Name: murine appendectomy model of chronic colitis associated colorectal cancer by precise localization of caecal patch
Uploaded: 2019-08-24T14:00:00
Description: Watch this Scientific Journal Video about Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch at JoVE.com

This work is partially supported by Fundamental Research Funds for the Central University (G2018KY0302), University Fundamental Research Development Fund (KT00062), National Natural Science Foundation of China (81870380), and Clinical Research Award of the First Affiliated Hospital of Xi&#8217;an Jiaotong University in China (NO.XJTU1AF-CRF-2015-029). The authors thank Dr. Chengxin Shi for his technical suggestions during the early exploration phase of the murine appendectomy model, as well as the pathologist Dr. Xi Liu for evaluation of H&#38;E staining results of colitis and colorectal tumors. Y.L. performed the surgery demonstration,&#160;did data analysis, and wrote the draft of the manuscript; J.L., G.L., Z.P., and Y.M. took part in the surgical preparation, tissue collections, and video production; M.Z. performed the flow cytometry and ELISA; Q.W. and H.X. provided the technical support of generating a clinically relevant murine model; R.X.Z. designed the study, supervised the research, and wrote and proofed the manuscript; J.S. reviewed the manuscript.

All animal procedures were approved by the Institutional Animal Care and Use Committee of Xi&#8217;an Jiaotong University (No. XJTULAC2019-1023).
1. Mice appendectomy
<ol>
	<li>House 8&#8211;10-week-old C57BL/6 male mice in a certified specific-pathogen free (SPF) environment for 1 week prior to surgery.</li>
	<li>Prepare the following sterile surgical instruments: one pair of micro-scissors, one pair of micro-forceps, two sizes (4-0 and 8-0) of sterilized non-absorbable sutures, an electric...

A murine appendectomy model of colitis-associated colorectal cancer was obtained using surgical steps with a high survival rate in mice. In most cases, since the caecum was positioned under the abdominal wall (Supplementary Table 1, Supplementary Table 2, and Figure 2), it was difficult to prejudge its location without laparotomy. In this surgical protocol, an easy step of touching the bump was introduced, and quantitative evaluation of the cecum location was also provided a...

The clinical appendectomy is a standard surgical procedure involving removal of the appendix mostly due to inflammation (e.g., appendicitis)1,2,3. However, the biological function of the vermiform human appendix remains controversial4,5,6. The appendix has been regarded as a vestigial remnant projecting from the cecum in the large bowel. Until recently, evolutionary, immunological, morphological, and microbiological studies have suggested that the appendix may possess distinct functions. These roles include the production of immunoglobins (e.g., IgA and IgG), a variety of B cells and T cells critical for adaptive immune responses within the gut-associated lymphoid tissues (GALTs), and replenishment of the large bowel with commensal microbiotas6,7,8,9,10,11,12.
Clinical epidemiological studies of patients with prior appendectomy or acute appendicitis have also revealed its potential roles in the pathogenesis of human diseases, such as inflammatory bowel disease (IBD), colorectal cancer, and non-gastrointestinal disorders (e.g., Parkinson&#8217;s disease and cardiovascular disease)13,14,15,16,17,18. For example, a large Asian population cohort study with 75,979 appendectomy patients recently showed a significant association between appendectomy and subsequent development of colorectal cancer, one of the most common malignancies with a high incidence and mortality14,19. Accordingly, establishing a suitable animal appendectomy model that resembles a human will be helpful to investigate the biological functions and molecular mechanisms of the appendix in the disease pathogenesis.
Many mammals possess an appendix or appendix-like organ, including primates, lagomorphs (e.g., rabbits), some rodents, and marsupials20. For small and commonly used laboratory animals, the rabbit possesses the vermiform appendix morphologically resembling the human21,22, but GALT in the rabbit is extremely large compared to that in humans, since the majority of lymphoid tissues are also found in Peyer&#8217;s patches located in both small and large intestines21. Additionally, the rabbit shows a different lymphoid follicular structure, T cell distribution, and immunoglobulin density from the human, which makes the studying of their appendices inappropriate21.
Mice are the most commonly used animal model to study human pathophysiology and test the various existing and novel therapuetics23,24,25. The single white large lymphoid cluster at the apex of the caecum in mice, known as the caecal patch, is thought to perform functions similar to the human appendix26,27,28. Yet, it is practically difficult to separate the caecal patch from caecum in mice. So far, the common surgical procedures for inducing appendicitis in a mouse model involve a relatively large incision (e.g., 1&#8211;2 cm) through the abdominal wall to gain access to the whole caecum (Supplemental Table 1)29,30,31,32,33,34,35,36.
Herein, to generate an appendectomy model associated with gastrointestinal disease, this report presents a facile surgical protocol for caecal patch removal in mice. This is followed by the combined administration of the genotoxic agent AOM and pro-inflammatory agent DSS for the induction of colitis-associated colorectal cancer similar to that seen in humans. IBD has been shown to be a risk factor of intestinal cancer37,38. The combination of AOM/DSS-induced chronic colitis-associated colorectal cancer has been well-established, and readers can refer to Neufert et al., and Thaker et al. for detailed procedures39,40. This reproducible and rapid murine appendectomy model can be used to study appendix-modulated bowel inflammation and colon microbiota, especially in the development and progression of IBD and colorectal cancer.

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			<td>Azoxymethane&#65288;AOM</td>
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murine appendectomy model of chronic colitis associated colorectal cancer by precise localization of caecal patch

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal cancer, inflammatory bowel disease, and Parkinson&#8217;s disease. To study the function of the appendix, a gut disease-associated murine appendectomy model has been established and its step-by-step protocol is described here. This report introduces a facile protocol for caecal patch removal in mice followed by the chemical induction of chronic colitis-associated colorectal cancer using a combination of dextran sulfate sodium (DSS) and azoxymethane (AOM). IgA specific cells and IgA concentration were significantly reduced upon removal of the caecal patch in male C57BL/6 mice&#160;compared to those in the sham group. Simultaneously administering 2% DSS and AOM resulted in nearly 80% mice survival in both sham and appendectomy groups without significant body weight loss. Histological results confirmed colonic inflammation and different degrees of adenocarcinoma. This model can be used for the study of the functional role of the appendix in maintaining gut microbiota homeostasis and pathogenesis of gut colitis and malignancies, as well as for the potential development of drug targeting therapies.

Establishment of murine appendectomy model
This murine appendectomy model of chronic colitis associated colorectal cancer can be generated by following the sequential surgical and induction steps as illustrated in Figure 1. The most frequent positions of caecum are in the left and right iliac fossa followed by the middle line of the abdomen (Figure 2). The successful rate of pre-localization of caecum prior to the abd...

Watch this Scientific Journal Video about Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch at JoVE.com

Murine Appendectomy Model of Chronic Colitis Associated Colorectal Cancer by Precise Localization of Caecal Patch

The presented protocol describes a facile surgical removal of the appendix (caecal patch) in a mouse followed by the induction of inflammatory bowel disease-associated colorectal cancer. This murine appendectomy model enables investigation of the biological role of the appendix in the pathogenesis of human gastrointestinal disease.

The human appendix has been recently implicated to play important biological roles in the pathogenesis of various complex diseases, such as colorectal ...

The overall protocol describes easily performed surgical procedures of removing the appendix known as caecal patch in mice. The murine appendectomy model can be used to study the biological roles of appendix in pathogenesis of the human gastrointestinal disease. The human appendix is a disputable organ.Recent studies have suggested that the appendix plays an important role in maintaining the gut health. To study the biological function of appendix, generating a disease-associated appendectomy animal model is critical. In this protocol, we will describe a simple and a safe surgical steps of removing appendix, known as caecal patch in mice.To ensure the success of this model, there are three key steps as you will see in the following video. First, locate the caecum of the mice before the surgery. Second, make a small incision in the abdomen.Third, disinfect and close the caecum stump. The first part, mice appendectomy. Prepare sterile surgical instruments and cleaning solutions before the surgery.Anesthetize and secure the mouse on a platform in a supine position by placing four strips of medical adhesive tape across the limbs to prevent postural movement during the surgery. Gently touch the whole abdomen of the mouse to find the feeling of bump. Cover the drape and disinfect the shaved area of mouse abdomen as wide as possible using two times of entoiodine.Make a longitudinal incision ranging from 0.5 centimeter to one centimeter at the midline of abdomen. According to the predetermined location of the caecum, seek the caecum and gently exteriorize it for approximately one centimeter long to identify the caecal patch. Use a pre-warmed and sterile physiological saline to hydrate the intestine.To prevent potential complications of post-operative bleeding and infection, ligate the mesenteric blood vessels of caecum using the 8-0 suture. Hydrate the caecum with the saline. Mark the resection region using the 4-0 suture with open loop near the apex of the caecum at the proximal colon.Cut off the caecal patch below the marked resection using micro-scissors and wipe out the residual caecal content with medical cotton swabs. Disinfect the stump of caecum with entoiodine. Close the stump with running suture using the 8-0 suture.Carefully remove the 4-0 thread loop, previously used for marking the resection at the stump of caecum. Sterilize the sutured position using entoiodine. Rehydrate the cutting area with the saline.Close the musculature layer with running suture using 8-0 suture thread. Close the skin layers using interrupted sutures with 4-0 suture thread. Disinfect the surgical cut twice with entoiodine.Then, remove iodine using 75%medical alcohol. Gently flip the mouse back. Subcutaneously inject 0.1 milligram per kilogram body weight of buprenorphine, and 0.4 milliliters of physiological saline.And rest the mouse on the heat pad, until it returns to consciousness. Put the mouse back to the sterile cage, and closely monitor the mouse for the signs of pain for three days post the surgery to ensure its recovery. The second part:Induction of Colitis-Associated Colorectal Cancer with Azoxymethane, AOM, and Dextran Sodium Sulfate, DSS, seven days post-appendectomy.Thaw one aliquot of AOM and dilute it to the 1 milligram per milliliter using 0.9%normal saline. The entire preparation process is carried out in the fume hood. Dissolve four grams DSS powder in 200 milliliters autoclaved water solution to prepare 2%DSS.Each cage contains five mice. Make an intraperitoneal injection of AOM with 0.01 milliliter per gram per mouse basing on its weight. Replace the autoclaved water with 2%DSS for five days.The mice will be fed with autoclaved water for additional two cycles. The third part:Results. Flow and ELISA results indicate the successful removal of the caecal patch.This procedure can contribute to significant reduction of IgA-specific plasma cells, an obvious change of secretory IgA in large bowel, compared to 3%DSS, which has high mortality rate and significant weight change because of severe bloody stool, anus prolapse, and abdominal abcess. The 2%DSS is suitable for the whole process for its high survival rate to nearly 80%and reasonable weight change in both sham and appendectomy groups. As shown in these images, we have successfully induced the colitis-associated colorectal tumors.In H&E staining images, black arrow indicates mucosal ulcer, arrowhead indicates adenocarcinoma, and a white arrow indicates neutrophil infiltration between glands. Different stages of adenocarcinoma can be seen in both sham and appendectomy groups. This cost-effective Murine Appendectomy Model can be used for answering some important questions such as:what's the role of appendix in the regulation of gut bacteria and how it participates in the immune response in the gut-associated lymphoid tissue.Remember, to avoid the damage in adhesion of intestine, the pre-location of caecum and small skin incision are important. In addition, we used the 2%DSS to induce colitis. But a higher concentration of DSS may be used for other mice strains.

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An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Murine Isolated Heart Model of Myocardial Stunning Associated with Cardioplegic Arrest

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful for modeling ischemic injury associated with numerous clinically relevant phenomenon including cardiac surgery with cardioplegic arrest and cardiopulmonary bypass, off-pump CABG, transplant, angina, brief ischemia, etc. The protocol presents a general method to model hypothermic hyperkalemic cardioplegic arrest and reperfusion in rodent hearts focusing on measurement of myocardial contractile function. In brief, a mouse heart is perfused in langendorff mode, instrumented with an intraventricular balloon, and baseline cardiac functional parameters are recorded. Following stabilization, the heart is then subject to brief infusion of a cardioprotective hypothermic cardioplegia solution to initiate diastolic arrest. Cardioplegia is delivered intermittently over 2 hr. The heart is then reperfused and warmed to normothermic temperatures and recovery of myocardial function is monitored. Use of this protocol results in reliable depressed cardiac contractile function free from gross myocardial tissue damage in rodents.

The goal of this protocol to assess myocardial stunning following ischemic cardioplegic arrest in rodents.

The following protocol is of use to evaluate impaired cardiac function or myocardial stunning following moderate ischemic insults. The technique is useful ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.