The authors would like to thank all EIB members for developing this technique. This research was supported in part by Grant-in-Aid for Scientific Research on Priority Areas "Membrane Traffic" (H.O.), and "Matrix of Infectious Phenomena" (K.H.), Young Scientists (S.F. and K. H.), and Scientific Research (H.O.), and Scientific Research on Innovative Areas "Intracellular Logistics" (H.O.), from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

1. Preparation of bacterial cells
<ol>
	<li>Streak glycerol stocked fluorescent bacteria (such as GFP expressing S. Typhimurium) on LB an agar plate containing 100 &mu;g/ml of ampicillin.</li>
	<li>Culture a single colony from LB agar overnight in 2 ml of new LB medium.</li>
	<li>Add 0.5 ml of bacterial culture to 4.5 ml of new LB medium and incubate until optical density of 1.0 at 600 nm is reached.</li>
	<li>Harvest bacterial cells by centrifugation (3,000 x g, 5 min, 4&deg;C).</li>
	<li>Discard the supernatant and wash twice with 5.0 ml of sterile phosphate buffer saline (PBS).</li>
	<li>Resuspend bacterial pellet with 5 ml of PBS, and use 50 &mu;l of the suspension containing approximately 107 colony forming unit (CFU) as the inoculum.</li>
	<li>In case of using fluorescently labeled bacteria, the bacterial cells were labeled by fluorescence labeling reagent according to standard protocol.</li>
</ol>
2. Anesthesia
<ol>
	<li>Fill a small plastic box (10 x 10 x 5 cm) with 5% (v/v) vaporized isoflurane mixed with air (flow rate: 200 ml/min).</li>
	<li>Anesthetize eight- to sixteen-weak-old male or female mice in the box.</li>
	<li>Move the mice to an autopsy table after anesthesia.</li>
	<li>Continuously anesthetize the mice by 2% (v/v) vaporized isoflurane mixed with air (flow rate: 200 ml/min) (Figure 1). This is a terminal procedure.</li>
</ol>
3. Ligated Peyer's patch loop assay
<ol>
	<li>Incise 1cm of the abdominal skin and then cut the abdominal peritoneum of an anesthetized mouse and take out the small intestine containing the Peyer's patch.</li>
	<li>Ligate the intestine with sewing yarn, taking care to avoid blood vessels. Note: only bind one side of the intestine, and leave the other side loose.</li>
	<li>Inject 50 &mu;l of bacterial suspension or PBS (control) with a syringe into ligated Peyer's patch loop on the loose side of the intestine (Figure 2).</li>
	<li>Bind loose side, and close the mouse's abdomen with a clip.</li>
	<li>After 1 h, remove the ligated Peyer's patch loop, and euthanize the mouse by cervical dislocation.</li>
	<li>Excise Peyer's patch from ligated Peyer's patch loop.</li>
	<li>Flashing the apical side of Peyer's patch by 1ml of PBS with using syringe attached to a needle to remove excess mucosal fluid and bacteria. Flashing the Peyer's patch an additional two times.</li>
</ol>
4. Whole mount staining and confocal microscopic analysis of Peyer's patch
<ol>
	<li>Fix Peyer's patch in BD Cytofix/Cytoperm solution on ice for 1 hr.</li>
	<li>Wash Peyer's patch three times with 1ml of BD Perm/Wash buffer for 5 min, and then block with 1 ml of blocking buffer containing 0.1% (w/v) saponin, 0.2% (w/v) BSA in PBS for 30 min on ice.</li>
	<li>Add 200-fold diluted anti-mouse GP2 monoclonal antibody (5 &mu;g/ml) to the specimen to detect M cells.</li>
	<li>Incubate for 2 hrs at room temperature or overnight at 4&deg;C.</li>
	<li>Wash three times with 1 ml of cold PBS, then add 200-fold diluted anti-rat IgG antibody conjugated with DyLight549 (20 &mu;g/ml)to the specimen.</li>
	<li>Incubate sample on ice for 2 hrs.</li>
	<li>Wash three times with 1 ml of cold PBS, then add 50-fold diluted Alexa 633 conjugated Phalloidin to the specimen to detect F-actin.</li>
	<li>Incubate on ice for 2 hrs.</li>
	<li>Wash lightly with 1 ml of cold PBS. Then place three to four pieces of circular cover glass on a slide, and embed the specimens in a 30% solution of glycerol in PBS on the slide (Figure 3).</li>
	<li>Observe specimens with a DM-IRE2 confocal laser scanning microscope or a DeltaVision Restoration deconvolution microscope.</li>
</ol>
5. Representative Results:
The specimen example of a mouse ligated Peyer's patch loop assay with GFP-S. Typhimurium was observed with a DeltaVision Restoration deconvolution microscope (Figure 4). GFP-S. Typhimurium is transcytosed by GP2+M cells. A mouse Peyer's patch intestinal loop assay can identify the kind of commensal or pathogenic bacteria that can be transcytosed by M cells.
<img alt="Figure 1" src="/files/ftp_upload/3225/3225fig1.jpg" /> 
Figure 1. Anesthesia of mice under vaporized isoflurane condition. Vaporized isoflurane was supplied by an anesthesia-dedicated apparatus (left side).
<img alt="Figure 2" src="/files/ftp_upload/3225/3225fig2.jpg" /> 
Figure 2. Summary of ligated Peyer's patch intestinal loop assay. The fluorescent bacterial suspension was injected by syringe into ligated Peyer's patch loop on the loose side of the intestine.
<img alt="Figure 3" src="/files/ftp_upload/3225/3225fig3.jpg" /> 
Figure 3. Mount of the specimens. The specimens were embedded in a 30% solution of glycerol in PBS on a special slide in which a perforated plastic plate (1mm thick) was bound by adhesive agent on a glass slide.
<img alt="Figure 4" src="/files/ftp_upload/3225/3225fig4.jpg" /> 
Figure 4. Example of a mouse ligated Peyer's patch loop assay with GFP-S. Typhimurium. GP2, which is reported as M cell specific marker5, was stained with anti-mouse GP2 antibody (Red).The specimen was observed with a DeltaVision Restoration deconvolution microscope. GFP-expressing S. Typhimurium was co-localized with GP2 on the apical plasma membrane of M cell and in the subapical cytoplasmic vesicles (arrowheads). Top, apical view. Bottom and side, lateral views. Scale bar: 10 &mu;m.

