Name: chronic salmonella infection induced intestinal fibrosis
Uploaded: 2019-09-22T14:00:00.000+00:00
Duration: 8 min 40 s
Description: Watch this Scientific Journal Video about Chronic Salmonella Infection Induced Intestinal Fibrosis at JoVE.com

We thank Ingrid Barta for histology services.

All animal protocols were approved by the Animal Care Committee of the University of British Columbia.
1. Preparation of Salmonella Typhimurium &#916;AroA cultures for oral gavage of mice
<ol>
	<li>From a frozen glycerol stock of S. Typhimurium &#916;AroA, prepare a streak plate using LB agar containing 100 &#956;g/mL streptomycin with a sterile inoculating loop. Incubate overnight at 37 &#730;C. Streak plates can be stored up to one week at 4 &#730;C.</li>
	<li>One day prior to infection, prepare antibiotic by dissolving 0.5 g of streptomycin in 2.5 mL of water. After filter sterilizing streptomycin solution, orally gavage the mice using a bulb-tipped 22G gavage needle and 1 mL syringe with 100 &#956;L of streptomycin solution (20 mg streptomycin/dose). Using an inoculating loop, inoculate 3 mL of LB broth (50 &#956;g/mL streptomycin) in a culture tube with a single colony. Incubate Salmonella culture aerobically at 37 &#730;C overnight with shaking at 200 RPM.</li>
	<li>On day of infection, prepare final infection dose by performing 2 consecutive 1/10 dilutions of overnight Salmonella cultures in sterile PBS. This would result in a 100 &#956;L inoculum containing approximately 3 x 106 CFU.</li>
	<li>Using a bulb-tipped 22G gavage needle and a 1 mL syringe, gavage each mouse with 100 &#956;L of the prepared Salmonella. 
	NOTE: Prepare Salmonella cultures using aseptic techniques. Final inoculation concentration of Salmonella may be verified by plating serial dilutions on LB agar with streptomycin. The S. Typhimurium &#916;AroA strain can be obtained by contacting Professor McNagny (kelly@brc.ubc.ca).</li>
</ol>
2. Assessment of Salmonella burdens in tissues
<ol>
	<li>Prepare 2 mL safe-lock, round bottom microtubes with 1 mL of sterile PBS and an autoclaved stainless steel bead. Pre-weigh the tubes prior to tissue collection.</li>
	<li>Resect cecal and splenic tissues from mice euthanized by carbon dioxide exposure. Collect tissue from individual animals in separate tubes. Weigh the tubes to determine tissue weights.</li>
	<li>Homogenize using a mixer mill apparatus for 15 min at 30 Hz. Transfer 900 &#956;L of PBS per well in a 96-well 2-mL megablock. Pipette 100 &#956;L of tissue homogenates into the first well, mix well, and perform serial dilutions by adding 100 &#956;L to subsequent wells until a 10-6 dilution is obtained. Plate 10 &#956;L of each dilution in triplicates onto LB agar containing 100 &#956;g/mL streptomcyin.</li>
	<li>Count and multiply average CFU by a factor of 100 since 10 &#956;L of the 1000 &#956;L sample was plated, and the appropriate dilution factor. Divide tissue weight total CFU by tissue weights to determine CFU per gram of tissue. 
	NOTE: Keep all samples on ice or at 4 &#730;C during tissue processing. Use wide-orifice pipette tips when performing serial dilutions with tissue homogenates. Prepare the 96-well megablock containing PBS in advance.</li>
</ol>
3. Picrosirius Red staining and quantification of collagen.
<ol>
	<li>Fix cecal tissues overnight in 10% buffered formalin and prepare for paraffin embedding. Cut 5-&#956;m sections for Picrosirius Red staining as described previously25.</li>
	<li>Capture composite images of whole cecal cross-sections on a brightfield microscope.</li>
	<li>Open Fiji (ImageJ) and drag and drop the .tif image file onto the toolbar.</li>
	<li>On the menu bar, select Image &#62; Type &#62; RGB Stack to split the image into red, green, and blue channels. Slide the horizontal bar at the bottom of the panel to set the channel to Green.</li>
	<li>Open Image &#62; Adjust &#62; Threshold tool. Adjust minimum and maximum limits to eliminate any background signals. Once the desired threshold is set, close the threshold tool and go to Analyze &#62; Set Measurements. Check off Area, Area Fraction, Limit to threshold, and Display label.</li>
	<li>Gate the tissue section with either Freehand selections or Polygon selections tool and measure the % area positive for collagen by clicking Analyze &#62; Measure.</li>
	<li>Normalize the absolute area positive for collagen staining to tissue area. 
	NOTE: Fiji (ImageJ) is an open-source program that can be downloaded at https://fiji.sc. Images with same capture conditions (i.e., brightness and focus) must have identical threshold limits for accurate quantification. If there is any background staining within the selected tissue, measure the absolute area and subtract it from the area positive for collagen from the entire tissue.</li>
</ol>

