Name: non-invasive assessment of the efficacy of new therapeutics for intestinal pathologies using serial endoscopic imaging of live mice
Uploaded: 2015-03-10T13:00:00.000+00:00
Duration: 9 min 1 s
Description: Watch this Scientific Journal Video about Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice at JoVE.com

We would like to thank CSL Ltd. for supporting the purchase of the endoscopy equipment. The research in the laboratory of ME is supported by the Ludwig Institute for Cancer Research, and the laboratories of TP and ME are supported by the Victorian State Government Operational Infrastructure Support and the National Health and Medical Research Council of Australia. ME is an NHMRC Senior Research Fellow.

The Walter and Eliza Hall Institute of Medical Research animal ethics committee approved each of the procedures described in these protocols.
1. Preparation of Experimental Mice
<ol>
	<li>Compose experimental cohorts of at least 4 gender matched 6-8 week old C57BL/6 mice (M. musculus) that are bred and housed in the same specific pathogen free (SPF) animal facility/room and provided with autoclaved food and water. The use of female mice will permit co-housing of mice from different lines, limit box numbers, and reduce cage-to-cage variation.</li>
	<li>Ensure that mice are at least 6 weeks of age to enable endoscopy procedures using the 3.0 mm diameter endoscopy sheath.</li>
	<li>Use mice of the same genetic background, and where possible, from cages stored on the same rack to reduce variations associated with the intestinal microflora. Variations in microflora between animal facilities should be taken into consideration when determining the appropriate control mice and genotypes for each experiment22.</li>
	<li>Mark mice by ear/toe clippings, tattoos, or similar to allow for easy identification.</li>
	<li>Weigh mice on Day 0, to determine baseline experimental weights.</li>
	<li>Perform endoscopy (see Section 5) of mice on Day 0 to record baseline colonic phenotypes.</li>
</ol>
2. Pre-clinical Trial in an Epithelial Damage and Acute Colitis Model
<ol>
	<li>Prepare a solution of the therapeutic agent to be tested. For therapeutics delivered by oral gavage (p.o.), prepare solutions to a maximum volume of 100 &#181;l. For therapeutics delivered by intraperitoneal (i.p.) injection, prepare solutions to a maximum volume of 200 &#181;l. 
	NOTE: Repeated i.p. or p.o. drug administration over prolonged periods of time can result in altered mouse behavior. Alternate the i.p. injection site to avoid prolonged discomfort. For p.o. administration, provide the mice with autoclaved sunflower seeds as a &#8216;treat&#8217; to minimize the adverse association with the gavage procedure.</li>
	<li>On Day 1, administer the therapeutic of interest (prepared in step 2.1) and appropriate vehicle controls. In the example provided (Figure 6A), 5 &#181;g of recombinant human (rh) iInterleukin (IL)-11 protein was dissolved in 200 &#181;l phosphate buffered saline (PBS) and administered i.p.</li>
	<li>Determine the timing and frequency of the administrations, which will be dependent on the pharmacokinetic profile established for the therapeutic reagent that is being tested. Figure 1 outlines prophylactic treatment using twice daily i.p. injections over the course of the experiment.</li>
	<li>Monitor, including stool consistency and the presence of blood, and weigh each mouse daily. On the days of treatment, weighing should coincide with administration of the therapeutic drug to minimize stress to the mice caused by repeated handling.</li>
	<li>On Day 3, prepare 2.5% (2.5 g/100 ml) DSS solution in the drinking water routinely provided to the mice by the animal facility. Approximately 5 ml/mouse/day of DSS solution is required for the experiment. The consumption of the DSS water may change if the ambient temperature of the animal facility fluctuates. 
	NOTE: DSS is an irritant and should be handled in accordance with MSDS instructions.</li>
	<li>Provide fresh DSS to the mice ad libitum in clean water bottles for 5 continuous days. On the evening of Day 5, provide normal drinking water in addition to mashed food pellets and a protein supplement provided in small petri dishes (100 g food pellets/10 g protein shake/10 ml water). This prevents dehydration and assists with minimizing severe weight-loss.</li>
	<li>Euthanize mice by CO2 intoxication and collect tissues for subsequent histological analysis on the morning of Day 8, or when the mice experience &#8805;15% weight-loss for more than three consecutive days, whichever occurs first. 
	NOTE: Adjust the DSS dose between 1-4% (w/v) depending on the microflora of the animal facility and the batch of DSS. Carry out, routine batch testing to establish the appropriate DSS dosage and circumvent batch-to-batch variation. The appropriate dosage should be based on weight-loss (not greater than 15% of original weight) and confirmed colitis histopathology.</li>
</ol>
