This work was supported by operating grants to KJ from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Crohn's and Colitis Foundation of Canada (CCFC). VM is supported by a CFRI Fellowship and GB a graduate studentship from the Canadian Institutes for Health Research (CIHR). BAV is a Children with Intestinal and Liver Disorders Foundation (CHILD) Chair of Pediatric IBD Research and the Canada Research Chair in Pediatric Gastroenterology and KJ is a Senior Clinician Scientist supported by CHILD and the Child and Family Research Institute (CFRI) Clinician Scientists Award Program, University of British Columbia.

Procedures involving animal subjects follow animal care guidelines outlined by the Institutional Animal Care and Use Committee (IACUC) at the University of British Columbia.
Procedures mentioned in this protocol will be described for both rats and mice, while the representative results will be presented from Sprague-Dawley rats.
1. Preparation of DNBS and Intrarectal Administration
<ol>
	<li>For rectal instillation of DNBS in rats, freshly prepare 15-30 mg of DNBS/250 &#181;l of 50% ethanol. For mice, prepare 2-6 mg of DNBS/100 &#181;l of 50% ethanol. Note: Optimize the exact DNBS concentration to be administered at each facility. Male Sprague-Dawley rats, and C57BL/6 mice are preferred for induction of DNBS colitis, however other mouse and rat strains can be used. Mice and rats treated only with 50% ethanol can be used as control groups however given the effects of ethanol are mild or absent, one can omit the inclusion of this group for a study12.</li>
	<li>Attach a 1 ml&#160;syringe to a 19 G needle that has a polyethylene catheter (PE-90 - rats or PE-50 - mice) fastened to its end.</li>
	<li>Lightly anesthetize the rat or mouse resting on a heating pad (e.g. by administering 1.5% isoflurane via nose cone, animals should still have blink and swallowing reflexes and display regular respiration). Apply tear gel to its eyes to avoid the eyes drying out.</li>
	<li>Gently apply pressure with fingertips to the posterior end of the animal to remove any stool that may be present in the distal colon.</li>
	<li>Gently insert the catheter intrarectally, with the catheter reaching approximately 8 cm proximal to the anus for rats, or 3-4 cm for mice. Inject a small amount of the solution while inserting the catheter to lubricate, allowing easier insertion. Note: If any resistance is felt while inserting the catheter, do not continue to insert as this may lead to perforation of the intestinal wall. Remove the catheter and attempt to gently reinsert, again lubricating the colon as proceeding.</li>
	<li>Slowly inject 250 (rats) or 100 (mice) &#181;l&#160;of DNBS, or an equal volume or 50% ethanol for controls. After injecting the DNBS, position the animal head-down for 90 sec to avoid loss of the DNBS.</li>
	<li>After DNBS administration, feed the animals 8% sucrose water in 0.2% saline to prevent dehydration during the first week.</li>
	<li>Carefully observe the animals, with daily monitoring for weight loss and signs of dehydration or distress for the duration of the experiment. Note: Weight loss may rapidly approach the CCAC recommendations of maximum permissible weight loss. Since this mostly derived from dehydration, weight loss should be used as an indicator for administering lactated Ringer&#39;s solution to limit dehydration. Please be sure to consult with the facility veterinarian on health issues as appropriate.</li>
</ol>
2. Collecting Tissues and Assessing Histological Damage in DNBS Challenged Mice
<ol>
	<li>At the desired time point after challenge with DNBS, euthanize animals by overdosing with isoflurane in an anesthetic chamber supplemented with sufficient oxygen. Observe the animal until no rising or falling of the chest is seen and there is no palpable heartbeat. Confirm the animal has been correctly euthanized by absence of a response to toe pinch or touching of the cornea, followed by cervical dislocation.</li>
	<li>Open the abdominal cavity and remove the entire large intestine. Open the intestine longitudinally; macroscopic mucosal ulcers should be observed approximately 5-6 cm proximal from the distal end of the colon in rats (2-3 cm in mice). Note: After removing the large intestine, assess macroscopic damage scores if desired. Collect segments of colon from corresponding anatomical location from control animals. Note: The individuals scoring the tissue for macroscopic damage scores should be blinded to the identity of the treatment groups. Score tissue sections using the following or similar criteria: severity and extent of ulceration (0-10), summed with scores for absence or presence of diarrhea (0 or 1), adhesions (0, 1, or 2) and maximal colonic wall thickness in millimeters11.</li>
	<li>Collect 0.5 cm sections proximal to the site of maximal macroscopic damage. Snap freeze one 0.5 cm section in liquid nitrogen to later assess myeloperoxidase (MPO) activity (a measure of neutrophil infiltration), and place the other section in 10% neutral buffered formalin for histological analysis. Note: Snap frozen tissues for myeloperoxidase assay can be stored at -80 &#176;C for up to 1 month.</li>
	<li>Allow the tissues collected for histological analysis in step 2.3 to fix in formalin overnight, and then wash with 70% ethanol. Embed tissues in paraffin and cut to desired thickness (3-5 &#181;m), stain with hemotoxylin and&#160;eosin (HE) for histological scoring.</li>
	<li>Have two observers score at least three tissue sections from each animal under a light microscope to determine the histological damage scores for each group. Note: The individuals scoring the tissue for histological damage scores should be blinded to the identity of the treatment groups. Score tissue sections using the following or similar criteria: extent of inflammatory cell infiltrate (0 = absent, 3 = transmural), loss of mucosal architecture (0 = absent, 3 = severe), presence or absence of crypt abscesses (0-1), and goblet cell depletion (0-3). Determine histological damage score as the sum of these scores11.</li>
</ol>
3. Performing Myeloperoxidase (MPO) Assay on DNBS Challenged Intestinal Tissue
<ol>
	<li>Assay MPO activity in the 0.5 cm colonic segments obtained in step 2.3. Homogenize tissues in MPO homogenization buffer (0.5% hexadecyltrimethyl-ammonium bromide in 50 mM potassium phosphate buffer (pH 6.0)) to give a 50 mg colon segment/ml&#160;of homogenization buffer suspension.</li>
	<li>Aliquot the homogenized solution into 1 ml&#160;portions and centrifuge at 4 &#176;C for 2 min at 10,000 x g.</li>
	<li>Mix 100 &#181;l&#160;of each aliquot with 2.9 ml&#160;of 50 mM phosphate buffer (pH 6.0) containing 0.167 mg/ml&#160;of o-dianisidinedihydrochloride and 0.0005% hydrogen peroxide in a cuvette.</li>
	<li>Measure the rate of absorbance using a spectrophotometer at 460 nm. Note: MPO activity is defined as the quantity of enzyme degrading 1 &#181;mol/min of H2O2 to H2O&#160;at room temperature and is expressed in units per mg of tissue.</li>
</ol>

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The authors declare that they have no competing financial interests.

The DNBS model described in this protocol is a valuable, inexpensive, and reproducible colitis model that can be utilized to study various aspects of IBD. When administered intrarectally to rats or mice, DNBS induces a substantial degree of inflammation and tissue injury in the colon, resembling human Crohn&#39;s disease in terms of its various histological features including transmural infiltration of polymorphonuclear cells and predominant NF-&#954;B-dependent Th1 activation.
This model has ...

Inflammatory bowel disease (IBD) is characterized by chronic relapsing inflammation in the gastro-intestinal (GI) tract, resulting in symptoms of diarrhea, weight loss, and abdominal pain. Ulcerative colitis (UC) and Crohn&#39;s disease (CD) are the two major forms of IBD, and can be distinguished by the location of the inflammation within the GI tract. In UC patients, the inflammation typically involves the rectum and extends contiguously up the colon for a variable extent affecting only the superficial mucosa. In contrast, CD can affect any part of the GI tract, although it predominantly affects the ileum and cecum. CD frequently manifests as transmural inflammation often associated with granulomas and leading to stricturing (fibrostenotic) and/or penetrating (fistulizing) disease. Although the etiology of IBD remains elusive, it is well accepted that IBD is multifactorial, involving interactions among the hosts immune system, genetic susceptibility and responses to environmental and microbial factors.
To date, different models of IBD have been proposed that display various clinical, histological and immune responses characteristic of UC and CD. The most commonly used models include genetically modified mice (IL-2, IL-10, SAMP/Yit), infection induced models (Citrobacter rodentium, Salmonella typhimurium), adoptive transfer models (CD45+ RB High, CD62L+ cell transfer into SCID mice) and chemically induced colitis models (Dextran Sodium Sulfate (DSS), Trinitrobenzene sulfonic acid (TNBS), and Dinitrobenzene sulfonic acid (DNBS)). Owing to their low cost and rapid onset of disease, chemical models are considered invaluable to the study of various aspects of IBD. Each of the chemical colitis models listed has advantages as well as limitations in some aspects of their clinical, immunological and histopathological relevance to IBD. DSS is one of the most common chemical methods employed to induce colitis in rodents1. Administration of 3-10% DSS (MW: 42 kDa) for 7-10 days in the drinking water of mice can induce symptoms and signs of colitis including weight loss, diarrhea with blood, colonic shortening, mucosal ulceration and neutrophil infiltration. This model is particularly useful for drug screening studies, as well as exploring the mechanisms of epithelial regeneration, the impact of innate immunity on mucosal homeostasis, and the role of inflammation in promoting intestinal dysplasia and adenocarcinoma development. There are however some drawbacks to the DSS model, including variation in the concentration of DSS needed to induce colitis in different animal facilities, as well as inconsistent water uptake by mice and thus inconsistent exposure to DSS, resulting in variation in the degree, extent and distribution of mucosal injury and ulceration in the colon. All these features lead to heterogeneity of results and limit the ability to compare results across studies from different research groups.
An alternative to the DSS model is the hapten-induced DNBS or TNBS models of colitis. This model employs rectal instillation of the mucosal sensitizing agents DNBS or TNBS, diluted in varying concentrations of ethanol. The administration of ethanol is a prerequisite to break the colonic mucosal barrier to allow penetration of DNBS or TNBS into the lamina propria. DNBS/TNBS will then haptenize the localized colonic and gut microbial proteins to become immunogenic, thereby triggering the host innate and adaptive immune responses. In general, this model is associated with severe and sometimes bloody diarrhea, weight loss and intestinal wall thickening however symptoms vary depending upon the type of rodent used, as well as the timing, dose and degree of exposure to the DNBS or TNBS used in the study. Important distinctions between rats and mice should be noted, with the benefits of lower cost for purchase and board, as well as lower body mass for decreasing per-animal costs of treatment in vivo. This should be set against the more rapid and severe course of colitis in mice, where the more fragile and responsive animals may quickly reach a humane endpoint.
The intestinal inflammation initially results from ethanol induced damage to intestinal epithelial cells, leading to increased epithelial permeability, microbial penetration into the mucosa, haptenization of host proteins, all of this resulting in infiltration of neutrophils, macrophages and Th1 T lymphocytes into the damaged mucosa. In comparison to DNBS, TNBS is considered as a hazardous chemical due to its highly oxidative properties that can pose a risk of explosion upon contact with bases such as sodium and potassium hydroxide. Therefore DNBS is currently regarded as a preferred choice of chemical over TNBS to induce colitis. In rodents, DNBS colitis is considered as one of the most convenient methods to study the following IBD associated modifiers of disease:
<ol>
	<li>Depression and reactivation of colitis: It has been shown that stress, anxiety and depression in IBD patients are frequently associated with disease relapse. DNBS colitis is a suitable model to study the role of depression and its consequences on reactivation of colitis in mice. The method typically involves initial induction of colitis by DNBS, followed by the resolution of the colitis by leaving the mice for 6-8 weeks. The mice are then administered with depression causing agents such as reserpine or by olfactory bulbectomy to induce depression followed by testing them for reactivation of colitis through challenge with a subcolitic dose of DNBS2.</li>
	<li>Stress and reactivation of colitis: Stress is another common environmental factor that has been linked to IBD pathogenesis. There is a growing body of evidence that suggests a strong association between chronic stress and the onset of symptoms of UC and CD in rodents as well as in humans and nonhuman primates3. DNBS colitis is a good model to study stress-associated reactivation of colitis in both mice and rats. The method is usually employed in a similar fashion as mentioned above for depression except in place of depression, the animals are exposed to stressors such as sonic and restraint stress4.</li>
	<li>Neurogenic inflammation: A number of studies have described either transient or permanent alterations in the enteric nervous system (ENS) structure and function, as seen in tissue samples from animal models and from patients with IBD. DNBS is a good model to explore the effects of inflammation on the ENS5 and to study both noradrenergic and cholinergic neural pathways6.</li>
	<li>Injury-repair mechanisms: During IBD pathogenesis, host-derived injury mediators such as reactive oxygen species (ROS), nitric oxide (NO), intercellular adhesion molecule 3 (ICAM-3), and P-selectin have all been shown to play a role in intestinal epithelial disruption. DNBS is an excellent model to study injuries caused by the up-regulation of these mediators as well as repair mechanisms that are regulated by using selective drugs or inhibitors7,8.</li>
	<li>Transmural inflammation: In addition to the above mentioned applications of DNBS, the model can also be applied to study transmural inflammation of the gut, a classical feature found in patients with CD. Both DNBS and TNBS-induced colitis are associated with significant infiltration of lymphocytes, into the colonic mucosa making these models particularly useful to study T-cell dependent immune mechanisms.</li>
</ol>
In comparison with the DSS model, the advantages of DNBS and TNBS-induced colitis include low cost, rapid development of colitis (usually requires 1-3 days to show reproducible ulceration and inflammation) and consistent localized damage to the distal colon. However the drawbacks are a requirement for a greater level of technical expertise, optimization of DNBS/ TNBS dose, and the need for anesthesia for rectal administration.
In this methodological paper we studied the effects of varying concentrations of n-6 and n-3 polyunsaturated fatty acids on altering the colonic mucosal response to DNBS-induced colitis in rats using the vegetable oils safflower oil (SO) and canola oil (CO), and fish oil (FO). It has been shown that n-6 and n-3 fatty acids are important mediators of intestinal inflammatory disease through their role as acyl moieties of cell membrane phosopholipids9. In contrast to the proinflammatory potential of n-6 fatty acids, n-3 fatty acids at sufficiently high intake are potentially potent anti-inflammatory agents. The anti-inflammatory actions of n-3 fatty acids are mediated directly through replacement of arachidonic acid as an eicosanoid substrate, inhibiting arachidonic acid metabolism and indirectly through alteration of proinflammatory gene expression and cell signaling. The n-3 fatty acids also give rise to resolvins, a family of anti-inflammatory mediators. High intake of n-3 fatty acids is associated with a decrease in production of proinflammatory eicosanoids, cytokines, chemokines, reactive oxygen species and expression of adhesion molecules. In this study, rats were fed ad libitum diets identical in all nutrients except fatty acids, with as a percent energy from fat, 20% SO, 20% CO, or 18% fish oil plus 2% safflower oil (FO)10-11. As a percentage&#160;of the daily energy, the SO diet provided 15% linoleic acid (LA), with &#60;0.06% &#945;-linolenic acid (ALA) and no eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), the CO diet had 4.2% LA and 1.9% ALA with no EPA or DHA, and the FO diet provided 1.4% EPA, 4.9% DHA, 0.32% LA, and 0.12% ALA. Three weeks after initiation of the lipid diets, the mice were administered intrarectal DNBS or 50% ethanol and sacrificed 5 days later. The inflammatory response was evaluated by assessment of weight loss, histological damage scores and tissue myeloperoxidase activity.

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<colgroup>
		<col />
		<col /> 
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1 mL syringe</td>
			<td style="width:121px;">BD Biosciences</td>
			<td style="width:119px;">309659</td>
			<td style="width:167px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">19 gauge needle</td>
			<td>BD Biosciences</td>
			<td>305187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing PE-50</td>
			<td>BD Biosciences</td>
			<td>427517</td>
			<td>for intra-rectal administration in mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylene tubing PE-90</td>
			<td>BD Biosciences</td>
			<td>427519</td>
			<td>for intra-rectal administration in rat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNBS</td>
			<td>MP Bio</td>
			<td>150959</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tear gel</td>
			<td>Novartis</td>
			<td>63601662596</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Formalin</td>
			<td>Fisher</td>
			<td>5F93-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">warming pad</td>
			<td>Kent Scientific</td>
			<td>TPZ-0510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>Fisher</td>
			<td>A-962.4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hexadecyltrimethyl-ammonium bromide</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td>H5882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium phosphate buffer solution</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td>79628-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">O-dianisidine dihydrochloride</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td>D3252</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">30% H2O2</td>
			<td style="width:121px;">Sigma-Aldrich</td>
			<td>31642</td>
			<td>hydrogen peroxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectrophotometer</td>
			<td>BIORAD</td>
			<td>Benchmarker Plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light Microscope</td>
			<td>Zeiss</td>
			<td>Axio Image.Z1</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Data analysis software program </td>
			<td>GraphPad Software</td>
			<td style="width:119px;"> GraphPad Prism Software Version 4.00 </td>
			<td><a href="http://www.graphpad.com/">San Diego California USA, www.graphpad.com</a></td>
		</tr>
	</tbody>
</table>

dnbs/tnbs colitis models: providing insights into inflammatory bowel disease and effects of dietary fat

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

Challenge of rats (Sprague-Dawley) or mice (C57BL/6) with DNBS typically results in transient weight loss and diarrhea, sometimes with blood present in the stool. However, factors such as genetics, diet and the intestinal microbiota can modify susceptibility to DNBS induced colitis. Therefore, animals included in the experiments should be from the same institution, either derived locally or ordered in from one animal supplier. All animals should be carefully monitored for the degree of weight loss and symptoms of distres...

Watch this Scientific Journal Video about DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat at JoVE.com

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

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

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

dnbstnbs-colitis-models-providing-insights-into-inflammatory-bowel

Research

JoVE Journal

Medicine

47.0K vistas.  BC Children's Hospital.  DNBS / TNBS colitis inducida ofrece un método alternativo y barato para estudiar la patobiología de la enfermedad inflamatoria del intestino. El protocolo descrito en este documento se describen la aplicación exitosa de DNBS para inducir la colitis en ratones y ratas y permite estudiar a fondo las respuestas intestinales mediados por el huésped. 

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

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are associated with obesity, though the precise underlying mechanisms that contribute to the disease are currently being delineated.2,3 Several small animal models of obesity have been developed and are employed in a variety of studies.4 A critical component to these experiments involves the collection of regional and/or total animal fat content data under varied conditions.
Traditional experimental methods available for measuring fat content in small animal models of obesity include invasive (e.g. ex vivo measurement of fat deposits) and non-invasive (e.g. Dual Energy X-ray Absorptiometry (DEXA), or Magnetic Resonance (MR)) protocols, each of which presents relative trade-offs. Current invasive methods for measuring fat content may provide details for organ and region specific fat distribution, but sacrificing the subjects will preclude longitudinal assessments. Conversely, current non-invasive strategies provide limited details for organ and region specific fat distribution, but enable valuable longitudinal assessment. With the advent of dedicated small animal X-ray computed tomography (CT) systems and customized analytical procedures, both organ and region specific analysis of fat distribution and longitudinal profiling may be possible. Recent reports have validated the use of CT for in vivo longitudinal imaging of adiposity in living mice.5,6 Here we provide a modified method that allows for fat/total volume measurement, analysis and visualization utilizing the Carestream Molecular Imaging Albira CT system in conjunction with PMOD and Volview software packages.

Fat content analysis is routinely conducted in studies utilizing murine obesity models. Emerging methods in small animal CT imaging and analysis are providing for longitudinal detail rich fat content analysis. Here we detail step by step procedures for performing small animal CT imaging, analysis, and visualization.

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

Ultrasound-guided Intracardiac Injection of Human Mesenchymal Stem Cells to Increase Homing to the Intestine for Use in Murine Models of Experimental Inflammatory Bowel Diseases

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have immunosuppressive potential and have been shown to suppress inflammation in mouse models of intestinal inflammation, even though the route of administration can limit their homing and effectiveness 1,3,4,5. Local application of MSCs to colonic injury models has shown greater efficacy at ameliorating inflammation in the colon. However, there is paucity of data on techniques to enhance the localization of human bone marrow-derived MSCs (hMSCs) to the small intestine, the site of inflammation in the SAMP-1/YitFc (SAMP) model of experimental Crohn&#39;s disease. This work describes a novel technique for the ultrasound-guided intracardiac injection of hMSCs in SAMP mice, a well-characterized spontaneous model of chronic intestinal inflammation. Sex- and age-matched, inflammation-free AKR/J (AKR) mice were used as controls. To analyze the biodistribution and the localization, hMSCs were transduced with a lentivirus containing a triple reporter. The triple reporter consisted of firefly luciferase (fl), for bioluminescent imaging; monomeric red fluorescent protein (mrfp), for cell sorting; and truncated herpes simplex virus thymidine kinase (ttk), for positron emission tomography (PET) imaging. The results of this study show that 24 h after the intracardiac administration, hMSCs localize in the small intestine of SAMP mice as opposed to inflammation-free AKR mice. This novel, ultrasound-guided injection of hMSCs in the left ventricle of SAMP mice ensures a high success rate of cell delivery, allowing for the rapid recovery of mice with minimal morbidity and mortality. This technique could be a useful method for the enhanced localization of MSCs in other models of small-intestinal inflammation, such as TNF&#916;RE6. Future studies will determine if the increased localization of hMSCs by intra-arterial delivery can lead to increased therapeutic efficacy.

Murine studies in models of colonic inflammation have demonstrated that a small percentage (1 - 5%) of mesenchymal stem cells (MSC) injected intravenously or intraperitoneally home to the inflamed colon1,2. This study shows that ultrasound-guided intracardiac injections of MSCs result in increased localization to the intestine.

Crohn&#39;s disease (CD) is a common chronic inflammatory disease of the small and large intestines. Murine and human mesenchymal stem cells (MSCs) have ...