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The authors have no financial conflicts of interest to disclose.

Our understanding of the pathogenesis of IBD has been greatly enhanced by mouse models of intestinal inflammation. Although such individual models do not recapitulate all features of the complex and multifactorial human disease, they have been useful in identifying key features of disease progression. Fibrotic strictures associated with IBD remains a major unmet clinical need as current treatments are ineffective in reversing disease development. Moreover, intestinal fibrosis is difficult to study in a laboratory setting due to limitations in current animal models. Chronic exposure to TNBS in BALB/c mice has been shown to induce robust collagen deposition in the colon that is driven by IL-13 and TGF-B1 signaling27,28. However, intestinal fibrosis is not commonly observed in other routinely used laboratory models of colitis such as DSS treatment, IL-10-deficient, or adoptive T cell transfer models. Grassl et al. demonstrated that chronic gastrointestinal infection of C57Bl6 with the attenuated &#916;AroA mutant Salmonella strain results in robust fibrosis in the mucosal and submucosal regions of the cecum12. They reported that peak fibrosis occurred three weeks after infection and was associated with Th1 and Th17 immunity and elevated pro-fibrotic factors TGF-B1, CTGF, and IGF-112. This pathology is reminiscent of CD as the severe inflammation and fibrosis is transmural. While IBD is typically progressive, one important limitation of the chronic Salmonella infection model is the transient nature of the fibrotic immunopathology. In C57Bl/6 mice, intestinal disease is typically resolved by week six after infection. Despite this shortcoming, the latter stages of this infection model can be utilized to identify factors or processes involved in promoting disease remission26.
Although bacterial pathogen-driven models of colitis are well studied, there is no association between Salmonella and CD. However, it has been proposed that the magnitude of fibrotic disease during the latter stages of chronic Salmonella colonization does not directly correlate with pathogen burdens suggesting that the pathology is &#8220;self-propagating&#8221; once severe intestinal inflammation is initiated by Salmonella29. In contrast, the adherent-invasive Escherichia coli (AIEC) pathovar has been strongly linked to the development of CD because of its high prevalence in the ileal mucosa of patients30. In addition, persistent AIEC colonization (up to 9 weeks) of the ileum, cecum and colon of several mouse strains including C57Bl/6 leads to robust matrix accumulation in the gut via flagellin expression thus demonstrating the fibrogenic induction potential of this pathobiont31,32. The recent development of infection driven models of intestinal fibrosis have provided robust experimental models of IBD. These have provided new systems to dissect the relationship between enteric bacterial species including their virulence factors, and host susceptibility factors that may enhance our understanding of IBD-associated fibrosis.

