05 female vs male; bp < .05 DSS vs water; cp < .05 each time point of treatment vs day 0. Females Water Males DSS WaterDSS WaterDSS Water Water DSS DSS Female Male Travelled distance Averagespeed RearingsCrossings 0916 23 30 37 0 10 20 30 40 Number b,cb,cb,cb,c b,c 0916 23 30 37 0 50 100 150 Number b,c b,c b,c b,cb,c 0916 23 30 37 0 500 1000 1500 2000 2500 Distance (cm) b,cb,cb,cb,cb,c 0916 23 30 37 9 10 11 12 13 14 Speed(cm/s) b,c b,c b,c b,c b,c Number 0916 23 30 37 0 10 20 30 40 b,c b,cb,c b,cb,c b,c b,c b,c b,c b,c Number 0916 23 30 37 0 50 100 150 b,c b,cb,cb,c b,c b,c b,cb,cb,c b,c Distance (cm) 0916 23 30 37 0 500 1000 1500 2000 2500 aaaaa b,c a,b,c b,c b,c b,cb,c b,ca,b,ca,b,ca,b,c a Speed(cm/s) 0916 23 30 37 9 10 11 12 13 14 b,cb,cb,cb,c b,c b,c b,cb,cb,cb,c (A) (B) (C) 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 9 of 17 SOTELOPARRILLA etal. total time of exploration, indicating that the rats did not lose their curiosity for exploration, an instinctive activity of animals in the face of novelty. In addition, we observed that the discrimination index did not change in rats that drank water compared to day zero with the other time points, indicating that the shortterm memory does not change under control conditions. A different effect was observed in DSS- treated rats displaying a decrease in the discrimination index throughout the entire treatment (Figure7A), indicating that the impairment in shortterm memory stayed at the same level without recovery in the remission periods of the colitis. Significant differences in this index as compared each time point with the day zero confirmed this effect. On the other hand, we did not find sex differences; however, the observed changes in both sexes have different tendencies (Figure7B). The reduction in the discrimination index in the females was more pronounced on days 9 (relapsing phase) and 16 (remitting phase) and then increased slightly along the development of colitis indicating an improvement in the memory, whereas in the males the values decreased from day 9 and remained that way throughout the treatment. Representative images of the heatmap plots from the rats on day 37 are shown in Figure7C. 3.6 | Proinflammatory cytokines are increased in the brain by chronic colitis establishment and differ by sex Next, we wondered whether mood, motor activity, and shortterm memory alterations observed in female and male rats with chronic colitis were associated with a neuroinflammation induced by the colitis in the brain regions involved in those tasks. For this purpose, we determined the mRNA abundance of the proinflammatory cytokines IL- 1β, IL- 6, and TNF- α, by realtime PCR, on day 37 in both sexes, in water (control) and DSS groups, when the chronic colitis is established, in hippocampus, motor cortex, and prefrontal cortex. The results revealed that chronic colitis increased the mRNA expression levels of the three cytokines in the three brain regions explored indicating neuroinflammation (Figure8). In addition, we found some sex differences in the relative amount of the three cytokines. In control rats, mRNA abundance of the three cytokines, in the hippocampus and those of TNF- α and IL- 1β in the prefrontal cortex, was higher in females than in males. However, in the motor cortex, we did not find differences between the sexes in any of the three cytokines analyzed (Figure9A). If we pay attention to the cytokine relative abundances in DSS- treated rats, we observe the samesex differences as previously mentioned under control conditions (Figure9A). We did not observe sex differences in the motor cortex. It is worth mentioning that of the three brain regions analyzed, the hippocampus showed the highest levels of cytokine expression in females in both control and inflammatory conditions. Nevertheless, to find out the effect of chronic colitis according to sex, we calculated the expression levels of the three cytokines, for each sex, as a fold change between the DSS group and their own water group, and the results were different. A significantly greater increase of IL- 1β in the hippocampus and of IL- 6 in prefrontal and motor cortices were found in the males compared to the females (Figure9B). Only TNF- α was significantly higher in females than in