<ol>
	<li>Fagarasan, S., Honjo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+IgA+synthesis:+regulation+of+front-line+body+defences.">Intestinal IgA synthesis: regulation of front-line body defences.</a> Nat. Rev. Immunol. 3, 63-72 (2003).</li><li>Hapfelmeier, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+microbial+colonization+of+germ-free+mice+reveals+the+dynamics+of+IgA+immune+responses.">Reversible microbial colonization of germ-free mice reveals the dynamics of IgA immune responses.</a> Science. 328, 1705-1709 (2010).</li><li>Owen, R. L., Jones, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+cell+specialization+within+human+Peyer's+patches:+an+ultrastructural+study+of+intestinal+lymphoid+follicles.">Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles.</a> Gastroenterology. 66, 189-203 (1974).</li><li>Neutra, M. R., Mantis, N. J., Kraehenbuhl, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaboration+of+epithelial+cells+with+organized+mucosal+lymphoid+tissues.">Collaboration of epithelial cells with organized mucosal lymphoid tissues.</a> Nat. Immunol. 2, 1004-1009 (2001).</li><li>Hase, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+through+glycoprotein+2+of+FimH(+)+bacteria+by+M+cells+initiates+mucosal+immune+response.">Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.</a> Nature. 462, 226-230 (2009).</li><li>Punyashthiti, K., Finkelstein, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteropathogenicity+of+Escherichia+coli.+I.+Evaluation+of+mouse+intestinal+loops.">Enteropathogenicity of Escherichia coli. I. Evaluation of mouse intestinal loops.</a> Infect. Immun. 4, 473-478 (1971).</li><li>Owen, R. L., Pierce, N. F., Apple, R. T., Cray, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cell+transport+of+Vibrio+cholerae+from+the+intestinal+lumen+into+Peyer's+patches:+a+mechanism+for+antigen+sampling+and+for+microbial+transepithelial+migration.">M cell transport of Vibrio cholerae from the intestinal lumen into Peyer's patches: a mechanism for antigen sampling and for microbial transepithelial migration.</a> J. Infect. Dis. 153, 1108-1118 (1986).</li><li>Owen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M+cells--entryways+of+opportunity+for+enteropathogens.">M cells--entryways of opportunity for enteropathogens.</a> J. Exp. Med. 180, 7-9 (1994).</li><li>Clark, M. A., Hirst, B. H., Jepson, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M-cell+surface+beta1+integrin+expression+and+invasin-mediated+targeting+of+Yersinia+pseudotuberculosis+to+mouse+Peyer's+patch+M+cells.">M-cell surface beta1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer's patch M cells.</a> Infect. Immun. 66, 1237-1243 (1998).</li><li>Jensen, V. B., Harty, J. T., Jones, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+the+invasive+pathogens+Salmonella+typhimurium,+Listeria+monocytogenes,+and+Shigella+flexneri+with+M+cells+and+murine+Peyer's+patches.">Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches.</a> Infect. Immun. 66, 3758-3766 (1998).</li><li>Nagai, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Peyer's+patches+in+the+induction+of+Helicobacter+pylori-induced+gastritis.">Role of Peyer's patches in the induction of Helicobacter pylori-induced gastritis.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 8971-8976 (2007).</li></ol>

No conflicts of interest declared.