3. Pre-clinical Trial in a Chronic Colitis or Colitis-associated Cancer Model
<ol>
	<li>For the chronic colitis model begin from step 3.7.</li>
	<li>For the colitis-associated cancer (CAC) model, on Day 1, inject each mouse with 10 mg/kg (w/w) Azoxymethane (AOM, 250 &#181;l, i.p.). 
	NOTE: AOM is a carcinogen and should be handled in accordance with MSDS instructions.</li>
	<li>Stock solutions of AOM are prepared as 10 mg/ml aliquots and stored at -20 &#176;C. On the day of injection, the AOM stock solution is thawed and diluted to 1 mg/ml in PBS. Repeated freezing and thawing of the AOM stock solution should be avoided.</li>
	<li>The AOM dose may be adjusted between 8-12 mg/kg. Routine batch testing is required to establish the appropriate AOM dosage in order to circumvent toxicity to mice due to batch-to-batch variation.</li>
	<li>During Days 1-7 following AOM administration weights will be stable and for this reason weight monitoring can be omitted during Days 1-7 to avoid handling animals that will excrete cytotoxic metabolites in their feces.</li>
	<li>On Day 7 all bedding needs to be changed following cytotoxic procedures. Once the bedding change is complete, the animals no longer require cytotoxic handling procedures, as they will no longer be excreting cytotoxic metabolites.</li>
	<li>On Day 8, prepare 2.5% (2.5 g/100 ml) DSS solution (as described in step 2.5) and provide to the mice ad libitum.</li>
	<li>Weigh mice daily throughout the experiment and monitor for signs of illness, including ruffled fur, hunching, bloody stools and reduced movement.</li>
	<li>On Day 13, remove the DSS solution and provide the mice with normal drinking water until Day 28. Days 8-28 constitute one &#8216;cycle&#8217; of the chronic colitis/CAC protocol.</li>
	<li>On Days 13-28, when the mice are given normal drinking water, provide the mice with mashed food pellets and a protein supplement provided in small petri dishes (100 g food pellets/10 g protein shake/10 ml water). This prevents dehydration and assists with minimizing severe weight-loss. 
	NOTE: The protein supplements in mash should not be provided when the mice are receiving DSS-containing drinking water in order to ensure consistency in DSS consumption.</li>
	<li>On Day 20 perform the second endoscopic examination to monitor individual mouse health (see Section 5).</li>
	<li>On Day 29 repeat the DSS cycle (Cycle 2: 2.5% (w/v) DSS provided during Days 29-33 and normal drinking water during Days 34-50).</li>
	<li>On Day 40 perform the third endoscopic examination to monitor individual mouse health and determine tumor burden (see Section 5).</li>
	<li>If no tumors are visible by endoscopy on Day 40, begin a third DSS cycle on Days 50-72 (Cycle 3: 2.5% (w/v) DSS provided during Days 50-55 and normal drinking water during Days 56-72). Generally, tumors are visible by Day 40 and therefore the third cycle of DSS can be omitted.</li>
	<li>Once tumors become visible by endoscopic examination, assign each mouse an endoscopic tumor score (see Section 5) and assign individual mice to cohorts with similar baseline tumor burdens.</li>
	<li>On Day 42 (or when tumors are visible), administer the therapeutic compound or relevant vehicle control (See Section 2.2). The timing of the doses will be dependent on the pharmacokinetic profile of the therapeutic. An example of an intervention treatment injected i.p. three times weekly in mice with established tumors is provided (Figure 2).</li>
	<li>Perform endoscopic exams once per week over the duration of the therapeutic treatment to monitor tumor burden. Generally, 4 weeks of a therapeutic treatment is sufficient to observe an objective treatment response. Following cessation of the treatment, a cohort of mice can also be monitored by endoscopy for tumor relapse.</li>
	<li>Euthanize mice by CO2 intoxication and collect tissues for biochemical and/or histological analysis on the morning of Day 72 or when mice experience &#8805;15% weight-loss for more than three consecutive days, whichever occurs first.</li>
</ol>
4. Pre-clinical Trial in a Sporadic Colorectal Cancer Model
<ol>
	<li>On Day 1, inject each mouse i.p. with 10 mg/kg AOM (see step 3.2). The AOM dose may be adjusted between 8-12 mg/kg. Routine batch testing is required to establish the appropriate AOM dosage in order to circumvent toxicity to mice due to batch-to-batch variation.</li>
	<li>Repeat the injection on the first day of each week for the following 5 weeks (Figure 3). The mice should be handled according to cytotoxic safety procedures for the entire 6 weeks.</li>
	<li>Weigh each mouse on the day of AOM injection to minimize distress to the mice caused by repeated handling. 
	NOTE: AOM is a carcinogen and should be handled in accordance with MSDS instructions.</li>
	<li>All animals should be handled according to cytotoxic safety procedures for the 6 weeks of AOM i.p. injections required for this protocol.</li>
