Special thanks to Sarah Kossak, Mitchell Guanzon, Sung Yeun Yang and Li Guo Jia, for their collaboration during the endoscopic assessment of their experimental animals.

1. Setting up the Endoscopy System
<ol>
	<li>Set up the endoscopy system before anesthetizing the mice, following the manufacturer&#39;s instructions. Use a video ureteroscope designed for humans for this protocol (Figure 1). The outer diameter of the insertion tube is 9.9 Fr (3.3 mm), its distal end is 8.5 Fr (2.8 mm), and the inner diameter of the instrumentation channel is 6 Fr (1.2 mm).</li>
	<li>Although the use of endoscopes have not been linked to disease transmission [9], sterilize the endoscope and lens with 70% ethanol10&#160;or with appropriate techniques when working with spore-forming microorganisms. Prepare bacteriostatic sterile lubricant and non-scratching wipe tissues to saturate the endoscope tip.</li>
	<li>Attach a 3 ml&#160;syringe full of air (or a suitable CO2&#160;gas mixture) to the top of the instrumentation channel.</li>
	<li>Turn on the power, and set up the white balance in normal white light, as well as narrow band imaging (NBI) mode, when applicable. Since 2001, NBI has provided diagnostic imaging benefits as it emphasizes fine blood vessel structures on mucous membranes, including ulcerative and tumorous lesions, which have characteristic abnormal vascularization11. NBI-filter mode restricts the wavelength of captured light to 390-445 nm and 530-550 nm, which highlights structures with hemoglobin.</li>
	<li>Print the assessment scoring form (Figure 2) to record your findings. Become familiar with expected normal and NBI images (Figure 3A) and the definition and grades of each criterion before the exam (see Section 6.1).</li>
	<li>Prepare a colorant-based solution on phosphate-buffered saline (PBS) for chromoendoscopy, if desired. In chromoendoscopy, the exam is performed using a dye as contrast medium to highlight mucosal changes. Use methylene blue (0.1-1%) and indigo carmine (0.1%) solutions (Figure 3B). Note that methylene blue is an oxidation-reduction agent that can produce oxidizing radicals, indirect cross-linking of amino acid residues, and DNA degradation12, which could interfere with sample analysis.</li>
</ol>
2. General Anesthesia of the Mouse
<ol>
	<li>Choose the anesthetic agent and administration route. Consult a veterinarian from an animal resource facility or Institutional Animal Care and Use Committee (IACUC) regarding potential interference of medications with the immune system and research goals. Some medications may influence cytokine profile expression in animals, including rodents13. Use injectable tribromoethanol for terminal procedures&#160;or isoflurane for survival endoscopies, along with appropriate controls.</li>
	<li>For induction, administer 4-5% of isoflurane in 100% oxygen at a rate of 0.2-0.5 L/min; use 0.5-2% for maintenance. This protocol is not appropriate for all animals. Consult your facility veterinarian for proper instructions and training. Abide by relevant IACUC recommendations to achieve the highest standards for animal welfare. Make sure that the mouse if fully anesthetized.</li>
	<li>&#8216;Strap&#8217; the anesthetized mouse to a supporting surface table in a dorsal or ventral position&#160;using laboratory adhesive tape. Beware of the anatomical orientation on your endoscope screen. Secure its lower extremities and tail. A band of tape can also be placed over the chest to prevent movement away from the respirator, but avoid restricting thoracic ventilatory movements.</li>
</ol>
3. Detailed Endoscopic Examination
<ol>
	<li>Look for perianal lesions before inserting the endoscope into the rectum, then advance the scope through the anus.</li>
	<li>Coat the endoscope with sterile surgical lubricant. Be sure not to coat the surface of the CCD (charge coupled device) sensor.</li>
	<li>Insert the endoscope and start recording. Recorded videos are useful for meticulous evaluation of endoscopy if needed after the examination. Try to perform the endoscopy in the least possible length of time to minimize intestinal/mucosal irritation.</li>
	<li>Apply air by using a syringe to slowly set apart the intestinal walls. Depending on the size of the mouse, use 1-2 ml&#160;of air. Avoid excessive air insufflation; it may cause respiratory distress and death of the anesthetized mouse.</li>
	<li>Advance the endoscope forward as you clear the way with air or gentle rotation of the scope. Do not advance the endoscope if you do not see the intestinal lumen. Perforations or rectal/colonic tears can occur at this stage, especially in young mice (&#60;8-10 weeks old). Take pictures as necessary.</li>
	<li>As the scope advances, assess if there is spontaneous bleeding (defined as naturally&#160;occurring mucosal hemorrhaging not associated with traumatic endoscopy). Spontaneous bleeding can be observed as the endoscope enters the rectum and colon.</li>
	<li>Assess the transparency of the colonic wall. Transparency is defined as the ability to visualize the intramural blood vessels in the colon and that of the other surrounding viscera (Figure 4). Gentle motion of the scope (back-forward, clockwise,&#160;counterclockwise) will help determining if blood vessels can be seen in the context of other viscera. Mesenteric adipose tissue can also be seen. Note if the rectum inside the pelvic cavity appears less transparent than the colonic wall in the abdominal cavity.</li>
	<li>Inspect to determine the type and number of focal lesions (edematous areas, erosions, ulcers, tumorous masses). This will be recorded in the scoring form using the colonic inflammation score and the decimal identifiers that have been developed.</li>
	<li>Once the area where the colon flexes caudally into the proximal colon is reached, withdraw the endoscope slowly and look for the presence of contact bleeding (which occurs due to mucosal friability after the endoscope is advanced or removed). It is not necessary to scrape the colon surface intentionally. Healthy colorectal mucosa does not bleed after proper endoscopic examination.</li>
</ol>
4. Biopsy
<ol>
	<li>When biopsies are needed, insert the biopsy forceps through the endoscope channel dedicated for instrumentation. Monitor the advancement of the forceps on the endoscope screen to prevent intestinal perforation.</li>
	<li>Open the biopsy cups of the forceps to about a 60-80&#176;&#160;cup-to-cup angle, and direct them to the area of interest. Excessively opened forceps or pressure may grasp extra layers of intestinal tissue, which results in a biopsy-induced perforation of healthy transparent intestinal walls. If perforation occurs, euthanize the animal during anesthesia following your IACUC recommendations. Perforations result in endotoxemia and peritonitis.</li>
	<li>Close the biopsy cups, and pull them out while keeping the forceps closed.</li>
	<li>Remove the specimen from the forceps by flushing the tissue with a solution that is gentle to the tissue and the forceps. RNA later and PBS are common working solutions; if needed, use the needle to dislodge the sample. Avoid eroding the forcep cups or cutting edges.</li>
</ol>
5. Recovery from Anesthesia
<ol>
	<li>Upon completion of the examination, provide a warm environment to the mouse until it recovers. Use either an electric light, or a warm pad set at 37 &#176;C. Avoid overheating; peripheral vasodilation compromises animal recovery. Monitor the mouse closely until full recovery.</li>
</ol>
6. Integrated Endoscopic Scoring System of Colorectal Health with Decimal Identifiers
<ol>
	<li>Use the scoring form (Figure 2) and the parameter definitions below (Endoscopic Assessment of Murine Colorectal
Inflammation and Tumors Using a Decimal-weighted Scoring System: Parameter Definitions, and Protocol) for examples and the optional 'Distal-Proximal Endoscopic Colitis scoring system' (D-PECS) as guidelines for nonbiased assessment and detailed record keeping.</li>
	<li>Score the four parameters of colorectal inflammation described on the scoring system form (Figure 2). Meticulously evaluate the perianal region, intestinal transparency, type of intestinal bleeding (if present), and presence and quantity of focal lesions (erosive, ulcerative, and/or tumorous). </li>
	<li>When assessing tumors for research purposes, examine the quantity, location, and size, and describe them using the colonic map and bar graph on the scoring form. The numerical sum of the scores obtained and number of lesions (when applicable) will determine the background colonic inflammation, the most predominant type of lesion, and the number of lesions.</li>
</ol>

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	<li>Huang, E. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonoscopy+in+mice.">Colonoscopy in mice.</a> Surg. Endosc. 16, 22-24 (2002).</li><li>Wirtz, S., Becker, C., Blumberg, R., Galle, P. R., Neurath, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+T+cell-dependent+experimental+colitis+in+SCID+mice+by+local+administration+of+an+adenovirus+expressing+IL-18+antisense+mRNA.">Treatment of T cell-dependent experimental colitis in SCID mice by local administration of an adenovirus expressing IL-18 antisense mRNA.</a> J. Immunol. 168, 411-420 (2002).</li><li>Funovics, M. A., Alencar, H., Su, H. S., Khazaie, K., Weissleder, R., Mahmood, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturized+multichannel+near+infrared+endoscope+for+mouse+imaging.">Miniaturized multichannel near infrared endoscope for mouse imaging.</a> Mol Imaging. 2, 350-357 (2003).</li><li>Becker, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+colitis+and+colon+cancer+development+in+mice+using+high+resolution+chromoendoscopy.">In vivo imaging of colitis and colon cancer development in mice using high resolution chromoendoscopy.</a> Gut. 54, 950-954 (2005).</li><li>Ravnic, D. J., Konerding, M. A., Huss, H. T., Wolloscheck, T., Pratt, J. P., Mentzer, S. 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Protoc. 1, 2900-2904 (2006).</li><li>Hensley, H. H., Merkel, C. E., Chang, W. C., Devarajan, K., Cooper, H. S., Clapper, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+imaging+and+size+estimation+of+colorectal+adenomas+in+the+multiple+intestinal+neoplasia+mouse.">Endoscopic imaging and size estimation of colorectal adenomas in the multiple intestinal neoplasia mouse.</a> Gastrointest. Endosc. 69, 742-749 (2009).</li><li>Banerjee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infection+control+during+GI+endoscopy.">Infection control during GI endoscopy.</a> Gastrointest. Endosc. 67, 781-790 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+and+disinfection+of+equipment+for+gastrointestinal+endoscopy.+Report+of+a+Working.">Cleaning and disinfection of equipment for gastrointestinal endoscopy. Report of a Working.</a> Party of the British Society of Gastroenterology Endoscopy Committee. Gut. 42, 585-593 (1998).</li><li>Sano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+diagnostic+method+based+on+color+imaging+using+narrow+band+imaging+(NBI)+system+for+gastrointestinal+tract.">New diagnostic method based on color imaging using narrow band imaging (NBI) system for gastrointestinal tract.</a> Gastrointest. Endosc. 53, 125-12 (2001).</li><li>Davies, J., Burke, D., Olliver, J. R., Hardie, L. J., Wild, C. P., Routledge, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylene+blue+but+not+indigo+carmine+causes+DNA+damage+to+colonocytes+in+vitro+and+in+vivo+at+concentrations+used+in+clinical+chromoendoscopy.">Methylene blue but not indigo carmine causes DNA damage to colonocytes in vitro and in vivo at concentrations used in clinical chromoendoscopy.</a> Gut. 56, 155-156 (2007).</li><li>Chiang, N., Schwab, J. M., Fredman, G., Kasuga, K., Gelman, S., Serhan, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthetics+impact+the+resolution+of+inflammation.">Anesthetics impact the resolution of inflammation.</a> PLoS One. 3, e1879 (2008).</li><li>Zilberman, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+digital+flexible+ureteroscope:+in+vitro+assessment+of+optical+characteristics.">The digital flexible ureteroscope: in vitro assessment of optical characteristics.</a> J. Endourol. 25, 519-522 (2011).</li><li>Machida, H., Sano, Y., Hamamoto, Y., Muto, M., Kozu, T., Tajiri, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Narrow-band+imaging+in+the+diagnosis+of+colorectal+mucosal+lesions:+a+pilot+study.">Narrow-band imaging in the diagnosis of colorectal mucosal lesions: a pilot study.</a> Endoscopy. 36, 1094-1098 (2004).</li><li>Dohoo, I., Martin, W., Stryhn, H., McPike, S. 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No financial or conflict of interests disclosed.

There are several considerations regarding modification and troubleshooting of the endoscopic technique in mice. Critical aspects of the procedure that need to be mastered during the use of flexible endoscopy include the adjustment of the air volume needed to prevent abdominal distension and respiratory compromise of the mouse, and minor technical aspects of view control with coordination of the angulation and torque controls. Torque control is important to minimize the risk of intestinal perforation. When handled proper...

Murine endoscopy has been performed as a useful research tool for over a decade1-3. To date, most studies employing murine endoscopy have used rigid endoscopes, although some have also used flexible sigmoidoscopy. Murine endoscopy provides immediate results and a more objective estimate of the extent of intestinal normalcy, severity of inflammation, and tumor progression compared to indirect measures, such as body weight loss, diarrhea, and histology (which is suboptimal when lesions are patchy), while offering insights on the overall health of the colon. Most notably, endoscopy allows for the repeated assessment of laboratory animal models over time, as opposed to traditional histological examination that requires the animal to be euthanized and colons to be harvested for analysis1. Despite these advantages, the use of murine endoscopic technology has not been widespread and is limited by the lack of standardized protocols for implementation and scoring of pathological findings. Properly implemented, murine endoscopy holds great promise to further facilitate our understanding and characterization of animal models of multiple chronic gastrointestinal disease states, including inflammatory bowel diseases, colitis, and colorectal tumors.
The utility of murine endoscopic technology hinges upon its reproducibility and objectivity, which requires the existence of standardized examination procedures and a consistent, non-redundant, and reliable scoring system for intestinal pathologies. At least three descriptive scoring systems for endoscopic assessment of colitis4-6, as well as for colonic tumors4,7,8 in mice have been published. However, these reported approaches and scoring systems are not readily comparable across studies. In many cases, there is no clear definition of the criteria used for classification of lesions, and when criteria are given, they vary widely. Moreover, there are no reported scoring systems that integrate the assessment of both colitis and tumors, two of the most common colonic pathologies that can occur simultaneously and have interactive effects on outcome, into a single measurement tool. Finally, the endoscopic scoring systems that do exist for colitis often have inflammatory categories with limited discrimination (i.e.&#160;narrow scoring options over short integer scales, often 1-4) to properly represent disease progression scenarios and enable the use of parametric statistical analysis.
In this paper, we describe the use of a flexible endoscope to assess the severity of murine colitis and colonic tumors&#160;and describe a standardized protocol for employing this technology in the anatomical assessment of the perianal region, rectum, and distal colon. We illustrate effective troubleshooting during endoscopy to minimize trauma and image artifacts, and we describe a reproducible scoring system based on validated published clinical parameters. The scoring system integrates evaluation of both intestinal inflammation and tumors using a high level of discrimination with 12 possible grades of inflammation, tumor mapping and plotting options, and a novel decimal unit system (i.e., decimal identifiers) to highlight findings with specific diagnostic value (i.e. tumor development, complications during endoscopy, etc.). The use of decimal identifiers allows for rapid tracking of relevant data sets for further downstream analysis. Finally, we perform reliability and validity testing of the scoring system in multiple murine models of colitis and colitis-associated cancer (dextran sulfate sodium (DSS) induced-colitis, Clostridium difficile infection, and azoxymethane/DSS-induced colonic cancer).

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			<td height="21" style="height:21px;width:240px;">Isoflurane, USP</td>
			<td style="width:160px;">Webster Veterinary&#160;</td>
			<td style="width:217px;"></td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Surgical lubricant</td>
			<td style="width:160px;">Savage laboratories</td>
			<td style="width:217px;">surgilube, 0281-0205-45</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Phosphate Buffered Saline</td>
			<td style="width:160px;">Thermo Scientific&#160;</td>
			<td style="width:217px;">SH30256</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">RNAlater</td>
			<td style="width:160px;">Ambion</td>
			<td style="width:217px;">AM7021</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:240px;">Methylene Blue 1% Aqueous Solution</td>
			<td style="width:160px;">Fisher Science Education</td>
			<td style="width:217px;">S96393</td>
			<td style="width:79px;"></td>
		</tr>
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			<td height="23" style="height:23px;width:240px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Flexible digital ureteroscope</td>
			<td style="width:160px;">Olympus America</td>
			<td style="width:217px;">URF-V</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Video system center</td>
			<td style="width:160px;">Olympus America</td>
			<td style="width:217px;">VISERA Pro OTV-S7 Pro</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Xenon light source</td>
			<td style="width:160px;">Olympus America</td>
			<td style="width:217px;">VISERA Pro CLV-S40 Pro</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Video recorder</td>
			<td style="width:160px;">MediCapture, Inc.</td>
			<td style="width:217px;">MediCap USB200</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Flexible biopsy cup forceps</td>
			<td style="width:160px;">Olympus America</td>
			<td style="width:217px;">FBC-3115</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Anesthesia machine</td>
			<td style="width:160px;">Euthanex Corporation</td>
			<td style="width:217px;">EZ-7000 Classic System</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

flexible colonoscopy in mice to evaluate the severity of colitis and colorectal tumors using a validated endoscopic scoring system

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

The protocol and images presented in this study were prepared with an Olympus URF-V flexible endoscope14&#160;and NBI11,15. Video images were saved in MPEG-2 format and pictures in JPG, TIF, PNG,&#160;or DICOM format with maximum 1,280 x 1,024 pixels, transferable to standard USB flash portable memories. NBI enhances the visual contrast of hemoglobin-containing tissues. Normal light and NBI imaging are key features to illustrate the normal anatomy of the colorectal mucosa and the differences between...

Watch this Scientific Journal Video about Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System at JoVE.com

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

flexible-colonoscopy-mice-to-evaluate-severity-colitis-colorectal

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JoVE Journal

Medicine

31.6K 次观看.  Case Western Reserve University School of Medicine, Cleveland. 在过去的几年中，新一代内窥镜已成为重要的诊断艾滋病的研究评估小鼠结肠炎和结肠肿瘤。我们在此提出一个详细的协议，用于炎症和大肠肿瘤在小鼠体内的内镜检查，以及使用十进制标识符记录结肠炎和结肠直肠肿瘤的内镜严重性一种新的评分系统。 

视频: Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Taste Exam: A Brief and Validated Test

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement classic taste-testing methods in two ways. First, we explain how to do a brief assessment of the mouth, including the tongue, to ensure that taste papillae are present and to note evidence of relevant disease. Second, we draw on genetics to validate taste test data by comparing reports of perceived bitterness intensity and inborn receptor genotypes. Discordance between objective measures of genotype and subjective reports of taste experience can identify data collection errors, distracted subjects or those who have not understood or followed instructions. Our expectation is that fast and valid taste tests may persuade researchers and clinicians to assess taste regularly, making taste testing as common as testing for hearing and vision. Finally, because many tissues of the body express taste receptors, taste responses may provide a proxy for tissue sensitivity elsewhere in the body and, thereby, serve as a rapid, point-of-care test to guide diagnosis and a research tool to evaluate taste receptor protein function.

This protocol measures human taste responses and includes a brief anatomical assessment, a short taste test, and a validation method using the subject&#39;s reported sensation and taste receptor genotype.

The emerging importance of taste in medicine and biomedical research, and new knowledge about its genetic underpinnings, has motivated us to supplement ...

The Pilocarpine Model of Temporal Lobe Epilepsy and EEG Monitoring Using Radiotelemetry System in Mice

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status epilepticus (SE) is frequently selected to recapitulate spontaneous recurrent seizures (SRS). Here we present a protocol of SE induction by intraperitoneal (i.p.) injection of pilocarpine and monitoring of chronic recurring seizures in live animals using a wireless telemetry video and electroencephalogram (EEG) system. We demonstrated notable behavioral changes that need attention after pilocarpine injection and their correlation with hippocampal neuronal loss at 7 days and 6 weeks post-pilocarpine. We also describe the experimental procedures of electrode implantation for video and EEG recording, and analysis of the frequency and duration of chronic recurrent seizures. Finally, we discuss the possible reasons why the expected results are not achieved in each case. This provides a basic overview of modeling chronic epilepsy in mice and guidelines for troubleshooting. We believe this protocol can serve as a baseline for suitable models of chronic epilepsy and epileptogenesis.

This manuscript describes a method of inducing status epilepticus by systemic pilocarpine injection and of monitoring spontaneous recurrent seizures in live animals using a wireless telemetry video and electroencephalogram system. This protocol can be utilized for studying the pathophysiologic mechanisms of chronic epilepsy, epileptogenesis, and acute seizures.

Temporal lobe epilepsy (TLE) is a common neurological disorder in adulthood. For translational studies of chronic epilepsy, pilocarpine-induced status ...

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.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Using a Knee Arthrometer to Evaluate Tissue-specific Contributions to Knee Flexion Contracture in the Rat

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ROM is called a joint contracture and results in increased morbidity. Due to the difficulty of reversing established knee contractures, early detection is important, and hence, knowing risk factors for their development is essential. The rat represents a good model with which the effect of an intervention can be studied due to the similarity of rat knee anatomy to that of humans, the rat's ability to tolerate long durations of knee immobilization in flexion, and because mechanical data can be correlated with histologic and biochemical analysis of knee tissue.
Using an automated arthrometer, we demonstrate a validated, precise, reproducible, user-independent method of measuring the extension ROM of the rat knee joint at specific torques. This arthrometer can be used to determine the effects of interventions on knee joint ROM in the rat.

The goal of the protocol is to measure the extension range of motion of the rat knee. The effects of various diseases that increase the stiffness of the knee joint and the effectiveness of treatments can be quantified.

Normal knee range of motion (ROM) is critical to well-being and allows one to perform basic activities such as walking, climbing stairs and sitting. Lost ...

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.

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 ...

Establishing In Situ Closed Circuit Perfusion of Lower Abdominal Organs and Hind Limbs in Mice

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small size of mouse organs, ex vivo perfusion of bone, bladder, skin, prostate, and reproductive organs is challenging or not feasible. Here, we report for the first time an in situ lower body perfusion circuit in mice that includes the above tissues, but bypasses the main clearance organs (kidney, liver, and spleen). The circuit is established by cannulating the abdominal aorta and inferior vena cava above the iliac artery and vein and cauterizing peripheral blood vessels. Perfusion is performed via a peristaltic pump with perfusate flow maintained for up to 2 h. In situ staining with fluorescent lectin and Hoechst solution confirmed that the microvasculature was successfully perfused. This mouse model can be a very useful tool for studying pathological processes as well as mechanisms of drug delivery, migration/metastasis of circulating tumor cells into/from the tumor, and interactions of immune system with perfused organs and tissues.

A protocol is described for in situ perfusion of the mouse lower body, including the bladder, the prostate, sex organs, bone, muscle and foot skin.

Ex vivo perfusion is an important physiological tool to study the function of isolated organs (e.g. liver, kidneys). At the same time, due to the small ...