The incubation time of ligated Peyer's patch with bacterial suspension is usually for 1 hr to observe the bacterial incorporation into M cells. In case of 4 hrs incubation, the bacteria are often detected in the T cell zone of Peyer's patches. As the vaporized isoflurane inhalant anesthesia could keep mice stable, the incubation time of ligated Peyer's patch with bacterial suspension is extendable to observe the bacteria which move into T-cell zone in Peyer's patch. The important point of this experiment is to take care ...

<table><tbody><tr><td>LB Agar Ampicillin- 100</td><td>SIGMA</td><td>L5667</td><td></td></tr><tr><td>Cy3 mono-Reactive Dye Pack</td><td>GE Healthcare</td><td>PA23001</td><td></td></tr><tr><td>Alexa Fluor 350 carboxylic acid, succinimidyl ester</td><td>Invitrogen</td><td>A10168</td><td></td></tr><tr><td>Inhalation anesthesia apparatus</td><td>Shinano Seisakusho</td><td>SN-487</td><td></td></tr><tr><td>Fixation and Permeabilization Solution</td><td>BD</td><td>554722</td><td></td></tr><tr><td>Anti mouse Glycoprotein 2 antibody</td><td>MBL</td><td>D278-3</td><td>200-fold dilution (5&amp;mu;g/ml)</td></tr><tr><td>Goat Anti-Rat IgG, F(ab&#039;)2 fragment specific</td><td>Jackson ImmunoResearch</td><td>112-505-006</td><td>200-fold dilution (20&amp;mu;g/ml)</td></tr><tr><td>Alexa Fluor 633 Phalloidin</td><td>Molecular Probes</td><td>A22284</td><td>50-fold dilution</td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica Microsystems</td><td>DMIRE2</td><td></td></tr><tr><td>DeltaVision Restoration deconvolution microscope</td><td>Applied Precision</td><td>DeltaVision Core</td><td></td></tr></tbody></table>

application of a mouse ligated peyer&#x2019;s patch intestinal loop assay to evaluate bacterial uptake by m cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes1, 2. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer's patches. This antigen transcytosis is mediated by specialized epithelial M cells3, 4. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane 5. Here, we present a method for the application of a mouse Peyer's patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described 6, 7. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer's patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer's patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer's patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.

Watch this Scientific Journal Video about Application of a Mouse Ligated Peyerâ€™s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells at JoVE.com

Application of a Mouse Ligated Peyer&#8217;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

M cells in a specialized follicle-associated epithelium covering Peyer&#x2019;s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Application of a Mouse Ligated Peyer&#x2019;s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed ...

application-mouse-ligated-peyers-patch-intestinal-loop-assay-to

Research

JoVE Journal

Immunology and Infection

19.4K Views.  RIKEN Research Center for Allergy and Immunology. M cells in a specialized follicle-associated epithelium covering Peyer’s patches play an important role for the mucosal immunosurveillance in gut-associated lymphoid tissue. Here we described the evaluation method for bacterial transcytosis by M cells in vivo. This method provides a method to understand M-cell function in the immune system.

Video: Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

Presented here is a unified description of techniques that can be used to develop, transform, administer, and test heterologous protein expression of the probiotic yeast Saccharomyces boulardii.

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Microscopic Observation of Lymphocyte Dynamics in Rat Peyer's Patches

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important phenomenon in the gut immunity. The stroma of secondary lymphoid organs, such as Peyer&#8217;s patches (PPs) or mesenteric lymph nodes, are where na&#239;ve lymphocytes sense antigens. Na&#239;ve lymphocytes circulate through the bloodstream to reach high endothelial venules, the portal of entry into PPs. Some immunomodulators are estimated to influence lymphocyte migration, but the precise evaluation of microcirculation dynamics is very difficult, and establishing a method to observe lymphocyte migration in vivo can contribute to the clarification of the precise mechanisms. We refined the method of collecting lymphocytes from the lymph duct and observing the detailed dynamics of gut-tropic lymphocytes in rat PPs. We chose confocal laser scanning microscopy to observe rat PPs in vivo and recorded it using time-lapse photography. We can now obtain clear images that can contribute to the analysis of lymphocyte dynamics.

Here, we describe a precise method for collecting thoracic duct lymphocytes and observing the migration of gut-tropic lymphocytes in rat Peyer&#8217;s patches for 3 hours using time-lapse photography. This technique can clarify how the dynamics of lymphocytes are affected under inflammatory conditions.

Na&#239;ve lymphocytes recirculate from the blood to the lymphoid tissues under physiological condition and it is commonly recognized as an important ...

Medicine

Murine Model for Studying Postoperative Ileus through Intestinal Manipulation

Postoperative Ileus Murine Model

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI is a consequence of mechanical damage during surgery, resulting in disruption of motility in the gastrointestinal tract. The mechanisms of POI are related to aberrant neuronal sensitivity, impaired epithelial barrier function, and increased local inflammation. However, the details remain enigmatic. Therefore, experimental murine models are crucial for elucidating the pathophysiology and mechanism of POI injury and for the development of novel therapies.
Here, we introduce a murine model of POI generated via intestinal manipulation (IM) that is similar to clinical surgery; this is achieved by mechanical damage to the small intestine by massaging the abdomen 1-3 times with a cotton swab. IM delayed gastrointestinal transit 24 h after surgery, as assessed by FITC-dextran gavage and fluorescence detection of the segmental digestive tract. Moreover, tissue swelling of the submucosa and immune cell infiltration were investigated by hematoxylin and eosin staining and flow cytometry. Proper pressure of the IM and a hyperemic effect on the intestine are critical for the procedure. This murine model of POI can be utilized to study the mechanisms of intestinal damage and recovery after abdominal surgery.

We describe a murine model of postoperative ileus generated via intestinal manipulation. Gastrointestinal transit function, pathologic changes, and immune cell activation were assessed 24 h after surgery.

Most patients experience postoperative ileus (POI) after surgery, which is associated with increased morbidity, mortality, and hospitalization time. POI ...

Mouse Ileal Loop Model: A Surgical Model to Study Intestinal Permeability in the Mouse

Source: Boerner, K., et al. <a href="https://www.jove.com/v/62093">Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.</a> J. Vis. Exp. (2021)
This video demonstrates the generation of an ileal loop model to study intestinal permeability in a mouse model. This method provides a live model to understand the epithelial barrier function and consequent immune response.

Model pętli jelita krętego myszy: model chirurgiczny do badania przepuszczalności jelit u myszy

Source: Boerner, K., et al. Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized ...

JoVE Encyclopedia of Experiments

Biology

Rodent Models

Adult

Oral Delivery of Transformed Yeast Cells into the Mouse Intestinal Immune System

After preparing yeast cultures according to the text protocol, use a hemocytometer to determine the cell concentration, and adjust to 10 to the ninth cells per milliliter. Pellet the cells by centrifugation at 2,500 times g for three minutes. Then, pour off the supernatant, and resuspend the cells by adding the appropriate volume of sterile water, and gently pipetting up and down.
Fix an appropriate gauge gavage needle onto a 1-milliliter sterile syringe, and load the yeast sample, eliminating any air bubbles. Load an additional syringe with sterile water to gavage control mice. Using the non-dominant hand, pick up the mouse to be gavaged, and with the index finger and thumb, tightly grasp the skin around the neck. Tuck the tail under the small finger. Be sure the grip is secure and prohibits the mouse from moving its head.
Next, gently insert the gavage needle into the mouse esophagus by angling the needle along the roof of the mouth and back of the throat, keeping slightly to the left of center. Wait for the mouse to swallow the bulb of the needle. If resistance is felt or the mouse gasps, gently remove the needle and try again to find the esophagus.
After the mouse has swallowed the bulb of the gavaged needle, gently depress the syringe plunger to administer the yeast directly into the mouse stomach. Gently remove the gavage needle from the mouse stomach and esophagus and return the mouse to the cage. Check that the mouse is breathing and moving normally to ensure the gavage was performed properly.
After sacrificing the mouse according to the text protocol, position it with the abdomen fully exposed and use 70% ethanol to spray the abdominal area. With scissors, make a transverse incision through the fur and skin. Then, manually pry the incision open to further expose the peritoneum, the thin serosa lining that covers the abdominal organs. Gently lift the peritoneum and make a transverse incision to expose the intestines.
Then, use blunt forceps to carefully tease the small intestine away from the mesenteric arteries, fat, and other tissues. Expose the small intestine from the stomach in the upper left quadrant of the mouse abdomen to the cecum, the large pocket of intestinal tissue at the start of the large intestine.
Next, isolate Peyer&#39;s patches by looking for 1 to 2-millimeter roughly circular patches of opaque tissue along the small intestine. Then, using curved dissection scissors, cut away the dome of the Peyer&#39;s patch, leaving margins to ensure that none of the surrounding tissue is collected.
Transfer the dissected Peyer&#39;s patches into complete IMDM. Then, pour the solution with suspended Peyer&#39;s patches onto a 40-micrometer cell strainer. After washing the Peyer&#39;s patches with complete IMDM, use a plunger from a 1-milliliter syringe to gently break up the tissue and collect the cells in a 50-milliliter tube. Pellet the strained cells at 1,800 RPM for seven minutes. Then wait and analyze the cells according to the text protocol.

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo