The evolution of the device started with somewhat different cutting prototype demonstrated at the Imperial Cancer Research Fund (ICRF) mouse club by Bass Hassan, following this there were several discussions with Rob Rudling and Nikki Mandir in the containment facility at ICRF Clare Hall. Jo Kitau of the mechanical workshop at ICRF constructed the initial versions and Andy Lucas designed and perfected the final model in the CRUK workshop in Oxford.

The various experimental procedures and the humane euthanasia are approved by the host institutions ethical committee and by the appropriate regulatory bodies. Euthanasia is normally achieved using CO2 asphyxiation followed by cervical dislocation.
1. Device
<ol>
	<li>Machine the main portion of the base of the device from out of a solid block of high-density acetyl resin, as described in this manuscript. At each end create 3 mm high-elevated strips, and part machine out of these 2.5 mm groves to take the rods (see Figure 2 and Figure 3).</li>
	<li>Make rods from a 2.4 mm diameter stainless steel with rounded-off ends. 
	NOTE: The main frame of the lid was machined out of Duralumin plate. A limited number of finished devices are available from Dr. Goodlad.</li>
</ol>
2. Use of the Device
<ol>
	<li>Dissect out the small intestine and colon from the abdominal cavity using scissors and gloved fingers. Remove the mesentery by holding it with curved forceps and gently pulling it away from the intestine.</li>
	<li>Rinse the intestines with cold phosphate buffered saline (PBS, pH 7.4) using a Gilson type pipette tip, which had been cut down at its wider end so that it can fit onto a 10 or 20 ml luer fitting plastic syringe.</li>
	<li>Lay out the small bowel (SB) on to a sheet of paper towel and divide into three equal sections in length (proximal, mid, and distal &#8211; SB1, SB2 and SB3) using scissors. Remove the cecum and lay out the colon.</li>
	<li>Make several very small cuts into the intestinal segments with scissors to allow the fluid to escape and place a sheet of paper towel over the intestines and gently run a finger over the preparations to squeeze out the fluid and blot dry.</li>
	<li>Lift the sections and insert the stainless steel rods that were previously wetted by soaking in a beaker of phosphate buffered saline. 
	NOTE: The intestine can be inverted if so desired (turned inside out).</li>
	<li>Label a piece of card or filter paper cut to size to fit into the base of the device. Pencil is best, as it is unaffected by virtually all solutions. Autopsy date, experimental code and animal identification number are suggested.</li>
	<li>Place the card or filter paper into the base of the device. Insert the rods and intestines into the slots in the base of the device. Make sure the proximal end of the segments is positioned in a standardized manner. The proximal ends should be near the card labels.</li>
	<li>Place the top piece of the device over the base. Use the angled bars to guide a scalpel blade to longitudinally cut the sections. 
	NOTE: It helps if the tissue is gently and carefully held with a finger to ensure that it does not move with the knife.</li>
	<li>Remove the top piece of the device. Carefully remove the filter paper and tissues (still on their rods). 
	NOTE: A piece of stiff card placed underneath the filter paper will aid lifting.</li>
	<li>Slightly wet (with a gloved fingertip dipped in PBS) the segments. Roll the rod, side to side to open up the gut and spread it flat. The tissue will then adhere to the filter paper.</li>
	<li>Visually examine the preparations for any gross lesions.</li>
	<li>Transfer the tissue adhered to the filter paper to a shallow bath (or sandwich box) containing fixative. 
	NOTE: After the tissue adheres to the filter paper or to card it will remain firmly attached. A wide range of fixatives can be used to preserve the preparation. It is recommended that Carnoy&#8217;s fluid should be used for the study of intestinal cell proliferations6, which is the ideal fixation for the best cell proliferation methods; however formalin or many other fixatives can also be used if other endpoints are required.</li>
	<li>After fixation (usually 3 hr), transfer the preparations into 70% ethanol. The samples can then be stored until required. The preparations can be studied en face and/or used for the preparation of histological samples for H&#38;E or other histological staining techniques, immunohistochemistry and/or in situ hybridization. 
	NOTE: Prolonged fixation can reduce immunohistochemical reactivity and/or in situ hybridization reactivity. Polypropylene sandwich boxes are ideal for storing a large number of the preparations.</li>
	<li>If required the fixed tissues from the various intestinal segments can be made into &#8216;Swiss rolls&#8217; and stained with the entire range of histological methods6 including those described above. To make a &#8216;Swiss roll&#8217; the intestinal segments are removed from the filter paper, rolled around a cocktail stick or a toothpick, secured with an entomological pin and processed for wax embedding6.</li>
	<li>If required, stain the bulk tissue with methylene blue to visualize aberrant crypt foci7.</li>
	<li>Score the fixed preparations at leisure.</li>
</ol>