Ulcerative colitis (UC) and Crohn&#8217;s disease (CD) are the two major forms of IBD and are characterized as chronic and relapsing inflammatory disorders of the gastrointestinal tract 1,2. These disorders have a major impact on the quality of life of patients. Symptoms of IBD include abdominal pain, diarrhea, nausea, weight loss, fever, and fatigue3. Recent studies have identified genetic and environmental factors that contribute to disease pathogenesis; it is thought that such risk factors contribute to the disruption of the epithelial barrier resulting in the translocation or oversampling of luminal antigens4. As a consequence, this initiates an aberrant inflammatory response to the commensal flora mediated by intestinal immune cells4. Features of IBD-associated complications may extend to sites beyond the GI tract affecting various organs including joints, skin, and liver1,2. Hallmarks of UC include severe and diffuse inflammation typically localized in the colon1. Disease pathology affects the mucosa and submucosa of the bowel resulting in superficial mucosal ulcerations1. In contrast, CD can affect any part of the GI tract although evidence of disease is commonly found in the colon and distal ileum2. Moreover, the inflammation in CD is transmural, affecting all layers of the bowel wall2.
Several IBD susceptibility genes that have been identified would indicate that dysregulation of the epithelial barrier or immunity are critical contributors to disease progression5. Mutations in nucleotide oligomerization domain 2 (NOD2) expressed by monocytes was found to be associated with increased susceptibility to CD; this highlights a link between altered innate immune detection of bacterial components and the disease6. More recent genome-wide association studies (GWAS) have revealed additional pathways potentially involved in the pathogenesis of IBD including genetic variations in: STAT1, NKX2-3, IL2RA, IL23R dependent pathways linked to adaptive immunity, MUC1, MUC19, and PTGER4 in intestinal barrier maintenance, and ATG16L-mediated autophagy7,8,9. While these population-based genetics studies have enhanced our understanding of IBD, susceptibility alleles alone are likely insufficient in initiating and sustaining chronic disease3. Other non-genetic factors including alterations in gut microbiome composition and a reduction in diversity have been associated with intestinal inflammation. However, it is unclear whether gut dysbiosis precedes or is the consequence of dysregulated immune responses3. Although the etiology of IBD remains unclear, our understanding of the pathogenesis of the disease has been enhanced by experimental mouse models of intestinal inflammation10,11. These models individually do not fully represent the complexity of the human disease, but they are valuable for elucidating pathophysiological pathways that could be relevant to IBD and for the validation of tentative therapeutic strategies10,11. Such mouse models typically rely on the initiation of inflammation by chemical induction or infection, immune cell transfer, or genetic manipulation. Moreover, these strategies often involve perturbations in epithelial integrity or modulation of innate or adaptive immunity.
Salmonella enterica serovars are intestinal pathogens that can infect humans and mice. After ingestion, Salmonella can colonize the gut by direct invasion of epithelia, M cells, or antigen presenting cells12. Mice infected orally with S. Typhimurium results in the colonization primarily of systemic sites such as the spleen and mesenteric lymph nodes with relatively low abundance in the GI tract12. However, pretreatment of mice with streptomycin enhances the efficiency of Salmonella colonization of the gut by diminishing the host protective effects of the normal microbiota13. Pathological features of this model include the disruption or ulceration of the epithelial barrier, granulocyte recruitment, and severe edema13. Alternatively, infection with the vaccine grade S. Typhimurium &#916;AroA mutant leads to chronic colonization of the cecum and colon that persists up to day 40 after infection14. The S. Typhimurium &#916;AroA strain has a defect in the biosynthesis of aromatic amino acids; this renders the mutant strain avirulent and can be utilized as a highly effective vaccine15. Oral infection in mice leads to a Th1- and Th17-cytokine associated inflammatory response, extensive tissue remodeling, and collagen deposition. Tissue pathology is associated with elevated levels of pro-fibrotic factor such as TGF-&#946;1, CTGF, and IGF14. The transmural fibrotic scarring reported in this model is reminiscent of stricture formations often observed in IBD. The induction of fibrosis by Salmonella requires virulence encoded by Salmonella pathogenicity islands (SPI)-1 and 2 12. Importantly, this S. Tymphimurium &#916;AroA infection model is a useful system for the study of fibrotic responses in mutant mice maintained on a C57/Bl6 background. The C57/Bl6 strain is extremely sensitive to S. Typhiumurim SL1344 infection due to a loss-of-function mutation in the gene encoding the natural resistance-associated macrophage protein (NRAMP)-116,17. We have found that IL-17A and ROR&#945;-dependent innate lymphoid cells are important contributors to pathogenesis in this model18.
A major complication of CD is the dysregulated and excessive deposition of extracellular matrix (ECM) including collagen2,19. Although the GI tract has a relatively high capacity for regeneration, fibrotic scarring can arise due to unresolved wound healing responses that are associated with chronic and severe inflammation20,21. In CD, this results in deleterious effects on tissue architecture leading to significant organ impairment21,22. The transmural nature of the inflammation observed in CD ultimately precedes the thickening of the bowel wall associated with symptomatic stenosis or stricture formation21. About a third of CD patients require intestinal resection for this complication22. There are no effective anti-fibrotic therapies in IBD given that the use of immunosuppressants such as azathioprine or anti-TNF&#945; biologics have no impact or only modestly reduced the requirement of surgical interventions19,23. While fibrosis is thought be the consequence of chronic inflammation, cells of mesenchymal origin such as fibroblasts and pericytes are thought to be the primary cellular sources of ECM in fibrotic scarring21,24. Chronic S. Typhimurium &#916;AroA infection is a robust mouse model of intestinal fibrosis that can offer insights into the pathogenesis of CD-like features.