	<li>During week 8 perform endoscopic examination to monitor for emerging tumors (see Section 5). Endoscopic examination should be performed every two weeks until tumors become visible. Generally, wild-type C57Bl/6 animals begin to develop tumors by approximately 40 weeks after the initial administration of AOM.</li>
	<li>Once tumors become visible by endoscopic examination, assign each mouse an endoscopic tumor score (see Section 5) and assign individual mice to a cohort with similar tumor burdens prior to the commencement of therapeutic treatment.</li>
	<li>During week 40 (or when tumors are first visible), administer the therapeutic compound or the relevant vehicle control.</li>
	<li>The timing of the doses will be dependent on the pharmacokinetic profile of the therapeutic (see step 2.1). An example of an intervention treatment injected i.p. 3 times weekly in mice with established tumors is provided (Figure 3).</li>
	<li>Perform endoscopic examination weekly for the duration of the therapeutic treatment periods to monitor tumor burden. Generally, 4 weeks of a therapeutic treatment is sufficient for an objective treatment response. Following treatment cessation, a cohort of mice can also be monitored by endoscopy for tumor relapse.</li>
	<li>Euthanize mice by CO2 intoxication and collect tissues for biochemical and/or histological analysis of the colon and tumors during week 45, or following 4 weeks of therapy.</li>
</ol>
5. Endoscopic Examinations
<ol>
	<li>The endoscopy equipment should be assembled according to standard procedures16.</li>
	<li>Sterilize and clean the endoscope probe with 70% ethanol or an anti-bacterial lubricant.</li>
	<li>Videos can be recorded using a laptop or desktop computer and standard media software, such as iMovie. A standard computer monitor, rather than a medical grade monitor, is sufficient to visualize the colon during the endoscopy procedure (Figure 4).</li>
	<li>Anesthetize groups of 5-6 mice simultaneously in a chamber with 3% isoflurane in 100% O2 at a rate of 0.2-0.4 L/min. Once the mice are anesthetized, which is confirmed by toe pinch; the isoflurane levels should be altered to 0.5-2% for maintenance.</li>
	<li>Remove an individual mouse from the chamber, and place it ventral side up with its head secured in a nose-cone. Full anesthesia should be monitored and the hind legs should be adjusted so that they are stretched out behind the mouse.</li>
	<li>Place a vet ointment on the eyes of the mice to prevent dryness.</li>
	<li>Hold the tail of the mouse where it meets the lower spine to clearly reveal the anus. Insert the rigid endoscope sheath into a beaker of water and adjust the airflow to allow the release of one small bubble at a time.</li>
	<li>The airflow will permit the colon to inflate. If the airflow is too strong, air will be pumped into the stomach of the mouse. No additional lubrication is required.</li>
	<li>Carefully insert the rigid endoscope sheath into the rectum. Generally, the endoscope can be inserted up to 3 cm, at which point the colon in mice curves and is not accessible to the rigid endoscope.</li>
	<li>A common difficulty during endoscopy procedures is blockage of access to the lumen of the colon due to fecal matter. Avoid this by gently massaging of the belly of mice prior to the procedure to encourage defecation, or carefully maneuvering the endoscope around the fecal matter during the procedure. Peristalsis can also occur, during which the endoscope should be held in position until the colon musculature relaxes.</li>
	<li>Initiate video recording at any stage of the endoscopy procedure. Disease scoring can be recorded by an experienced assistant during the procedure, or at a later time point from the video files.</li>
	<li>Following the endoscopic exam, return the mice to their cage and monitor them during recovery from anesthesia. Animals are generally awake and mobile within 2 min following removal from the isoflurane administering nose cone.</li>
	<li>Ensure that these procedures are performed by an experienced scientist. Each endoscopy procedure will take approximately 2 min per mouse.</li>
</ol>
6. Disease Scoring
<ol>
	<li>To monitor colitis, score videos for the Murine Endoscopic Index of Colitis Severity (MEICS)16, which documents changes in (i) the thickness of the colon wall indicated by transparency, (ii) stool consistency, (iii) blood vessel integrity and presence, (iv) ulcers and areas of regeneration that present in a granular fashion, (v) bleeding indicated by fibrin deposits (Figure 5a).</li>
	<li>To monitor tumor burden, score videos for tumor incidence and size. Tumor size is determined by the diameter of the colonic lumen occupied by the tumor16 (Figure 5b). It is common to use both colitis and tumor scoring parameters for a single mouse. Using endoscopy, individual tumors and the health of the colonic mucosa can be monitored over time, providing a read-out for the success of new therapeutics in a single animal.</li>
	<li>(Optional) Still images can be extracted from the videos for generation of representative figures (Figure 6).</li>
</ol>

<ol>
	<li>Tenesa, A., Dunlop, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+aetiology+of+colorectal+cancer+from+genome-wide+association+studies.">New insights into the aetiology of colorectal cancer from genome-wide association studies.</a> Nat Rev Genet. 10, 353-358 (2009).</li><li>Rustgi, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+hereditary+colon+cancer.">The genetics of hereditary colon cancer.</a> Genes Dev. 21, 2525-2538 (2007).</li><li>Rutter, M., 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=Severity+of+inflammation+is+a+risk+factor+for+colorectal+neoplasia+in+ulcerative+colitis.">Severity of inflammation is a risk factor for colorectal neoplasia in ulcerative colitis.</a> Gastroenterology. 126, 451-459 (2004).</li><li>Eaden, J. A., Abrams, K. R., Mayberry, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+risk+of+colorectal+cancer+in+ulcerative+colitis:+a+meta-analysis.">The risk of colorectal cancer in ulcerative colitis: a meta-analysis.</a> Gut. 48, 526-535 (2001).</li><li>Lakatos, P. L., Lakatos, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+for+colorectal+cancer+in+ulcerative+colitis:+changes,+causes+and+management+strategies.">Risk for colorectal cancer in ulcerative colitis: changes, causes and management strategies.</a> World J Gastroenterol. 14, 3937-3947 (2008).</li><li>Xiang, B., Snook, A. E., Magee, M. S., Waldman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+Colorectal+cancer+immunotherapy."> Colorectal cancer immunotherapy.</a> Discov Med. 15, 301-308 (2013).</li><li>Feng, Q. 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=Anti-EGFR+and+anti-VEGF+agents:+Important+targeted+therapies+of+colorectal+liver+metastases.">Anti-EGFR and anti-VEGF agents: Important targeted therapies of colorectal liver metastases.</a> World J Gastroenterol. 20, 4263-4275 (2014).</li><li>Dinarello, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+Agents:+Present+and+Future.">Anti-inflammatory Agents: Present and Future.</a> Cell. 140, 935-950 (2010).</li><li>Grivennikov, S. I., 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=Adenoma-linked+barrier+defects+and+microbial+products+drive+IL-23/IL-17-mediated+tumour+growth.">Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.</a> Nature. 491, 254-258 (2012).</li><li>Putoczki, T., Thiem, S., Loving, A., Busuttil, R. A., Wilson, N. A., Ziegler, P., Nguyen, P., Preaudet, A., Farid, R., Edwards, K., Boglev, Y., Luwor, R. B., Jarnicki, A. J., Horst, D., Boussioutas, A., Heath, J., Sieber, O., Nash, A., Greten, F., McKenzie, B. S., Ernst, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-11+is+the+dominant+IL-6+family+cytokine+during+gastrointestinal+tumorigenesis+and+can+be+targeted+therapeutically.">Interleukin-11 is the dominant IL-6 family cytokine during gastrointestinal tumorigenesis and can be targeted therapeutically.</a> Cancer Cell. 24, 257-271 (2013).</li><li>Perse, M., Cerar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dextran+sodium+sulphate+colitis+mouse+model:+traps+and+tricks.">Dextran sodium sulphate colitis mouse model: traps and tricks.</a> J Biomed Biotechnol. 2012, 718617 (2012).</li><li>Rose, W. A., Sakamoto, K., Leifer, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+role+of+dextran+sulfate+sodium+for+in+vivo+modeling+of+intestinal+diseases.">Multifunctional role of dextran sulfate sodium for in vivo modeling of intestinal diseases.</a> BMC Immunol. 13, 41 (2012).</li><li>Laroui, 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=Dextran+sodium+sulfate+(DSS)+induces+colitis+in+mice+by+forming+nano-lipocomplexes+with+medium-chain-length+fatty+acids+in+the+colon.">Dextran sodium sulfate (DSS) induces colitis in mice by forming nano-lipocomplexes with medium-chain-length fatty acids in the colon.</a> PLoS One. 7, e32084 (2012).</li><li>Miyazawa, F., Olijnyk, O. R., Tilley, C. J., Tamaoki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+dextran+sulfate+and+Escherichia+coli+ribosomes.">Interactions between dextran sulfate and Escherichia coli ribosomes.</a> Biochim Biophys Acta. 145, 96-104 (1967).</li><li>Peterson, L. W., Artis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+epithelial+cells:+regulators+of+barrier+function+and+immune+homeostasis.">Intestinal epithelial cells: regulators of barrier function and immune homeostasis.</a> Nat Rev Immunol. 14, 141-153 (2014).</li><li>Neufert, C., Becker, C., 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=An+inducible+mouse+model+of+colon+carcinogenesis+for+the+analysis+of+sporadic+and+inflammation-driven+tumor+progression.">An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression.</a> Nat Protoc. 2, 1998-2004 (2007).</li><li>Schwitalla, 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=Loss+of+p53+in+Enterocytes+Generates+an+Inflammatory+Microenvironment+Enabling+Invasion+and+Lymph+Node+Metastasis+of+Carcinogen-Induced+Colorectal+Tumors.">Loss of p53 in Enterocytes Generates an Inflammatory Microenvironment Enabling Invasion and Lymph Node Metastasis of Carcinogen-Induced Colorectal Tumors.</a> Cancer Cell. 23, 93-106 (2013).</li><li>Fiala, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigations+into+the+metabolism+and+mode+of+action+of+the+colon+carcinogens+1,2-dimethylhydrazine+and+azoxymethane.">Investigations into the metabolism and mode of action of the colon carcinogens 1,2-dimethylhydrazine and azoxymethane.</a> 40, 2436-2445 (1977).</li><li>Chen, J., Huang, X. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signal+pathways+in+azoxymethane-induced+colon+cancer+and+preventive+implications.">The signal pathways in azoxymethane-induced colon cancer and preventive implications.</a> Cancer Biol Ther. 8, 1313-1317 (2009).</li><li>Bollrath, J., 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=gp130-mediated+Stat3+activation+in+enterocytes+regulates+cell+survival+and+cell-cycle+progression+during+colitis-associated+tumorigenesis.">gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis.</a> Cancer Cell. 15, 91-102 (2009).</li><li>Becker, C., Fantini, M. C., 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=High+resolution+colonoscopy+in+live+mice.">High resolution colonoscopy in live mice.</a> Nat Protoc. 1, 2900-2904 (2006).</li><li>Ivanov, I. I., 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=Induction+of+intestinal+Th17+cells+by+segmented+filamentous+bacteria.">Induction of intestinal Th17 cells by segmented filamentous bacteria.</a> Cell. 139, 485-498 (2009).</li><li>Thiem, 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=mTORC1+inhibition+restricts+inflammation-associated+gastrointestinal+tumorigenesis+in+mice.">mTORC1 inhibition restricts inflammation-associated gastrointestinal tumorigenesis in mice.</a> J Clin Invest. 123, 767-781 (2013).</li><li>Stuart, 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=Therapeutic+inhibition+of+Jak+activity+inhibits+progression+of+gastrointestinal+tumors+in+mice.">Therapeutic inhibition of Jak activity inhibits progression of gastrointestinal tumors in mice.</a> Mol Cancer Ther. 13, 468-474 (2014).</li><li>De Robertis, M., 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+AOM/DSS+murine+model+for+the+study+of+colon+carcinogenesis:+From+pathways+to+diagnosis+and+therapy+studies.">The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies.</a> J Carcinog. 10, 9 (2011).</li><li>Viennois, E., Chen, F., Laroui, H., Baker, M. T., Merlin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dextran+sodium+sulfate+inhibits+the+activities+of+both+polymerase+and+reverse+transcriptase:+lithium+chloride+purification,+a+rapid+and+efficient+technique+to+purify+RNA.">Dextran sodium sulfate inhibits the activities of both polymerase and reverse transcriptase: lithium chloride purification, a rapid and efficient technique to purify RNA.</a> BMC Res Notes. 6, 350 (2013).</li><li>Kerr, T. A., 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=Dextran+sodium+sulfate+inhibition+of+real-time+polymerase+chain+reaction+amplification:+a+poly-A+purification+solution.">Dextran sodium sulfate inhibition of real-time polymerase chain reaction amplification: a poly-A purification solution.</a> Inflamm Bowel Dis. 18, 344-348 (2012).</li></ol>

The authors have nothing to disclose.

The three protocols that are described outline methods of reliable and reproducible induction of colonic disease pathology in mice. When combined with routine endoscopic monitoring and the intervention strategies outlined here, these protocols will provide powerful pre-clinical insight into the efficacy of therapeutics. Our laboratories routinely use all of these protocols to monitor the success of novel therapeutics10,23,24.
There are a number of considerations when choosing a pre-clinical animal model to test new therapeutics. These include relevance of the model to the human disease, and the contribution of the tumor microenvironment to the proposed action of the therapeutic target. Here we provide three protocols for therapeutic intervention in established intestinal disease models. These models are reproducible and the delivery of reagents to induce disease is easy to manage. Importantly, the models are highly relevant to multiple facets and stages of colitis onset, and of tumor initiation and progression. Researchers should take into consideration the genetic background of the mouse strains used when designing experiments, as the susceptibility to disease induced by DSS and/or AOM can vary considerably25. In addition, different microbial communities may have different metabolic capacities in the context of AOM, which is metabolized by bacteria. We caution against using different cohorts of mice that were born into different animal facilities (including commercial vendors) in a single experiment. Similarly, the different microflora in mice used from different facilities may elicit different host responses to DSS-induced epithelial barrier damage11. Moreover, the appropriate analysis of tissue (for example RNA purification) should also be considered, since the ability of DSS to inhibit reverse transcriptase will impact on subsequent molecular analysis26,27.
Mouse endoscopy is a cutting edge technique to repeatedly monitor disease onset and progression in an individual mouse. The ability to record videos and extract still images permits easy monitoring of multiple disease parameters and tumors. In addition to improving animal welfare, endoscopic monitoring also reduces the need for multiple cohorts of experimental mice, which traditionally were culled at different time-points to track disease outcome. The MEICS scoring system is not a substitute for histopathological analysis, but provides an alternative means to monitor animal health and mucosal damage in live mice. Mouse endoscopy is a specialized laboratory technique, and all procedures should be performed by trained personnel to ensure appropriate manipulation and handling of the mice, as well as to provide consistent quality in the images used for disease scoring. In the hands of qualified personal, we have found that endoscopy induces little or no damage to the tumors that would cause intra-tumoral bleeding. For the therapeutic protocols outlined, we consider endoscopy highly advantageous, since it provides a way to determine the initial tumor burden, and allows us to group cohorts of animals with similar tumor burdens prior to the administration of a therapeutic drug. Sequential monitoring of the mice enables researchers to determine the efficacy of novel therapies early on, with the option of terminating unsuccessful experiments in a timely manner.
As our understanding of inflammatory bowel disease and colorectal cancers advance, new targets for therapy will be identified. Appropriate animal models will be integral to ensuring that the most promising new therapies are moved towards clinical trials.

Colorectal cancer (CRC) is the 4th most common cause of malignancy worldwide1. Despite the significant progress in our understanding of the familial basis of this disease, genetic predisposition only contributes to ~20% of CRC cases2. The remainder are attributed to numerous extrinsic and environmental factors, including chronic inflammation. In humans, the link between chronic inflammation and colon cancer is evident in ulcerative colitis (UC) patients, who have a greater risk of developing colitis-associated colon cancer (CAC), depending on the duration, extent and severity of the inflammatory disease3-5. Accordingly, novel therapies are in development to control the immune response and the associated production of growth promoting factors by the inflammatory tumor microenvironment6-8. There is an increasing requirement for appropriate pre-clinical animal models to characterize the therapeutic efficacy of these drugs against the development and progression of disease.
Mouse models have unequivocally demonstrated that the inflammatory microenvironment contributes to CRC progression, even in the absence of overt inflammation9,10. These models include the use of the polysaccharide dextran sulfate sodium (DSS), provided in the drinking water of mice, to model epithelial injury and acute and chronic inflammatory bowel disease (IBD)11,12. Although the mechanism by which DSS induces mucosal damage and colitis is not completely understood, some studies suggest that DSS inhibits cellular reverse transcriptase and ribonuclease activities within cells, or promotes the formation of nano-lipid complexes that fuse with the colonic membrane leading to epithelial damage13,14. Modifications to the standard DSS model have also provided significant insight into the mechanisms by which colonic epithelial cells maintain tissue homeostasis and regulate mucosal immune responses15.
Intraperitoneal administration of Azoxymethane (AOM) alone, or in combination with DSS, provides a model for examining the interplay between somatic mutations in the epithelial mucosa and the inflammatory and stromal microenvironment16,17. AOM is a metabolite of the carcinogen 1,2-dimethylhydrazine (DMH) that does not directly result in DNA mutations. Instead, AOM is hydrolyzed to methylazoxymethanol (MAM) by the cytochrome isoform CYP2E1 in the liver, where MAM is conjugated with glucuronic acid and then transported to the intestine through bile secretions18. It is thought that the bacterial &#946;-glucuronidase contributes to the degradation of MAM resulting in DNA alkylation and the accumulation of mutations in epithelial cells19. Most AOM-induced colonic tumors harbor missense mutations in the gene encoding &#946;-catenin, rendering the protein resistant to proteasomal degradation, which results in aberrant activation of the canonical Wnt-signaling pathway20. When the activity of AOM is combined with the mucosal damage elicited by DSS, the ensuing wound healing response creates a microenvironment that is conducive to the growth and expansion of the mutagenized epithelium. In a variation of this model, repetitive administration of AOM alone over a period of several weeks can be used to model sporadic colorectal cancer, in the absence of DSS-induced colitis10,17. These two complimentary models provide experimental settings to study CAC and sporadic CRC respectively, both of which are associated with a pro-inflammatory tumor microenvironment10.
The use of serial endoscopy in mice was pioneered by Becker and colleagues21, and enables longitudinal monitoring of colitis and tumor progression. Here we provide three pre-clinical protocols based on DSS-induced mucosal damage and/or AOM-mediated tumor induction to reproducibly induce specific colonic pathologies. The first protocol describes inducing acute mucosal damage in response to DSS administration to elicit many of the histopathological features associated with IBD. The second protocol is based on three consecutive cycles of DSS administration to mimic the flares of inflammation commonly observed in IBD patients, and can be carried out in conjunction with AOM-induced mutations. The final protocol is based on AOM-induced sporadic epithelial mutations. For each of these protocols, we expand on the relevant standard procedures to include prophylactic and therapeutic intervention methods that we have developed to monitor the efficacy of new drugs.

<table><tbody><tr><td>Dextran sulfate sodium (MW 36,000-50,000)</td><td>MP Biochemicals</td><td>160110</td><td>Requires batch testing.</td></tr><tr><td>Azoxymethane</td><td>Sigma</td><td>A5486-100MG</td><td>Requires batch testing.</td></tr><tr><td>Vanilla protein shake</td><td>N/A</td><td>N/A</td><td>Available from hospital pharmacies.</td></tr><tr><td>Isoflurane</td><td>PPC</td><td>M60303</td><td>This is a restricted reagent, which should be stored under lock and key.</td></tr><tr><td>70% ethanol</td><td>N/A</td><td>N/A</td><td>Standard lab reagent.</td></tr><tr><td>Coloview miniendoscopic system</td><td></td><td></td><td></td></tr><tr><td>Endovision Tricam</td><td>Karl Storz</td><td>20212001-020</td><td></td></tr><tr><td>Xenon 175 light source with anti-fog pump</td><td>Karl Storz</td><td>20134001</td><td></td></tr><tr><td>HOPKINS straight Forward Telescope</td><td>Karl Storz</td><td>64301AA</td><td></td></tr><tr><td>Endoscopic sheath (total diameter 3 mm)</td><td>Kalr Stroz</td><td>61029C</td><td></td></tr><tr><td>Fiber optic light cable</td><td>Kalr Stroz</td><td>69495ND</td><td></td></tr><tr><td>Computer and media player software</td><td>Apple</td><td></td><td>iMovie</td></tr><tr><td>Scale</td><td>Any</td><td></td><td>Any scale suitable for weighing mice.</td></tr></tbody></table>

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.

Weight-loss is used as a standard parameter to monitor for disease associated with colitis, which is routinely used to monitor the overall health of mice. Animals generally maintain their weight while DSS-containing water is administered, and only start to loose weight when they are returned to normal drinking water. Acceptable weight-loss parameters should be established in accordance with the institution&#8217;s animal ethics committee. In order to prevent the dehydration associated with prolonged diarrhea, use the routine provision of mash (food pellets mashed and mixed with drinking water) supplemented with a protein shake in addition to normal drinking water.
As an alternative anesthesia, ketamine/xylazine or similar agents can be used, if isoflurane equipment is not available. Due to the rigid nature of the endoscope, these procedures only allow for visualization of the most distal 3 cm of the mouse colon. Newer endoscopes with additional capabilities (including flexibility and fluorescence) are available depending on the needs of the experiment. However, since the deleterious effects of DSS are primarily limited to the distal colon of mice, with milder pathology observed in the middle colon, the rigid endoscope does not hinder monitoring of the mucosal health of individual mice. Although we describe a protocol for prophylactic treatment of acute DSS-induced colitis, this protocol can easily be modified to test intervention treatment strategies. The efficacy of a drug designed to alleviate epithelial damage and colitis can be monitored longitudinally in individual mice and quantified based on the scoring parameters described (Figure 5). This is advantageous over traditional experimental designs, which require culling of mice at specific time points during the experimental protocol, and does not permit characterization of the disease response to a treatment over time.
Clinical studies in humans have highlighted considerable variability in how different tumors in an individual patient respond to treatments. The procedures that are described here provide a means to monitor overall tumor burden, as well as the treatment response of individual tumors over the course of an experiment. It is important to consider that the intervention protocols that are outlined for the cancer models do not take into account the effects of a therapy on tumor initiation. Prophylactic protocols, with the treatment provided prior to when tumors become visible by endoscopy, are required to obtain this information. The protocols outlined here provide information on therapeutic effects on the on the progression (measured by tumor size) of individual tumors. Tumor regression may also be indicated by a reduction in the number of visible tumors.
<img alt="Figure 1" src="/files/ftp_upload/52383/52383fig1highres.jpg" /> 
Figure 1: Monitoring the efficacy of a prophylactic treatment therapy in a model of acute colonic damage. The experimental protocol (a) requires 8 days from start to completion. Therapeutics are administered (b) from Day 1 for prophylactic treatments. DSS is provided in the drinking water (c) from Day 3 of the experimental protocol. Endoscopy is performed (d) to monitor the disease progression in the animals. Suggested timepoints include Day 0 (untreated) and Day 2 (health monitoring), Day 5 and 8 (to determine disease burden). The experiment is terminated (e) the morning of Day 8. In a wild-type mouse (f) the progression of disease increases over time.
<img alt="Figure 2" src="/files/ftp_upload/52383/52383fig2highres.jpg" /> 
Figure 2: Monitoring the efficacy of intervention therapy in a model of colitis-associated cancer. The experimental protocol (a) requires 72 days from start to completion. Therapeutics (b) are administered to mice with established tumors from Day 46 for intervention treatments. AOM (c) is injected on Day 1, and DSS is provided in the drinking water over the course of three cycles of the experimental protocol, beginning on Day 8. Endoscopy (d) is performed to monitor the disease progression in the animals. Suggested timepoints include Day 0 (untreated), Day 20 (health monitoring), and Day 40 (to group animals according to tumor burden). Endoscopy is performed weekly over the course of the therapeutic treatment to monitor disease outcomes. The experiment (e) is terminated the morning of Day 72. In a wild-type mouse (f) tumor burden increases from Day 40 onwards.
<img alt="Figure 3" src="/files/ftp_upload/52383/52383fig3highres.jpg" /> 
Figure 3: Monitoring the efficacy of intervention therapy in a model of spontaneous colorectal cancer. The experimental protocol (a) requires &#62;50 weeks from start to completion. Therapeutics (b) are administered to mice with established tumors for intervention treatments. AOM (c) is injected on Day 1, and weekly thereafter for 6 consecutive injections over the course of the experimental protocol. Endoscopy (d) is performed to monitor the disease progression in the animals. Suggested timepoints include Day 0 (untreated) and Week 8 (tumor monitoring) and biweekly there after (to establish tumor burden). Endoscopy is performed weekly over the course of treatment to monitor therapeutic outcomes. The experiment (e) is terminated in Week 50. In a wild-type mouse (f) tumor burden increases from Week 40 onwards.
<img alt="Figure 4" src="/files/ftp_upload/52383/52383fig4highres.jpg" width="650" /> 
Figure 4: Equipment set-up. The experimental set-up (a) for the endoscopic unit, with individual pieces of equipment indicated. The rigid endoscope (b), with individual components indicated. Scale bar = 2.5 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52383/52383fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52383/52383fig5highres.jpg" width="650" /> 
Figure 5: Scoring disease parameters by endoscopy. An outline (a) of the Murine Endoscopic Index of Colitis Severity (MEICS). An outline (b) of the individual tumor scoring parameters. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52383/52383fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52383/52383fig6highres.jpg" width="650" /> 
Figure 6: Representative therapeutic treatments. Representative weight-loss, endoscopic images and scores for: (a) Acute DSS-induced mucosal damage. (b) Tumors that developed following the AOM/DSS protocol. (c) Tumors that developed following the sequential AOM protocol. N = 3 mice per group. *P &#60;0.05, ***P &#60;0.001 (Student&#8217;s T-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52383/52383fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice at JoVE.com

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

non-invasive-assessment-efficacy-new-therapeutics-for-intestinal

Nanyang Technological University

Research

JoVE Journal

Medicine

10.0K Views.  The Walter and Eliza Hall Institute for Medical Research. 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.

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

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

Cancer Research

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

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

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

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

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).

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 ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Immunology and Infection

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Neuroscience

Induction of Intestinal Graft-versus-host Disease and Its Mini-endoscopic Assessment in Live Mice

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) face and is frequently associated with a poor clinical outcome. While, for instance, GvHD manifestations of the skin are usually responsive to established immune-suppressive therapies and are, hence, not taking a fatal course, the presence and the intensity of intestinal GvHD, especially of the mid-to-lower parts of the gut, strongly influence the outcome and overall survival of patients with acute GvHD. Therapeutic options are essentially limited to the classic immune-suppressive agents yielding only moderate disease-mitigating effects. Hence, detailed knowledge about the tissue-resident immune cascade, changes in the intestinal microbiota, and the stromal response prior, upon, and after intestinal GvHD onset are urgently needed to understand the events and mechanisms underlying its pathogenesis and to develop innovative therapeutic options. Murine models of GvHD are frequently employed to identify and functionally assess molecules and pathways putatively driving intestinal GvHD. However, means to specifically monitor and evaluate intestinal inflammation over time are essentially lacking since established scores to assess and grade acute GvHD are routinely comprised of various parameters which rather reflect&#160;systemic GvHD manifestations. The detailed evaluation of intestinal GvHD has been restricted to studies using euthanized mice, thereby essentially excluding longitudinal (i.e., kinetic) analyses of the colonic compartment under a given experimental condition (e.g., antibody-mediated blockade of a proinflammatory cytokine) in live mice (i.e., in vivo). The mini-endoscopic in situ assessment of the distal colon of allo-HCT-treated mice described here allows a) a detailed macroscopic evaluation of different aspects of intestinal inflammation and b) the option to collect tissue samples for downstream analyses at various time points over the course of the observation period. Overall, the mini-endoscopic approach provides a major advance in preclinical noninvasive monitoring and assessment of intestinal GvHD.

Here, we present a protocol that describes allogeneic hematopoietic stem cell transplantation and allows repetitive mini-endoscopic evaluations of the distal colon in situ for the presence, characteristics, and severity of colonic inflammation within live mice suffering from intestinal graft-versus-host disease.

Acute graft-versus-host disease (GvHD) represents the most severe complication that patients previously undergoing allogeneic hematopoietic stem cell ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal ...

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video