The authors thank Nico Zantl, Klaus Pfeffer, and Bernhard Holzmann. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Germany (GRK-840; Vo 450/10-1).

1. Preparation of the Mouse
<ol>
<li>Anesthetize the mouse by intraperitoneal injection of the narcotic fluid (see table of reagents) and place it in supine position.</li>
<li>The feet of the mouse need to be fixed with tape on the plate to ensure a stable position of the animal during the operation.</li>
</ol>
2. Operation
<ol>
<li>Take an i.v. cannula, which is commonly used for i.v. injections in humans, and carve its plastic tube circularly 2mm from its tip. We use three different sizes of cannulas to control mortality rates (BD Venflon 18GA (1,2x45mm, 80 mL/min); 16GA (1,7x45mm, 180 mL/min) 14GA (2,0x45mm, 270 mL/min)).</li>
<li>After thorough disinfection of the abdominal skin, incise it along its midline about 15mm. Open the peritoneal cavern by incising the abdominal muscles and the peritoneum along the linea alba.</li>
<li>Identify the cecal pole and pull out carefully cecum, terminal ileum and ascending colon out of the abdomen by the use of cotton swabs.</li>
<li>15mm distal from the ileocecal valve, one has to pierce the wall of the ascending colon with a 7/0 suture. Thereby, lesions of the gut vessels have to be avoided. The suture is fixed on the colonic wall by two surgical knots.</li>
<li>Now puncture the ascending colon with the prepared cannula 1-2 mm proximal from the 7/0 suture. Carefully insert the cannula into the colon until the furrow in the plastic tube is on a level with the serosa.</li>
<li>Put the free ends of the 7/0 suture around the cannula and place a two-fold knot exactly into the prepared furrow of the plastic tube.</li>
<li>Now, take the needle of the 7/0 suture and stitch it through the antimesenteric wall of the colon. Consecutively, two surgical knots have to be performed to additionally fix the plastic tube in the colonic wall. Cut the suture ends.</li>
<li>Retrieve the iron part of the cannula a little and cut the plastic tube off close (1mm) to the fixing 7/0 suture.</li>
<li>Now one has to carefully milk stool from the cecum towards the colon stent by the use of cotton swabs until a small tip of stool appears on top of the stent.</li>
<li>Put the gut back into the peritoneal cavern and performe fluid resuscitation by intraperitoneal administration of 0.5 mL of saline solution.</li>
<li>Close the peritoneum with continuous suture (4/0).</li>
<li>Close the skin with singular sutures (4/0).</li>
</ol>
3. Postoperative Care
<ol>
<li>Put the animal back in its cage, which should contain enough food and water. For analgesia, intraperitoneal administration of a powerful analgesic substance (we use buprenorphine) should be regularly performed.</li>
<li>Within the first two days after the operation, one has to control the animals every 6 hours.</li>
</ol>
4. Sham CASP
<ol>
<li>Perform steps 1.1-2.4.</li>
<li>Do not puncture the colon. Just perform steps 2.6-2.8.</li>
<li>Perform steps 2.10-3.2.</li>
</ol>
5. CASPI
<ol>
<li>Perform CASP using a 14G cannula following steps 1.1-3.1</li>
<li>5h after CASP, one has to operate the animal again. Prepare and anesthetize the mouse again according to steps 1.1-1.2</li>
<li>Open the sutures of the abdominal wall.</li>
<li>Pull out the ascending colon with the stent inserted.</li>
<li>Carefully cut the sutures fixing the stent and remove the stent.</li>
<li>Close the defect in the colon with single inverting sutures (7/0).</li>
<li>Put the gut back into the abdominal cavity and flush the latter twice with 10 mL of saline solution.</li>
<li>Close the peritoneum with continuous suture (4/0).</li>
<li>Close the skin with singular sutures (4/0).</li>
<li>Follow steps 3.1-3.2</li>
</ol>
6. Representative Results
Within a few hours following the operation, animals show clinical signs of a beginning sepsis. Typical symptoms of the disease are reduced mobility, horrent coat, sweating, decreased food intake, loss of weight and also reduced getaway behaviour. Animals developing a severe peritonitis with consecutive systemic infection normally die within 48h. Depending on the inserted stent size, distinct mortality rates can be generated. A 14G stent results in 100% mortality, 16G gives 70% mortality, and mortality rates of 50% can be reached by using an 18G stent (Figure 1a). The Sham operation results in a survival rate of 100%. Usually, all animals die inbetween the first 48 hours after CASP. Thus, all animals that are alive at this time point can be regarded as survivors. However, we recommend an additional observation period of at least 72 hours to detect rare "late deaths" after CASP.
The outcome of CASPI depends on the period of time after CASP when the stent is removed. An intervention 3h after CASP results in a survival rate of 45%. If the stent is removed 5h after CASP, only 10% of the animals survive the procedure. A stent removal after 9h results in a mortality of 100% (Figure 1b). Sham CASPI leads to 100% survival. For CASPI settings, we recommend an observation period of 10 days because there usually are animals, which die later than 48 hours after CASP.
Macroscopic inspection of the abdominal situs 24 hours after CASP shows typical signs of peritonitis: edema, vasodilatation, erythematic gut wall, sharp demarcation of Peyer's patches, gut paralysis, free fluid, and septic secretion (Figure 2).
The systemic infection can be shown by bacteriological analysis. 12 hours after CASP, massive bacterial amounts can be detected in peritoneal lavage, blood, liver, lung, spleen, and kidneys. Qualitative analysis of bacteria reveals systemic infection by typical gut bacteria like E. coli, bacteroides species, enterococcus species, etc. (Figure 3).
CASP-induced sepsis manifests in local and also systemic release of pro- and anti-inflammatory cytokines and chemokines. 12 hours following CASP, significant amounts of TNF, IL-1&beta;, IL-6, IL-10, MCP-1 and others can be measured by ELISA in blood but also in supernatants of organ suspensions, e.g. liver, lung, spleen, kidney (Fig 4).
When establishing CASP in your laboratory, survival rates, bacteriology and cytokine release should be taken as control parameters for correct performance of CASP. Later on, these parameters can be taken as standard read out system for all experimental settings.
<img src="/files/ftp_upload/2299/2299fig1.jpg" alt="Figure 1" /> Figure 1a: Survival after CASP. Survival rates following CASP surgery depend on the diameter of the inserted stent. 14G CASP results in a mortality rate of 100%, 16G CASP results in 70% mortality, and 18G CASP leads to 50% mortality. Sham CASP is associated with a survival of 100%. n=20/group. B: Survival rates following CASPI surgery depend on the time of stent removal. Stent removal at 3h after 14G CASP leads to a mortality rate of 55%. Stent removal at 5h after 14G CASP results in a mortality rate of 90% whereas stent removal at 9h after 14G CASP results in 100% mortality. Sham CASPI (5h) results in a survival of 100%. n=10/group (Sham CASPI; CASPI 9h), n=20/group (3h CASPI; 5h CASPI).
<img src="/files/ftp_upload/2299/2299fig2.jpg" alt="Figure 2" /> Figure 2: Abdominal situs 24h after CASP. The abdominal wall was removed 24h after 16G CASP or sham CASP, respectively. The situs of the sham operated mouse looks physiological. In contrast, CASP results in edema, hyperemia, dilatated bowels, and demarcation of Peyer's patches demonstrating severe peritonitis.
<img src="/files/ftp_upload/2299/2299fig3.jpg" alt="Figure 3" /> Figure 3: Bacteriology. CASP leads to severe infection of several compartments and organs by gut bacteria within 12h after operation. Bacterial numbers are given as colony forming units (cfu) per mL, respectively. Bacterial cultures taken from sham operated mice are completely sterile (not shown). 16G CASP, n=5/group.
<img src="/files/ftp_upload/2299/2299fig4.jpg" alt="Figure 4" /> Figure 4: Cytokines. CASP triggers a systemic immune response as several cytokines and chemokines are detectable in plasma and also in organ supernatants. The figure exemplarily shows the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) as well as the predominantly anti-inflammatory IL-10. 12h after CASP, remarkable levels can be detected in plasma, liver, lung, and spleen. However, IL-10 cannot be detected in lung and spleen after CASP. Sham operation does not lead to any cytokine secretion (not shown). 16G CASP, n=5/group.

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No conflicts of interest declared.

CASP is a polymicrobial model of abdominal sepsis that meets most criteria postulated for a valuable sepsis model: CASP can be performed in small animals, is highly reproducible, easily to monitor by standard parameters, provides good read out possibilities, and closely mimics clinical settings of abdominal sepsis. In a comparative study we could show that CASP leads to diffuse peritonitis with early and steadily increasing systemic infection and inflammation (systemic inflammatory response syndrome), whereas cecal ligation and puncture (CLP) reveals a model of intra-abdominal abscess formation with sustained and minor signs of systemic inflammation (1). The technique of CASPI presents a possibility to simulate the clinical course of peritonitis with consecutive surgical focus sanitation, which is the most important therapeutic principle in humans. CASP can be applied for all questions around sepsis or peritonitis and is therefore useful for all disciplines dealing with sepsis research, for example immunology, pharmacology, surgery, intensive care medicine, etc. A selection of publications concerning different subjects of sepsis using the CASP model is given below (1-14).
When starting with CASP, one should be aware that some practice is necessary to perform the operation exactly and quickly. Surgical experience may be helpful but is not required. We recommend detection of letality as a parameter for correct performance of CASP. One should start with 14G CASP as a large stent is easier to handle than a thinner one. A trained CASP surgeon is able to operate one mouse within 10 minutes. Before starting any experiments, you should make sure that you are near that period. When you gained some experience with the surgical procedure, you should conduct a survival kinetic to check your skills. 14G CASP should result in a 100% lethality. Beginners in our laboratory have to prove that they are able to generate a 100% lethality with 14G CASP before they start with experiments. Additionally, bacteriology and cytokine measurements should be performed to verify sepsis.
If you have problems in the beginning with the microsurgical steps of CASP, the use of magnifying glasses or a surgical microscope may be helpful. But in principal, CASP can be well performed without these utilities. For reproduction of our survival rates, it is absolutely necessary that the same materials, especially the stents (BD Venflon, see below), are used. Otherwise, substantial variations of the results are possible. Additionally, we suggest that there can be variations of the results depending on, for example, the individuality of the surgeon. However, the influence of factors like that should be small.
In summary, CASP is a practicable, simple, reproducible and valuable sepsis model that mimics closely the clinical situation of abdominal sepsis. It is therefore suitable for all fields of sepsis research.

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		<div>
		<table border="1">
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Narcotic fluid</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Mix 0.5 mL Rompun with 4.0 mL Ketanest, and add 5.5 mL of saline solution (0.9%). 
For complete anesthesia, inject intraperitoneally 10 &mu;L/g body weight</td>
</tr>
<tr>
<td>Rompun (20 mg/ml Xylazine)</td>
<td>&nbsp;</td>
<td>Bayer AG 51368 Leverkusen, Germany</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ketanest (25mg/ml Esketamine)</td>
<td>&nbsp;</td>
<td>Pfizer Pharma GmbH, 10785 Berlin, Germany</td>
<td>647028001E</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 18GA (1,2x45mm, 80ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391457</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 16GA (1,7x45mm, 180ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391455</td>
<td>&nbsp;</td>
<tr>
<td>BD Venflon 14GA (2,0x45mm, 270ml/min)</td>
<td>&nbsp;</td>
<td>BD Biosciences, Franklin Lakes, NJ USA 07417</td>
<td>391456</td>
<td>&nbsp;</td>
<tr>
<td>Catgut Polyester 4/0 white, non absorbable</td>
<td>&nbsp;</td>
<td>Catgut GmbH, 08258 Markneukirchen, Germany</td>
<td>17218113</td>
<td>&nbsp;</td>
<tr>
<td>Mariderm black 7/0, non absorbable</td>
<td>&nbsp;</td>
<td>Catgut GmbH, 08258 Markneukirchen, Germany</td>
<td>18104900</td>
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colon ascendens stent peritonitis (casp) - a standardized model for polymicrobial abdominal sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

Watch this Scientific Journal Video about Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis at JoVE.com

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. This article describes the surgical procedure of CASP. The CASP model and its variants allow the systematic investigation of various problems concerning the subject of sepsis.

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

colon-ascendens-stent-peritonitis-casp-standardized-model-for

Research

JoVE Journal

Immunology and Infection

19.4K vistas.  University of Greifswald. El stent de colon ascendens Peritonitis (CASP) es un modelo altamente estandarizados para la sepsis abdominal polimicrobiana en los roedores. En este artículo se describe el procedimiento quirúrgico del CASP. El modelo CASP y sus variantes permiten la investigación sistemática de los diversos problemas relacionados con el tema de la sepsis.

Video: Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method. Current protocols to assess autophagy in vivo in the context of CLP-induced sepsis are presented here: A protocol for measuring autophagy using (GFP)-LC3 mice, and a protocol for measuring autophagosome formation by electron microscopy.

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

This manuscript describes a detailed protocol for using high frequency ultrasound imaging to measure luminal diameter, pulse propagation velocity, distensibility and radial strain on a mouse model of abdominal aortic aneurysm.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

A Contemporary Warming/Restraining Device for Efficient Tail Vein Injections in a Murine Fungal Sepsis Model

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve warming of the animal to promote vasodilation, which aids in both the identification of the blood vessels and positioning of the needle into the vessel lumen while securely restraining the animal. Although tail vein injections are common procedures in many protocols and are not considered highly technical if performed correctly, accurate and consistent injections are crucial to obtain reproducible results and minimize variability. Conventional methods for inducing vasodilation prior to tail vein injections generally depend on the use of a heat source such as a heat lamp, electrical/rechargeable heat pads, or pre-heated water at 37 &#176;C. Despite being readily accessible in a standard laboratory setting, these tools evidently suffer from poor/limited thermo-regulatory capacity. Similarly, although various forms of restraining devices are commercially available, they must be used carefully to avoid trauma to the animals. These limitations of the current methods create unnecessary variables in experiments or result in varying outcomes between experiments and/or laboratories.
In this article, we demonstrate an improved protocol using an innovative device that combines an independent, thermally regulated, warming device with an adjustable restraining unit into one system for efficient streamlined tail vein injection. The example we use is an intravenous model of fungal bloodstream infection that results in sepsis. The warming apparatus consists of a heat-reflective acrylic box installed with an adjustable automatic thermostat to maintain the internal temperature at a pre-set threshold. Likewise, the width and height of the cone restraining apparatus can be adjusted to safely accommodate various rodent sizes. With the advanced and versatile features of the device, the technique shown here could become a useful tool across a range of research areas involving rodent models that employ tail vein injections.

Here, we present an effective and efficient method for rodent tail vein injections using a uniquely designed warming/restraining device. By streamlining the initiation of vasodilation and restraining processes, this protocol allows accurate and timely intravenous injections of large groups of animals with minimal distress.

In rodent models, tail vein injections are important methods for intravenous administration of experimental agents. Tail vein injections typically involve ...

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 ...

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

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

Characterizing Salmonella Typhimurium-induced Septic Peritonitis in Mice

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

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

Evaluation of a Reliable Biomarker in a Cecal Ligation and Puncture-Induced Mouse Model of Sepsis

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous efforts to elucidate the pathophysiology of sepsis using various animal models; the mouse model of sepsis induced by cecal ligation and puncture (CLP) is widely used in laboratories. The three technical aspects that affect the severity and replicability of the CLP model are the percentage of cecum ligated, the size of the needle used for cecal puncture, and the volume of feces squeezed into the abdominal cavity. The rapid and specific diagnosis of sepsis is a crucial factor that affects the outcome. The gold standard for sepsis diagnosis is microbial culture; however, this process is time-consuming and sometimes inaccurate. The detection of sepsis-specific biomarkers is fast, but the existing biomarkers are unsatisfactory due to a short half-life, non-specificity, and insufficient sensitivity. Therefore, there is an urgent need for a reliable biomarker of sepsis in the early stages. Previous publications suggest that excessive neutrophil extracellular traps (NETs) occur in sepsis. Citrullinated histone H3 (CitH3), as a NET component, is elevated both in septic animals and patients, and the presence of CitH3 is a reliable diagnostic biomarker of sepsis. The present study aimed to describe a standardized mouse model of CLP-induced sepsis and establish a reliable blood biomarker of sepsis. Our work may contribute to the early and accurate diagnosis of sepsis in the future.

This protocol presents the operative details of cecal ligation and puncture (CLP) in a mouse model of sepsis. CLP is one of the most widely used techniques to create an animal model of sepsis. Therefore, a standardized CLP protocol is required for the attainment of reliable research results.

Sepsis is a severe life-threatening and rapidly developing disease that causes millions of deaths annually worldwide. Researchers have made tremendous ...