<table><tbody><tr><td>2 ml round bottom safe lock tubes</td><td>Eppendorf</td><td>22363344</td><td></td></tr><tr><td>Stainless steel beads</td><td>Qiagen</td><td>69989</td><td></td></tr><tr><td>PBS</td><td>Gibco</td><td>10010031</td><td></td></tr><tr><td>Large-Orifice Pipet Tips</td><td>Fisher</td><td>2707134</td><td></td></tr><tr><td>2 mL megablock plates</td><td>Sarstedt</td><td>82.1972.002</td><td></td></tr><tr><td>Gavage needles</td><td>FST</td><td>18061-22</td><td></td></tr><tr><td>Streptomycin sulfate</td><td>Sigma</td><td>S9137</td><td></td></tr><tr><td>Mixer mill</td><td>Retsch</td><td>MM</td><td></td></tr></tbody></table>

chronic salmonella infection induced intestinal fibrosis

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and dysregulated repair. In inflammatory bowel disease (IBD), fibrosis leads to recurrent stricture formations for which there is no effective therapy other than surgical resection. Due to its late onset, the processes that drive fibrosis is less studied and largely unknown. Therefore, fibrotic complications represent a major challenge in IBD. In this protocol, a robust in vivo model of intestinal fibrosis is described where streptomycin pre-treatment of C57Bl/6 mice followed by oral gavage with vaccine grade Salmonella Typhimurium &#916;AroA mutant leads to persistent pathogen colonization and fibrosis of the cecum. Methodologies for preparing S. Typhimurium &#916;AroA for inoculation, quantifying pathogen loads in the cecum and spleen, and evaluating collagen deposition in intestinal tissues are explained. This experimental disease model is useful for examining host factors that either enhance or exacerbate CD-like intestinal fibrosis.

Streptomycin treatment followed by oral infection with S. Typhimurium &#916;AroA leads to robust intestinal inflammation and fibrosis especially in the cecum (Figure 1). Typical pathogen burdens of 108 to 109 CFU per 1 g of cecum and 104 CFU per 1 g of spleen can be recovered from infected animals (Figure 2). Assessment of fibrosis in picrosirius red stained cecal sections indicate peak fibrosis 21 days after infection while much of the pathology is resolved by day 42 pi (Figure 3 and Figure 4). Collagen deposition is most pronounced in the submucosa of the intestine while fibrosis in the mucosa is milder.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60068/60068fig01.jpg" /> 
Figure 1. Diagram of cecal sectioning for histology, Salmonella burden assessment, and cytokine quantification. 
Segment 1 representing the cecal tip may be used for gene expression analysis while segment 2 will be fixed in 10% buffered formalin for histology and Segment 3 will be homogenized for bacterial enumeration.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60068/60068fig02.jpg" /> 
Figure 2. Pathogen burdens in ceca and spleens. 
Salmonella CFU per weight of tissue during course of infection. This figure has been modified from Lo et al.26. <a href="https://www.jove.com/files/ftp_upload/60068/60068fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60068/60068fig03.jpg" /> 
Figure 3. Picrosirius red stained cross sections of intestinal tissue. 
Bright field images of ceca from uninfected animals and animals 21 and 42 days after S. Typhimurium &#916;AroA infection. Scale bar, 200 &#956;m. This figure has been modified from Lo et al.26.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60068/60068fig04.jpg" /> 
Figure 4. Quantification of collagen deposition by morphometric analyses. 
(A) PSR+ staining normalized to tissue area. Significance determined by one-way ANOVA Kruskal-Wallis test with Dunns post test. **, P &#60; 0.01, N.S., P &#62; 0.05. This figure has been modified from Lo et al.26. (B) Example of collagen quantification in cecal tissue using Fiji. <a href="https://www.jove.com/files/ftp_upload/60068/60068fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Chronic Salmonella Infection Induced Intestinal Fibrosis at JoVE.com