males in the motor cortex. The results indicated that chronic colitis triggered neuroinflammation with differing profiles between sexes. Females had higher baseline cytokine levels, but males reached greater neuroinflammation. TABLE 1 Differences in behavior parameters between males and females under control and inflammatory conditions. Day Group Traveled distance (cm) Time in the center (%) Transitions to the center (number) Traveled distance in the center (cm) Still time to the corners (s) 9 Water 534 ± 51 0.7 ± 0.2 10.1 ± 1.3 65 ± 8 69 ± 7.7 DSS 623 ± 78 1.2 ± 0.2 6.8 ± 1 51 ± 5 77 ± 10 16 Water 575 ± 75 0.8 ± 0.2 8.6 ± 2 78 ± 11 63 ± 5 DSS 445 ± 57 0.5 ± 0.2 7 ± 1.4 51 ± 8 42 ± 11 23 Water 448 ± 47 1.1 ± 0.2 8.3 ± 1.4 64 ± 9 65 ± 7.7 DSS 579 ± 67 0.5 ± 0.2 6.7 ± 1.4 67 ± 9 79 ± 12 30 Water 526 ± 67 0.4 ± 0.3 8.8 ± 1.2 73 ± 13 58 ± 8 DSS 632 ± 70 0.9 ± 0.2 7.1 ± 2 66 ± 10 71 ± 12 37 Water 616 ± 70 0.7 ± 0.3 9.2 ± 1.8 62 ± 9 38 ± 7 DSS 595 ± 80 0.5 ± 0.5 9.8 ± 1.5 50 ± 12 46 ± 21 Note: Data are expressed as means ± SEM (n = 8–15). ANOVA analysis did not find significant differences between groups. 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 16 of 17 | SOTELOPARRILLA etal. Pablo García- Miranda https://orcid. org/0000-0003-2588-6904 María L. Calonge https://orcid. org/0000-0002-2002-024X María D. Vázquez- Carretero https://orcid. org/0000-0001-9373-5679 María J. Peral https://orcid.org/0000-0003-0343-7550 REFERENCES 1. Ungaro R, Mehandru S, Allen PB, Peyrin- Biroulet L, Colombel JF. Ulcerative colitis. Lancet. 2017;389:1756-1770. 2. Porter RJ, Kalla R, Ho GT. Ulcerative colitis: recent advances in the understanding of disease pathogenesis. F1000Research. 2020;9:294. 3. Fracas E, Costantino A, Vecchi M, Buoli M. Depressive and anxiety disorders in patients with inflammatory bowel diseases: are there any gender differences? Int J Environ Res Public Health. 2023;20(13):6522. 4. Barberio B, Zamani M, Black CJ, Savarino EV, Ford AC. Prevalence of symptoms of anxiety and depression in patients with inflammatory bowel disease: a systematic review and metaanalysis. Lancet Gastroenterol Hepatol. 2021;6:359-370. 5. Goodhand JR, Wahed M, Mawdsley JE, Farmer AD, Aziz Q, Rampton DS. Mood disorders in inflammatory bowel disease: relation to diagnosis, disease activity, perceived stress, and other factors. Inflamm Bowel Dis. 2012;18:2301-2309. 6. Tribbick D, Salzberg M, Connell W, et al. Differences across illness perceptions in inflammatory bowel disease and their relationships to psychological distress and quality of life. Gastroenterol Nurs. 2017;40:291-299. 7. Masanetz RK, Winkler J, Winner B, Günther C, Süß P. The gut–immune–brain axis: an important route for neuropsychiatric morbidity in inflammatory bowel disease. Int J Mol Sci. 2022;23:11111. 8. Hassan AM, Jain P, Reichmann F, etal. Repeated predictable stress causes resilience against colitisinduced behavioral changes in mice. Front Behav Neurosci. 2014;6:8. 9. Nyuyki KD, Cluny NL, Swain MG, Sharkey KA, Pittman QJ. Altered brain excitability and increased anxiety in mice with experimental colitis: consideration of hyperalgesia and sex differences. Front Behav Neurosci. 2018;12:58. 10. Salameh E, Meleine M, Gourcerol G, et al. Chronic colitisinduced visceral pain is associated with increased anxiety during quiescent phase chronic colitisinduced visceral pain is associated with increased anxiety during quiescent phase. Am J Physiol Gastrointest Liver Physiol. 2019;316:692-700. 11. Carloni S, Bertocchi A, Mancinelli S, etal. Identification of a choroid plexus vascular barrier closing during intestinal inflammation. Science. 2021;374(6566):439-448. 12. Zhou Y, Ji G, Yang X, Chen Z, Zhou L. Behavioral abnormalities in C57BL/6 mice with chronic ulcerative colitis induced by DSS. BMC Gastroenterol. 2023;23(1):84. 13. Painsipp E, Herzog H, Sperk G, Holzer P. Sexdependent control of murine emotionalaffective behaviour in health and colitis by peptide YY and neuropeptide y. Br J Pharmacol. 2011;163:1302-1314. 14. Chen J, Winston JH, Fu Y, etal. Genesis of anxiety, depression, and ongoing abdominal discomfort in ulcerative colitislike colon inflammation. Am J Physiol Regul Integr Comp Physiol. 2015;308:18-27. 15. Jain P, Hassan AM, Koyani CN, etal. Behavioral and molecular processing of visceral pain in the brain of mice: impact of colitis and psychological stress. Front Behav Neurosci. 2015;9:177. 16. Gadotti VM, Andonegui G, Zhang Z, etal. Neuroimmune responses mediate depressionrelated behaviors following acute colitis. iScience. 2019;16:12-21. 17. Matisz CE, Vicentini FA, Hirota SA, Sharkey KA, Gruber AJ. Behavioral adaptations in a relapsing mouse model of colitis. Physiol Behav. 2020;216:112802. 18. Salvo E, Stokes P, Keogh CE, etal. A murine model of pediatric inflammatory bowel disease causes microbiotagut- brain axis deficits in adulthood. Am J Physiol Gastrointest Liver Physiol. 2020;319:361-374. 19. Sohrabi M, Pecoraro HL, Combs CK. Gut inflammation induced by dextran sulfate sodium exacerbates amyloidβ plaque deposition in the AppNL−G−F mouse model of Alzheimer's disease. J Alzheimers Dis. 2021;79:1235-1255. 20. Abautret- Daly Á, Dempsey E, Parra- Blanco A, Medina C, Harkin A. Gutbrain actions underlying comorbid anxiety and depression associated with inflammatory bowel disease. Acta Neuropsychiatrica. 2018;30:275-296. 21. Fei HX, Li LL, Biao XW, et al. Chronic colitis exacerbates NLRP3- dependent neuroinflammation and cognitive impairment in middleaged brain. J Neuroinflammation. 2021;18:153. 22. Cluny NL, Nyuyki KD, Almishri W, etal. Recruitment of α4β7 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxietylike behavior. J Neuroinflammation. 2022;19:73. 23. Keightley PC, Koloski NA, Talley NJ. Pathways in gutbrain communication: evidence for distinct gut- to- brain and brain- to- gut syndromes. Aust N Z J Psychiatry. 2015;49:207- 214. 24. Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH. Mechanisms underlying inflammation in neurodegeneration. Cell. 2010;140:918-934. 25. Butovsky O, Weiner HL. Microglial signatures and their role in health and disease. Nat Rev Neurosci. 2018;19:622-635. 26. Craig CF, Filippone RT, Stavely R, Bornstein JC, Apostolopoulos V, Nurgali K. Neuroinflammation as an etiological trigger for depression comorbid with inflammatory bowel disease. J Neuroinflammation. 2022;19:4. 27. Carrascal L, Vázquez- Carretero MD, García- Miranda P, et al. Acute colon inflammation triggers primary motor cortex glial activation, neuroinflammation, neuronal hyperexcitability, and motor coordination deficits. Int J Mol Sci. 2022;23:5347. 28. Wirtz S, Popp V, Kindermann M, et al. Chemically induced mouse models of acute and chronic intestinal inflammation. Nat Protoc. 2017;12:1295-1309. 29. Carvajal AE, Vázquez- Carretero MD, García- Miranda P, Peral MJ, Calonge ML, Ilundain AA. Reelin expression is upregulated in mice colon in response to acute colitis and provides resistance against colitis. Biochim Biophys Acta Mol basis Dis. 2017;1863:462-473. 30. Mathis A, Mamidanna P, Cury KM, etal. DeepLabCut: markerless pose estimation of userdefined body parts with deep learning. Nat Neurosci. 2018;21:1281-1289. 31. Nath T, Mathis A, Chen AC, Patel A, Bethge M, Mathis MW. Using DeepLabCut for 3D markerless pose estimation across species and behaviors. Nat Protoc. 2019;14:2152-2176. 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | 17 of 17 SOTELOPARRILLA etal. 32. Sturman O, von Ziegler L, Schläppi C, et al. Deep learningbased behavioral analysis reaches human accuracy and is capable of outperforming commercial solutions. Neuropsychopharmacology. 2020;45:1942-1952. 33. Lauer J, Zhou M, Ye S, et al. Multianimal pose estimation, identification and tracking with DeepLabCut. Nat Methods. 2022;19:496-504. 34. Wickham H. ggplot2. 2016. Accessed April 12, 2024. doi:10.1007/978- 3- 319- 24277- 4 35. Bábíčková J, Tóthová Ľ, Lengyelová E, etal. Sex differences in experimentally induced colitis in mice: a role for estrogens. Inflammation. 2015;38:1996-2006. 36. Shah SC, Khalili H, Gower- Rousseau C, etal. Sexbased differences in incidence of inflammatory bowel diseases—pooled analysis of populationbased studies from Western countries. Gastroenterology. 2018;155:1079-1089.e3. 37. Dockman RL, Carpenter JM, Diaz AN, Benbow RA, Filipov NM. Sex differences in behavior, response to LPS, and glucose homeostasis in middleaged mice. Behav Brain Res. 2022;418:113628. 38. Tsao CH, Wu KY, Su NC, Edwards A, Huang GJ. The influence of sex difference on behavior and adult hippocampal neurogenesis in C57BL/6 mice. Sci Rep. 2023;13:17297. 39. Scholl JL, Afzal A, Fox LC, Watt MJ, Forster GL. Sex differences in anxietylike behaviors in rats. Physiol Behav. 2019;211:112670. 40. Zhang T, Ding C, Xie T, etal. Skeletal muscle depletion correlates with disease activity in ulcerative colitis and is reversed after colectomy. Clin Nutr. 2017;36:1586-1592. 41. Zaltman C, Braulio VB, Outeiral R, Nunes T, Natividade de Castro CL. Lower extremity mobility limitation and impaired muscle function in women with ulcerative colitis. J Crohns Colitis. 2014;8:529-535. 42. Emge JR, Huynh K, Miller EN, et al. Modulation of the microbiotagut- brain axis by probiotics in a murine model of inflammatory bowel disease. Am J Physiol Gastrointest Liver Physiol. 2016;310:989-998. 43. Dempsey E, Abautret- Daly Á, Docherty NG, Medina C, Harkin A. Persistent central inflammation and region specific cellular activation accompany depression- and anxietylike behaviours during the resolution phase of experimental colitis. Brain Behav Immun. 2019;80:616-632. 44. Bercik P, Park AJ, Sinclair D, et al. The anxiolytic effect of Bifidobacterium longum NCC3001 involves vagal pathways for gutbrain communication. Neurogastroenterol Motil. 2011;23:1132-1139. 45. Burgueño JF, Lang JK, Santander AM, etal. Fluid supplementation accelerates epithelial repair during chemical colitis. PLoS One. 2019;14:e0215387. 46. Knight P, Chellian R, Wilson R, Behnood- Rod A, Panunzio S, Bruijnzeel AW. Sex differences in the elevated plusmaze test and large open field test in adult Wistar rats. Pharmacol Biochem Behav. 2021;204:173168. 47. Lovick TA, Zangrossi H. Effect of estrous cycle on behavior of females in rodent tests of anxiety. Front Psychiatry. 2021;12:711065. 48. Bangasser DA, Cuarenta A. Sex differences in anxiety and depression: circuits and mechanisms. Nat Rev Neurosci. 2021;22:674-684. 49. Jang SE, Lim SM, Jeong JJ, etal. Gastrointestinal inflammation by gut microbiota disturbance induces memory impairment in mice. Mucosal Immunol. 2018;11:369-379. 50. Mitchell J, Kim SJ, Howe C, etal. Chronic intestinal inflammation suppresses brain activity by inducing neuroinflammation in mice. Am J Pathol. 2022;192:72-86. 51. Castaneda AE, Tuulio- Henriksson A, Aronen ET, Marttunen M, Kolho KL. Cognitive functioning and depressive symptoms in adolescents with inflammatory bowel disease. World J Gastroenterol. 2013;19:1611-1617. 52. Golan D, Gross B, Miller A, etal. Cognitive function of patients with Crohn's disease is associated with intestinal disease activity. Inflamm Bowel Dis. 2016;22:364-371. 53. Petruo VA, Zeißig S, Schmelz R, Hampe J, Beste C. Specific neurophysiological mechanisms underlie cognitive inflexibility in inflammatory bowel disease. Sci Rep. 2017;7:13943. 54. Tadin Hadjina I, Zivkovic PM, Matetic A, etal. Impaired neurocognitive and psychomotor performance in patients with inflammatory bowel disease. Sci Rep. 2019;9:13740. 55. Hopkins CW, Powell N, Norton C, Dumbrill JL, Hayee H, Moulton CD. Cognitive impairment in adult inflammatory bowel disease: a systematic review and metaanalysis. J Acad Consult Liaison Psychiatry. 2021;62:387-403. 56. Bisgaard TH, Allin KH, Keefer L, Ananthakrishnan AN, Jess T. Depression and anxiety in inflammatory bowel disease: epidemiology, mechanisms and treatment. Nat Rev Gastroenterol Hepatol. 2022;19(11):717-726. 57. Guo B, Zhang M, Hao W, Wang Y, Zhang T, Liu C. Neuroinflammation mechanisms of neuromodulation therapies for anxiety and depression. Transl Psychiatry. 2023;13:5. 58. Villa A, Gelosa P, Castiglioni L, etal. Sexspecific features of microglia from adult mice. Cell Rep. 2018;23(12):3501-3511. SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. How to cite this article: Sotelo- Parrilla G, Ruiz- Calero A, García- Miranda P, Calonge ML, Vázquez- Carretero MD, Peral MJ. Motor, mood, and memory impairments persist during remission periods in chronic colitis and are influenced by neuroinflammation and sex. The FASEB Journal. 2024;38:e70133. doi:10.1096/fj.202400837R 15306860, 2024, 20, Downloaded from https://faseb.onlinelibrary.wiley.com/doi/10.1096/fj.202400837R by Readcube (Labtiva Inc.), Wiley Online Library on [14/11/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License