Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.
Inflammatory bowel diseases (IBD), including Crohn's disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and ω3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.
Colitis is an inflammatory condition of the colon that can cause diarrhea and abdominal pain. Colitis can be acute, in response to infection or to stress, or it can develop as a chronic disease, such as in ulcerative colitis (UC), which belongs to the group of inflammatory bowel diseases (IBD). Although the clinical signs of UC have been well described, the pathogenesis is still poorly understood1. It is accepted among experts that UC is a multifactorial disease, with genetic mutations and aberrant immune responses playing a major role2. However, environmental factors, such as style of living and nutrition, also contribute to disease development and progression3.
Unfortunately, IBD is not curable, but there are many treatment options that aim to alleviate clinical symptoms. Current treatments include anti-inflammatory drugs, such as sulfasalazine and corticosteroids; immunosuppressants, such as azathioprine; monoclonal antibodies that capture pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), or block adhesion molecules, such as integrins, to reduce excessive leukocyte recruitment; and inhibitors that target kinases that trigger pro-inflammatory pathways, such as Janus kinase (JAK)4. Not all patients respond to all treatments, so therapeutic strategies must be individualized5. Furthermore, most of these therapeutic drugs interfere with the metabolism and immune responses, often causing severe side effects. For this reason, alternative treatment options have been investigated.
Alternative treatments include probiotics and nutritional supplements, which have been applied in animal models and clinical trials with varying levels of success6,7. We recently found that the application of different single nutritional supplements, such as anti-oxidative vitamins or ω3-poly-unsaturated fatty acids (PUFAs), is inferior to the application of a combination of such supplements in alleviating colitis and cardiovascular disease symptoms8,9. These studies were performed in mice, so clinical studies must be performed in humans to determine whether these findings will also be applicable to humans. Before clinical studies are initiated, the effectiveness and safety of new treatment options must be evaluated in animal models.
For IBD, the dextran sulfate sodium (DSS) model has been widely used to study the mechanisms of disease development and the beneficial effects of drugs and nutritional supplements6,10. In most studies, only an acute disease over a time period of seven days is induced; nevertheless, the clinical signs observed in these animals closely resemble those observed in IBD patients (i.e., bloody diarrhea, weight loss, epithelial dysfunction, and immune cell infiltration)10. DSS induces erosions in the mucosa, resulting in barrier dysfunction and in increased intestinal epithelial permeability11. The exact mechanism remains unknown. However, a study suggests that DSS interacts with medium chain-length fatty acids to form nano-lipocomplexes that are able to enter epithelial cells and to induce inflammatory signaling pathways12. In this article, we describe in detail how colitis is induced and analyzed in mice, how nutritional supplements are applied by gavage to ensure a constant dosage in each animal, and how the effects of such supplements on various colitis symptoms are examined.
All animal experiments have been approved by the Institutional Animal Care and Use Committee of Cinvestav.
1. Preparation of DSS Drinking Water and the Induction of Colitis
2. Application of Nutritional Supplements by Gavage
3. Determination of the Disease Activity Index
NOTE: The disease activity index is the combination of the scores for weight loss, perianal bleeding, and stool consistency, which are obtained daily13.
4. Determining Intestinal Epithelial Permeability In Vivo by Evans Blue Assay
5. Tissue Collection
6. Analysis of Colon Histology by Hematoxylin-eosin Staining
7. Analyzing Junction Composition by Immunofluorescence
8. Determination of Oxidative Stress by Oxidation of Ethidium
9. Analysis of Leukocyte Recruitment by MPO Assay
10. Evaluating the Expression of Pro-inflammatory Cytokines by PCR
Diet Supplements Can Protect from DSS Colitis
Anti-inflammatory and anti-oxidative diet supplements, such as vitamin C, vitamin E, the nitric oxide (NO)-source L-arginine, and ω3-polyunsaturated fatty acids (ω3-PUFAs), have been used with varying success in animal models to alleviate signs of inflammatory diseases3,6,15. Malnutrition, oxidative stress, and the production of pro-inflammatory cytokines can trigger both acute and chronic colitis3. In a previous study, we proved that a combination of micronutrients showed protective effects against DSS-induced colitis in vivo. Here, we provide detailed protocols on how these effects were measured8. First, we analyzed the effects of nutritional supplements on disease progression by determining the disease activity index (DAI), consisting of the three parameters of weight loss, stool consistency, and perianal bleeding. Representative results showed that DSS treatment led to a continuously-increasing DAI, as expected, reaching a maximum of 8.2 ± 0.46 on day seven (Figure 1A). Anti-inflammatory diet supplementation delayed the onset of colitis, but at later time points, when colitis was very strong, the protective effect was marginal and no longer statistically significant, suggesting that diet supplements are only effective when the colitis is not yet too strong. Importantly, dietary intervention could prevent the DSS-induced increase in intestinal epithelial permeability, which is an important feature of colitis (Figure 1B). DSS colitis led to reduced colon lengths, and this shortening was also ameliorated by the anti-inflammatory and anti-oxidative diet supplement (Figure 1C). Thus, clinical symptoms of colitis can indeed be alleviated by changes in diet composition.
Colon Tissue Damage and Disassembly of TJ and AJ during Colitis Is Attenuated by Anti-inflammatory and Anti-oxidative Diet Supplements
To analyze why colitis progression was less severe in animals receiving a diet supplement, tissue morphology was investigated after hematoxylin/eosin (H/E) staining of paraffin-embedded distal colon tissues. Control samples showed a normal morphology (Figure 2A, upper panel), whereas animals treated with 3.5% DSS showed typical signs of colitis (i.e., leukocyte infiltration, edema formation, crypt dysplasia, and ulcerations; Figure 2A, middle panel). Of note, parallel treatment with diet supplementation clearly alleviated colitis-induced morphological changes, with an overall morphology that was more comparable to the control group (Figure 2A, bottom panel). These observations were very similar to what has been reported for co-treatments with probiotic bacteria13 and clearly show that changes in diet can counteract the development of colitis.
We knew already that colitis-induced intestinal epithelial permeability could be counteracted by diet supplementation. Increased permeability is a hallmark of IBD, and it is in large part caused by the disassembly of the stabilizing adherens junctions (AJ) and tight junctions (TJ). Destabilization of epithelial cell contacts by the internalization of junctional proteins is induced by pro-inflammatory cytokines, which are abundantly produced during colitis16. We found, through the immunofluorescent staining of distal colon tissue, that the TJ molecule zonula occludens-1 (ZO-1) and the AJ molecule E-cadherin were internalized during DSS-induced colitis and that co-localization of these proteins was partly lost at crypt epithelial cell contacts (Figure 2B). Importantly, internalization of E-cadherin and ZO-1 was reduced when DSS-treated mice were co-treated with an anti-inflammatory and anti-oxidative diet supplement (Figure 2B). Overall, crypt and junction structures were more comparable to controls when the diet supplement was applied, suggesting that the observed protective effect was at least in part due to the preservation of tissue architecture.
Dietary Intervention Can Counteract Inflammatory Neutrophil Recruitment, Oxidative Stress, and the Production of Pro-inflammatory Cytokines
Another hallmark of IBD is uncontrolled neutrophil infiltration. Recruited neutrophils contribute to tissue damage by producing and releasing reactive oxygen species (ROS) and proteases17. Using an MPO activity assay, we found strong neutrophil recruitment in the colon in response to DSS, which was counteracted by diet supplementation (Figure 3A).
Oxidative stress is another important feature of colitis, caused by the release of ROS from recruited neutrophils. Thus, we analyzed the production of ROS in colon cross-sections. Figure 3B demonstrates that DSS treatment strongly increased oxidative stress. Of note, changes in diet completely prevented DSS-induced oxidative stress (Figure 3B), which is most likely a consequence of reduced neutrophil recruitment (Figure 3A).
Pro-inflammatory cytokines and chemokines are strongly produced by various cell types during colitis18. Such inflammatory mediators contribute to neutrophil recruitment and junction protein internalization-features that we already knew could be attenuated by changes in diet. Analysis of mRNA expression by RT-PCR revealed that DSS increased the expression of IL1β, IL-6, and keratinocyte-derived chemokine (KC), as expected (Figure 3C), and that anti-inflammatory diet supplementation diminished this DSS-induced increase (Figure 3C). These data imply that diet supplements can indeed serve to counteract clinical signs of IBD.
Figure 1. Nutritional Supplements Can Alleviate Disease Activity Index, Intestinal Epithelial Permeability, and Colon Shortening. (A) The disease activity index (DAI, values from 0 – 12) consists of the three parameters of weight loss, stool consistency, and perianal bleeding and was recorded daily in the experimental groups of control (ctrl), colitis, and colitis + nutritional supplement (colitis+suppl.). The control group did not show any signs of colitis, resulting in a DAI of 0 throughout the experimental period. n = 5 per group. (B) Intestinal epithelial permeability was measured by Evans blue leakage in the abovementioned experimental groups and was expressed as the absorption at 620 nm per g of tissue. n = 8 per group. All data are shown as the mean ± the standard deviation of the mean (SDM). *p< 0.05; **p< 0.01; ***p< 0.001. Statistics were calculated by one-way ANOVA. (C) Representative images of whole colons of the respective experimental groups after 7 days of treatment. The scale on the left is in cm. Please click here to view a larger version of this figure.
Figure 2. Tissue Morphology and Junction Architecture Are Better Preserved during Colitis when Applying Nutritional Supplements. (A) 5-µm paraffin cross-section of tissue biopsies from the distal colons of the respective experimental groups were stained with hematoxylin/eosin and analyzed for typical signs of colitis, such as edema formation (arrows), leukocyte infiltration (arrowheads), and ulceration (*). Overall tissue morphology of the colitis+suppl. group more resembles that of the control than the colitis group, proving an overall protective effect against DSS colitis. Images are representative of 3 independent tissue preparations per group. Images were taken using a 10X objective. Bar = 100 µm. (B) 8 μm colon cryo-sections of the respective groups were stained with antibodies against ZO-1 (green) and E-cadherin (red). Co-localization (yellow in the merged images; arrows) of E-cadherin and ZO-1 at crypt epithelial cell contacts, which is lost during colitis, is mostly preserved in the colitis + suppl. group. Images are representative of 3 independent tissue preparations. Bar = 50 µm. Please click here to view a larger version of this figure.
Figure 3. Neutrophil Recruitment, Oxidative Stress, and the Production of Pro-inflammatory Cytokines can be Reduced by Nutritional Supplements. (A) Myeloperoxidase (MPO) activity was measured to analyze the amount of leukocyte recruitment into colon tissues in the experimental groups: control (ctrl), colitis, and colitis + nutritional supplement (colitis+suppl.). n = 5 per group; *p< 0.05. Data are shown as the mean ± SDM. Statistics were calculated by one-way ANOVA. (B) The fluorescence of oxidized ethidium, depicted in red as the measure of oxidative stress in 8-µm cryo-sections of colon tissues, was recorded by laser confocal microscopy. Images are representative of 3 independent tissue preparations. Bar = 100 µm. (C) The production of the pro-inflammatory cytokines IL1β and IL-6 and the chemokine KC was determined by semi-quantitative RT-PCR in a 1.5% agarose gel (black and white were reverted for the sake of clarity). β-actin served as the housekeeping gene. Representative images of three independent cDNA preparations are shown. Please click here to view a larger version of this figure.
In order to use nutritional supplements in clinical studies, the benefits and safety of such supplements must be carefully evaluated in vivo in animal studies. In the case of colitis, several appropriate animal models that resemble the clinical signs of IBD have been established, including chemical models using DSS, TNBS, or acetic acid; knock-out (KO) models such as IL10-KO; and immune-cell-mediated colitis using adoptive T-cell transfer19-21. The DSS model of colitis is a rapid and reliable method of inducing colitis that resembles many disease symptoms of human IBD and has been used in a plethora of studies investigating the molecular mechanisms of colitis22. Moreover, DSS colitis is reversible and thus also allows for the study of tissue healing after the colitogenic stimulus has been removed. The provided protocol describes in detail the DSS colitis model that has been previously optimized in our laboratory8.
A disadvantage is that the optimization of the DSS concentration must be done in every laboratory, since environmental factors greatly affect disease development and progression23. Additionally, DSS must be in a molecular weight range of 40 – 50 kDa to induce colitis. For the unexperienced researcher, it will be important to develop an eye for an unbiased assessment of the DAI parameters of stool consistency and perianal bleeding. If the assessment is performed by an unexperienced researcher, it is recommendable to have a colleague confirm the assessment, ideally in a blinded fashion.
To analyze the intestinal epithelial permeability for macromolecules, three major methods have been used throughout the literature: the Evans blue dye assay, as described here; the instillation of 4-kDa FITC-dextran in an intestinal loop; and the feeding of 4-kDa FITC-dextran13,24,25. In the latter assay, FITC-dextran is fed by gavage, and the amount of FITC dextran that crossed the epithelium and leaked into the blood stream is measured fluorometrically from serum samples. In this assay, the permeability of the entire gastrointestinal tract is measured, since the tracer passes all the way from the esophagus to the rectum and most of the tracer will pass before reaching the colon. In the loop model, permeability is measured in a confined intestinal region used for the loop (usually the small intestine)25. In contrast, the Evans blue assay has the advantage that only epithelial permeability in the colon is measured, since the tracer is directly instilled into the entire colon. Thus, using this assay, we can conclude that the colonic epithelium is protected by the diet supplement.
It is always important to record the colon weight and length after extraction and before using the tissues for other assays, because some data (e.g., leaked Evans blue in the permeability assay) are expressed per g of tissue. Furthermore, the weight-to-length ratio is an independent read-out of tissue damage26. In general, tissue morphology is analyzed using paraffin-embedded tissues stained with hematoxylin and eosin, as described here. Paraffin embedding has the advantage that the tissue morphology is much better conserved compared to other embedding methods. This allows for the unequivocal identification of different cell types within the mucosa (i.e., immune cells, epithelium, goblet cells, etc.) and the analysis of tissue alterations during colitis, such as edema formation, leukocyte infiltration, and epithelial apical erosions. On the other hand, paraffin has the disadvantage that immunofluorescent staining can only be performed after time-consuming epitope recovery. Therefore, we always prepare different frozen tissue biopsies embedded in OCT compound. Such tissue samples can be easily sectioned using a cryostat and show low levels of autofluorescence. We use ethanol for fixation by dehydration because neither does it interfere with actin filaments (as methanol does), nor does it trigger autofluorescence (as PFA does). Using these techniques, we found that an anti-inflammatory and anti-oxidative diet supplement can improve tissue morphology and junction architecture during colitis (compare Figure 2).
Excessive neutrophil recruitment is a critical event during colitis that is induced by the production of chemoattractants in response to inflammation17. Neutrophil recruitment is a physiological event because neutrophils contribute to the removal of the inflammatory cue and to subsequent wound healing. However, if this innate immune response is not properly controlled and terminated, neutrophils in the mucosa can contribute to tissue damage by releasing proteases and reactive oxygen species. Neutrophils contain MPO, which can be easily measured and quantified using a colorimetric assay, where the amount of MPO is directly proportional to the number of neutrophils. Figure 3A shows that neutrophil recruitment increases during colitis and that dietary intervention can prevent this excessive and uncontrolled immune response. In accordance with the abundant neutrophil presence in the mucosa, oxidative stress increases during colitis (Figure 3B).
Our diet supplementation clearly protects the mucosa from oxidative stress, which is most likely a consequence of reduced neutrophil recruitment. As mentioned, neutrophils are attracted by chemokines and the production of chemokines is induced by pro-inflammatory cytokines that are secreted by various cell types during colitis. Thus, we assumed that reduced neutrophil recruitment is a consequence of reduced cytokine and chemokine production. Figure 3C demonstrates that the pro-inflammatory cytokines IL-1β and IL-6, as well as the chemokine KC, are upregulated during DSS-induced colitis. The diet supplement reversed the levels of IL-6 back to control levels and ameliorated the increased production of IL-1β and KC. Of note, KC is the most important chemoattractant for neutrophils in mice.
Thus, it is tempting to speculate that the observed preservation of tissue morphology is due to reduced neutrophil infiltration, which is, in turn, a consequence of the reduced production of KC. Analyzing RNA production in DSS-treated tissues by RT-PCR is problematic when residual DSS is present in the isolated RNA. This is because DSS inhibits both reverse transcriptases and Taq-polymerases. If no amplification can be achieved, it will be necessary to further purify the RNA samples by LiCl-mediated precipitation, as described elsewhere27.
In summary, we describe in detail different methods for the analysis of tissue damage during DSS colitis and how this damage can be ameliorated by dietary supplements. Knowledge gained from such animal experiments can justify the establishment of patient cohorts to investigate the protective effects of the analyzed supplements in human IBD. Data obtained from clinical studies may then open new avenues to develop alternative treatment strategies for the management of IBD.
The authors have nothing to disclose.
This work was supported by grants from the Mexican Council for Science and Technology (Conacyt, 207268 and 233395 to Michael Schnoor). KFCO is a recipient of a Conacyt stipend (396260) to obtain an MSc degree.
0.3% of gelatin | Bioxon | 158 | |
10% formaldehyde | J.T. Baker | 2106-03 | |
96-well plate with flat bottom | Corning | 3368 | |
Absolute ethanol | J.T. Baker | 9000-03 | |
Absolute xylene | J.T. Baker | 9490-03 | |
ABTS | Sigma Aldrich | H5882 | |
Bovine Serum Albumin | Sigma-Aldrich | 9048-46-8 | |
ColoScreen | Helena Laboratories | 5072 | |
Corabion (Kindly provided by Merck, Naucalpan, Mexico) | Merck | ||
Dextran Sulfate Sodium Salt | Affymetrix | 9011-18-1 | (M.W. 40,000-50,000) |
Dihydroethidium | Life Technology | D11347 | |
Eosin-Y | J.T. Baker | L087-03 | |
Evans blue | Sigma-Aldrich | 314-13-6 | |
Feeding needle | Cadence Science Inc. | 9921 | |
Glass slide rack with handle | Electron Microscopy | 70312-16 | |
Glass Slides | Corning | 2947 | |
Harris hematoxylin | Sigma-Aldrich | H3136 | |
Hexadecyltrimethyammonium (HTAB) | Sigma Aldrich | H6269 | |
Histosette (Embedding cassette) | Simport | M498.2 | |
Hydrochloric acid 1 M | J.T. Baker | 9535-62 | |
Hydrogen peroxide | Sigma Aldrich | H3410 | |
Ketamine | PiSA Agropecuaria | Q-7833-028 | |
Liquid paraffin | Paraplast | 39501-006 | |
Lithium carbonate | Sigma-Aldrich | 2362 | |
N,N-dimethylformamide | J.T. Baker | 68-12-2 | |
N-acetylcysteine | Sigma-Aldrich | A9165 | |
Plastic cubes | Electron Microscopy | 70181 | |
Poly-L-Lysine Solution | Sigma-Aldrich | 25988-63-0 | |
Prism 5 statistical software | GraphPad Software | Prism 5 | |
Saline Solution 0.9% NaCl | CS PiSA | Q-7833-009 | |
Sodium citrate | Sigma-Aldrich | W302600 | |
Synthetic resin | Poly Mont | 7987 | |
Taq DNA polymerase | Invitrogen | 11615-010 | |
Tissue-tek. O.C.T Compound | Sakura Finetek | 4583 | |
Tuberculine Syringe | BD Plastipak | 305945 | |
Tween 20 | Sigma-Aldrich | 9005-64-5 | |
VECTASHIELD Antifade Mounting Medium with DAPI | Vector Laboratories | H-1200 | |
Xylazine | PiSA Agropecuaria | Q-7833-099 |