Chronic Salmonella Infection Induced Intestinal Fibrosis

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn&#8217;s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Tissue fibrosis characterized by the pathological accumulation of extracellular matrix such as collagen is the outcome of persistent inflammation and ...

chronic-salmonella-infection-induced-intestinal-fibrosis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

6.9K Views.  University of British Columbia. This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn’s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Video: Chronic Salmonella Infection Induced Intestinal Fibrosis

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

This protocol outlines the steps required to produce a robust model of infectious disease and colitis, as well as the methods used to characterize Citrobacter rodentium infection in mice. C. rodentium is a gram negative, murine specific bacterial pathogen that is closely related to the clinically important human pathogens enteropathogenic E. coli and enterohemorrhagic E. coli. Upon infection with C. rodentium, immunocompetent mice suffer from modest and transient weight loss and diarrhea. Histologically, intestinal crypt elongation, immune cell infiltration, and goblet cell depletion are observed. Clearance of infection is achieved after 3 to 4 weeks. Measurement of intestinal epithelial barrier integrity, bacterial load, and histological damage at different time points after infection, allow the characterization of mouse strains susceptible to infection.
The virulence mechanisms by which bacterial pathogens colonize the intestinal tract of their hosts, as well as specific host responses that defend against such infections are poorly understood. Therefore the C. rodentium model of enteric bacterial infection serves as a valuable tool to aid in our understanding of these processes. Enteric bacteria have also been linked to Inflammatory Bowel Diseases (IBDs). It has been hypothesized that the maladaptive chronic inflammatory responses seen in IBD patients develop in genetically susceptible individuals following abnormal exposure of the intestinal mucosal immune system to enteric bacteria. Therefore, the study of models of infectious colitis offers significant potential for defining potentially pathogenic host responses to enteric bacteria. C. rodentium induced colitis is one such rare model that allows for the analysis of host responses to enteric bacteria, furthering our understanding of potential mechanisms of IBD pathogenesis; essential in the development of novel preventative and therapeutic treatments.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

This protocol  outlines the steps required to produce a robust model of infectious disease and  colitis, as well as the methods used to characterize ...

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Medicine

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Biology

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.

Disruption of bile flow results in severe inflammatory cholestatic liver injury with a characteristic time-dependent sequence of morphological alterations. Here we present a protocol for the surgical ligation of the common bile duct in mice that allows to induce a strong fibrotic response after 21 to 28 days.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well ...

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not routinely employed in animal models. Conventional experimental readouts often rely on post mortem and ex vivo analyses, which prevent intra-individual follow-up examinations and increase the number of study animals needed. Fluorescence-mediated tomography enables the non-invasive, repetitive, quantitative, three-dimensional assessment of fluorescent probes. It is highly sensitive and permits the use of molecular makers, which allows for the specific detection and characterization of distinct molecular targets. In particular, targeted probes represent an innovative tool for analyzing gene activation and protein expression in inflammation, autoimmune disease, infection, vascular disease, cell migration, tumorigenesis, etc. In this article, we provide step-by-step instructions on this sophisticated imaging technology for the in vivo detection and characterization of inflammation (i.e., F4/80-positive macrophage infiltration) in a widely used murine model of intestinal inflammation. This technique might also be used in other research areas, such as immune cell or stem cell tracking.

Target-specific probes represent an innovative tool for analyzing molecular mechanisms, such as protein expression in various types of disease (e.g., inflammation, infection, and tumorigenesis). In this study, we describe a quantitative three-dimensional tomographic assessment of intestinal macrophage infiltration in a murine model of colitis using F4/80-specific fluorescence-mediated tomography.

Murine models of disease are indispensable to scientific research. However, many diagnostic tools such as endoscopy or tomographic imaging are not ...

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has hindered the development of a preventative drug. Primarily, finding a suitable animal model is challenging in understanding the mechanism of Crohn&#39;s-associated intestinal fibrosis pathology. Here, we adopted an effective method where TNBS chemical exposure to mice rectums produces substantially deep ulceration and chronic inflammation, and the mice then chronically develop intestinal fibrosis. Also, we describe a technique where a rapamycin injection shows inhibitory effects on TNBS-mediated fibrosis in the mouse model. To assess the underlying mechanism of fibrosis, we methodically discuss a procedure for purifying Cx3Cr1+ cells from the lamina propria of TNBS-treated and control mice. This detailed protocol will be helpful to researchers who are investigating the mechanism of fibrosis and pave the path to find a better therapeutic invention for Crohn&#39;s-associated intestinal fibrosis.