<ol><li>Goodlad, R. A. Cancer Handbook. Alison, M. R. , John Wiley and Sons, Ltd. 243-262 (2007).</li>
<li>Ward, J. M., Treuting, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rodent+intestinal+epithelial+carcinogenesis%3A+pathology+and+preclinical+models%5BTitle%5D)+AND+%22Toxicol+Pathol%22%5BJournal%5D)">Rodent intestinal epithelial carcinogenesis: pathology and preclinical models.</a> Toxicol Pathol. 42, 148-161 (2014).</li>
<li>Sundberg, J. P., Hogenesch, H., Nikitin, A. Y., Treuting, P. M., Ward, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Training+mouse+pathologists%3A+ten+years+of+workshops+on+the+Pathology+of+Mouse+Models+of+Human+Disease%5BTitle%5D)+AND+%22Toxicol+Pathol%22%5BJournal%5D)">Training mouse pathologists: ten years of workshops on the Pathology of Mouse Models of Human Disease.</a> Toxicol Pathol. 40, 823-825 (2012).</li>
<li>Wasan, H. S., Novelli, M., Bee, J., Bodmer, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dietary+fat+influences+on+polyp+phenotype+in+multiple+intestinal+neoplasia+mice%5BTitle%5D)+AND+%22Proc+Natl+Acade+Sci+USA%22%5BJournal%5D)">Dietary fat influences on polyp phenotype in multiple intestinal neoplasia mice.</a> Proc Natl Acade Sci USA. 94, 3308-3313 (1997).</li>
<li>Rudling, R., Hassan, A. B., Kitau, J., Mandir, N., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+device+to+rapidly+prepare+whole+mounts+of+murine+intestine%5BTitle%5D)+AND+%22Cell+Prolif%22%5BJournal%5D)">A simple device to rapidly prepare whole mounts of murine intestine.</a> Cell Prolif. 39, 415-420 (2006).</li>
<li>Alferez, D., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((To+best+measure+cell+proliferation+in+samples+from+the+intestine%5BTitle%5D)+AND+%22Cell+Prolif%22%5BJournal%5D)">To best measure cell proliferation in samples from the intestine.</a> Cell Prolif. 40, 231-240 (2007).</li>
<li>Park, H. S., Goodlad, R. A., Wright, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+incidence+of+aberrant+crypt+foci+and+colonic+carcinoma+in+dimethylhydrazine-treated+rats+varies+in+a+site-specific+manner+and+depends+on+tumor+histology%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">The incidence of aberrant crypt foci and colonic carcinoma in dimethylhydrazine-treated rats varies in a site-specific manner and depends on tumor histology.</a> Cancer Res. 57, 4507-4510 (1997).</li>
<li>Park, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+epidermal+growth+factor+and+dimethylhydrazine+on+crypt+size%2C+cell+proliferation%2C+and+crypt+fission+in+the+rat+colon.+Cell+proliferation+and+crypt+fission+are+controlled+independently%5BTitle%5D)+AND+%22Am+J+Pathol%22%5BJournal%5D)">Effects of epidermal growth factor and dimethylhydrazine on crypt size, cell proliferation, and crypt fission in the rat colon. Cell proliferation and crypt fission are controlled independently.</a> Am J Pathol. 151, 843-852 (1997).</li>
<li>Femia, A. P., Dolara, P., Caderni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mucin-depleted+foci+%28MDF%29+in+the+colon+of+rats+treated+with+azoxymethane+%28AOM%29+are+useful+biomarkers+for+colon+carcinogenesis%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Mucin-depleted foci (MDF) in the colon of rats treated with azoxymethane (AOM) are useful biomarkers for colon carcinogenesis.</a> Carcinogenesis. 25, 277-281 (2004).</li>
<li>Ruehl-Fehlert, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revised+guides+for+organ+sampling+and+trimming+in+rats+and+mice--part+1%5BTitle%5D)+AND+%22Exp+Toxicol+Pathol%22%5BJournal%5D)">Revised guides for organ sampling and trimming in rats and mice--part 1.</a> Exp Toxicol Pathol. 55, 91-106 (2003).</li>
<li>Goodlad, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibiting+vascular+endothelial+growth+factor+receptor-2+signaling+reduces+tumor+burden+in+the+ApcMin%2F%2B+mouse+model+of+early+intestinal+cancer%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Inhibiting vascular endothelial growth factor receptor-2 signaling reduces tumor burden in the ApcMin/+ mouse model of early intestinal cancer.</a> Carcinogenesis. 27, 2133-2139 (2006).</li>
<li>Mandir, N., Englyst, H., Goodlad, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resistant+carbohydrates+stimulate+cell+proliferation+and+crypt+fission+in+wild-type+mice+and+in+the+Apc%28Min%2F%2B%29+mouse+model+of+intestinal+cancer%2C+association+with+enhanced+polyp+development%5BTitle%5D)+AND+%22Br+J+Nutr%22%5BJournal%5D)">Resistant carbohydrates stimulate cell proliferation and crypt fission in wild-type mice and in the Apc(Min/+) mouse model of intestinal cancer, association with enhanced polyp development.</a> Br J Nutr. 100, 711-721 (2008).</li>
</ol>

The authors have nothing to disclose.

The device greatly speeds up the process so that two operators can fully autopsy a mouse within 6 min. This will include a visual check for obvious gross lesions and weighing and fixing the major organs. It will also allow the operators to rinse, blot and weigh the stomach, caecum, small intestine and colon and prepare the whole mounts. The flattened tissue can be fixed very quickly and thus avoid degradation artifacts.
One of the major advantages of the device (apart from speed) is that the d...

The mouse and the genetically modified mouse1-3 are an invaluable model for the study of various pathologies especially cancer and its relationship to inflammation in the gastrointestinal tract. This often requires the preparation of whole mounts of the small intestine and/or colon.
The preparation of whole mounts can be done with a precision pair of offset scissors but it can take about 20 min&#160;per mouse4. This is not very practical for comparative studies, which require a significant amount of mice (say 10 per group subject to statistical analysis). In addition, the length of time taken increases the risk of tissue degradation. An attempt to alleviate this problem led to the trial of various cutting preparation aids. The first was based on a series of metal plates, with cutting groves, but the plates obscured the tissue and the cutting was thus difficult to control.
A conventional six-sided pencil finally provided inspiration for the cutting device, as we could then see how a half pencil shape would make a good guide for a scalpel (Figure 1). This resulted in a design for a frame with four cutting guides5. The effectiveness of this device was dramatic as the preparations in Figure 4 demonstrate the difference between the scissor preparation and the device preparation.
The tops of the original devices was constructed from several pieces of metal, but later ones, and the current model was machined out of a solid piece of Duralumin. The base was machined out of highest quality high-density resin.
The device has proved to be of great use for the preparation of whole mounts of mouse intestine as it greatly speeds up and improves the quality and consistency of the preparations, which have a wide range of applications.

<table><tbody><tr><td>Delrin</td><td>Ryan Plastics, Earls Barton, UK</td><td></td><td>High-density acetal resin similar material would suffice</td></tr><tr><td>Duralumin plate</td><td>Metal Supplies Ltd, Park Road,Dukinfield. SK16 5LP UK</td><td></td><td>Aluminium allow dating back to 1909 so alterative suppliers are available&amp;nbsp;</td></tr><tr><td>Finished device(s) ready for use</td><td>Contact Dr Goodlad&amp;nbsp; r.goodlad@imperial.ac.uk</td><td></td><td>Contact&amp;nbsp; r.goodlad@imperial.ac.uk for supply details</td></tr></tbody></table>

a simple device to rapidly prepare whole mounts of the mouse intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

Polyps are easy to identify under a stereomicroscope with a cold light source to side illuminate the tissue (Figure 4). It is helpful to mark the card with pencil marks when a polyp is observed in order that scoring can be discussed later and different operators can reach agreement. The tissue can also be bulk stained [with methylene blue] to identify aberrant crypt foci8 or mucin depleted foci (MDFs)9 by illumination from above or by trans illumination (Figure 5).<...

Watch this Scientific Journal Video about A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine at JoVE.com

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

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Research

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Medicine

19.6K Views.  National Institutes of Health. The use of a simple device to cut and ‘roll’ mouse intestines to rapidly prepare whole mount preparations is described.

Video: A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Developmental Biology

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the complex cellular and molecular of intestinal pathology. Although the tunica mucosa is often the organ of interest in many inflammatory diseases, recent works demonstrated that the muscularis externa (ME) is also a highly immunocompetent organ that harbours a dense network of resident immunocytes.1,2 These works were performed within the standardized model of intestinal manipulation (IM) that leads to inflammation of the bowel wall, mainly limited to the ME. Clinically this inflammation leads to prolonged intestinal dysmotility, known as postoperative ileus (POI) which is a frequent and unavoidable complication after abdominal surgery.3 The inflammation is characterized by liberation of proinflammatory mediators such as IL-64 or IL-1&beta; or inhibitory neurotransmitters like nitric oxide (NO).5 Subsequently, tremendous numbers of immunocytes extravasate into the ME, dominated by polymorphonuclear neutrophils (PMN) and monocytes and finally maintain POI.2 Lasting for days, this intestinal paralysis leads to an increased risk of aspiration, bacterial translocation and infectious complications up to sepsis and multi organ failure and causes a high economic burden.6
In this manuscript we demonstrate the standardized model of IM and in vivo assessment of gastrointestinal transit (GIT) and colonic transit. Furthermore we demonstrate a method for separation of the ME from the tunica mucosa followed by immunological analysis, which is crucial to distinguish between the inflammatory responses in these both highly immunoactive bowel wall compartments. All analyses are easily transferable to any other research models, affecting gastrointestinal function.

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Inflammation of the gastrointestinal tract is a common reason for a variety of human diseases. Animal research models are critical in investigating the ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunology and Infection

Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves required for peristalsis and mediate neuronal input from the enteric nervous system1. Different subtypes of ICC form distinct networks in the muscularis of the GI tract 2,3. Loss or injury to these networks is associated with a number of motility disorders4. ICC cells express the KIT receptor tyrosine kinase on the plasma membrane and KIT immunostaining has been used for the past 15 years to label the ICC network5,6. Importantly, normal KIT activity is required for ICC development5,6. Neoplastic transformation of ICC cells results in gastrointestinal stromal tumor (GIST), that frequently harbor gain-of-function KIT mutations7,8. We recently showed that ETV1 is a lineage-specific survival factor expressed in the ICC/GIST lineage and is a master transcriptional regulator required for both normal ICC network formation and for of GIST tumorigenesis9. We further demonstrate that it cooperates with activating KIT mutations in tumorigenesis. Here, we describe methods for visualization of ICC networks in mice, largely based on previously published protocols10,11. More recently, the chloride channel anoctamin 1 (ANO1) has also been characterized as a specific membrane marker of ICC11,12. Because of their plasma membrane localization, immunofluorescence of both proteins can be used to visualize the ICC networks. Here, we describe visualization of the ICC networks by fixed-frozen cyrosections and whole mount preparations.

Visualization of the Interstitial Cells of Cajal ICC Network in Mice

The interstitial cells of Cajal (ICC) are the pacemaker cells of the gastrointestinal (GI) tract. They form complex networks between smooth muscle cells and post-ganglionic neuronal fibers to regulate GI contractility. Here, we present immunofluorescence methods cross-sectional and whole-mount visualization of murine ICC networks.

The interstitial cells of Cajal (ICC) are mesenchymal derived "pacemaker cells" of the gastrointestinal (GI) tract that generate spontaneous slow waves ...

Biology

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

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 ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Efficient Swiss Roll Method for High-Quality Immunostaining of Intestinal Tissue

Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ileum. Each anatomical region of the intestine has a unique function that is reflected by differences in cellular structure. Investigating changes in the intestine requires an in-depth analysis of different tissue regions and cellular alterations. To study the intestine and visualize large pieces of tissue, researchers commonly use a technique known as intestinal Swiss rolls. In this technique, the intestine is divided into each anatomical region and fixed in a flat orientation. Then, the tissue is carefully rolled and processed for paraffin embedding. Proper tissue fixation and orientation is an often-overlooked laboratory technique but is critically important for downstream analysis. Additionally, improper Swiss rolling of intestinal tissue can damage the fragile intestinal epithelium, leading to poor tissue quality for immunostaining. Ensuring well-fixed and properly oriented tissue with intact cellular structures is a crucial step that ensures optimal visualization of intestinal cells. We present a cost-effective and simple method for making Swiss rolls to include all sections of the intestine in a single paraffin-embedded block. We also describe optimized immunofluorescence staining of intestinal tissue to study various aspects of the intestinal epithelium. The following protocol provides researchers with a comprehensive guide to obtaining high-quality immunofluorescence images through intestinal tissue fixation, Swiss-roll technique, and immunostaining. Employing these refined approaches preserves the intricate morphology of the intestinal epithelium and fosters a deeper understanding of intestinal physiology and pathobiology.

Author Spotlight: Effective Immunofluorescent Staining Protocol for Intestinal Cell Biology Research

The intestine is vital for digestion and absorption. Each region-duodenum, jejunum, ileum, colon-serves distinct functions due to unique cellular structures. Studying intestinal physiology demands meticulous tissue analysis. This protocol outlines tissue fixation and processing using the Swiss roll technique, ensuring accurate immunostaining through proper tissue preservation and orientation.

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ...

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

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