In this study, we describe a detailed procedure of TNBS-mediated intestinal fibrosis, which exhibits comparable pathophysiology to Crohn&#39;s fibrosis. We also discuss this approach in light of rapamycin facilitated inhibitory effects on intestinal fibrosis.

Significant studies have been carried out to understand effective management of intestinal fibrosis. However, the lack of better knowledge of fibrosis has ...

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting approximately 20 per 1,000,000 in Europe and USA. CD is characterized by transmural inflammation, intestinal fibrosis, and luminal stenosis. Although anti-inflammatory therapies may help control inflammation, they have no efficacy on fibrosis and stenosis in CD. The pathogenesis of CD is not well understood. Current studies focus mainly on delineating dysregulated gut immune response mechanisms. While CD-associated transmural inflammation, intestinal fibrosis, and luminal stenosis all represent mechanical stress to the gut wall, the role of mechanical stress in CD is not well defined. To determine if mechanical stress plays an independent pathogenic role in CD, a protocol of TNBS-induced CD-like colitis model in rodents has been developed. This TNBS-induced transmural inflammation and fibrosis model resembles pathological hallmarks of CD in the colon. It is induced by intracolonic instillation of TNBS into the distal colon of adult Sprague-Dawley rats. In this model, transmural inflammation leads to stenosis at the TNBS instillation site (Site I). Mechanical distention is observed in the portion proximal to the instillation site (Site P), representing mechanical stress but not visible inflammation. Colonic portion distal to inflammation (Site D) presents neither inflammation nor mechanical stress. Distinctive changes of gene expression, immune response, fibrosis, and smooth muscle growth at different sites (P, I, and D) were observed, highlighting a profound impact of mechanical stress. Therefore, this model of CD-like colitis will help us better understand CD&#39;s pathogenic mechanisms, particularly the role of mechanical stress and mechanical stress-induced gene expression in immune dysregulation, intestinal fibrosis, and tissue remodeling in CD.

The present protocol describes the development of a Crohn's-like colitis model in rodents. Transmural inflammation leads to stenosis at the TNBS instillation site, and mechanical enlargement is observed in the segment proximal to the stenosis. These changes allow studying mechanical stress in colitis.

Inflammatory bowel diseases (IBD) such as Crohn&#39;s disease (CD) are chronic inflammatory disorders of the gastrointestinal tract affecting ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection or damage. Currently, the widely used mice models of sepsis include lipopolysaccharide (LPS)-induced endotoxemia, cecal ligation and puncture (CLP), and monobacterial infection model systems. This protocol describes a method to study the host responses during Salmonella Typhimurium infection-induced septic peritonitis in mice. S. Typhimurium, a Gram-negative intracellular pathogen, causes typhoid-like disease in mice.
This protocol elaborates the culture preparation, induction of septic peritonitis in mice through intraperitoneal injection, and methods to study systemic host responses. Furthermore, the assessment of bacterial burden in different organs and the flow cytometric analysis of increased neutrophil numbers in the peritoneal lavage is presented. Salmonella Typhimurium-induced sepsis in mice leads to an increase in proinflammatory cytokines and rapid infiltration of neutrophils in the peritoneal cavity, leading to lower survival.
Every step in this protocol has been optimized, resulting in high reproducibility of the pathogenesis of septic peritonitis. This model is useful for studying immunological responses during bacterial sepsis, the roles of different genes in disease progression, and the effects of drugs to attenuate sepsis.

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.

Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of the infection ...

Chronic Salmonella Infection Induced Intestinal Fibrosis

Summary

Explore More Videos

Chapters in this video

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4⁺CD45RB^high T Cells into Immunodeficient Mice

Chronic Salmonella Infected Mouse Model

Bile Duct Ligation in Mice: Induction of Inflammatory Liver Injury and Fibrosis by Obstructive Cholestasis

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Fluorescence-mediated Tomography for the Detection and Quantification of Macrophage-related Murine Intestinal Inflammation

Mechanistic Insight into the Development of TNBS-Mediated Intestinal Fibrosis and Evaluating the Inhibitory Effects of Rapamycin

A TNBS-Induced Rodent Model to Study the Pathogenic Role of Mechanical Stress in Crohn's Disease

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice