Name: murine fecal isolation and microbiota transplantation
Uploaded: 2023-05-26T13:30:00
Description: Watch this Scientific Journal Video about Murine Fecal Isolation and Microbiota Transplantation at JoVE.com

This study was supported by Vanderbilt Clinical and Translational Science Award Grant UL1TR002243 (to A.K.) from the National Center for Advancing Translational Sciences; American Heart Association Grant POST903428 (to J.A.I.); and National Heart, Lung, and Blood Institute Grants K01HL13049, R03HL155041, R01HL144941 (to A.K.), and NIH grant 1P01HL116263 (to V.K.). Figure 1 was created using Biorender.

Vanderbilt University&#39;s Institutional Animal Care and Use Committee approved all procedures described in this manuscript. C57B1/6 male mice at 3 months of age, purchased from The Jackson Laboratory, were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals.
1. Collection, storage, and processing of human fecal samples
<ol>
	<li>Collect a stool sample, using a sterile container if the subject is in the clinic. Refrigerate the stool ...

<ol>
	<li>Virani, S. 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=Heart+disease+and+stroke+statistics-2021+update:+a+report+From+the+American+Heart+Association.">Heart disease and stroke statistics-2021 update: a report From the American Heart Association.</a> Circulation. 143 (8), 254 (2021).</li><li>Wu, 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=The+gut+microbiota+in+prediabetes+and+diabetes:+a+population-based+cross-sectional+study.">The gut microbiota in prediabetes and diabetes: a population-based cross-sectional study.</a> Cell Metabolism. 32 (3), 379-390 (2020).</li><li>Crovesy, L., Masterson, D., Rosado, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profile+of+the+gut+microbiota+of+adults+with+obesity:+a+systematic+review.">Profile of the gut microbiota of adults with obesity: a systematic review.</a> European Journal of Clinical Nutrition. 74 (9), 1251-1262 (2020).</li><li>Avery, E. G., 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+gut+microbiome+in+hypertension:+recent+advances+and+future+perspectives.">The gut microbiome in hypertension: recent advances and future perspectives.</a> Circulation Research. 128 (7), 934-950 (2021).</li><li>Perez-Matute, P., Iniguez, M., de Toro, M., Recio-Fernandez, E., Oteo, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+fecal+transplantation+from+a+lean+state+potentiates+caloric+restriction+effects+on+body+weight+and+adiposity+in+obese+mice.">Autologous fecal transplantation from a lean state potentiates caloric restriction effects on body weight and adiposity in obese mice.</a> Scientific Reports. 10 (1), 9388 (2020).</li><li>Zoll, 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=Fecal+microbiota+transplantation+from+high+caloric-fed+donors+alters+glucose+metabolism+in+recipient+mice,+independently+of+adiposity+or+exercise+status.">Fecal microbiota transplantation from high caloric-fed donors alters glucose metabolism in recipient mice, independently of adiposity or exercise status.</a> American Journal of Physiology. Endocrinology and Metabolism. 319 (1), 203-216 (2020).</li><li>Hvas, C. L., 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=Fecal+microbiota+transplantation+is+superior+to+fidaxomicin+for+treatment+of+recurrent+Clostridium+difficile+infection.">Fecal microbiota transplantation is superior to fidaxomicin for treatment of recurrent Clostridium difficile infection.</a> Gastroenterology. 156 (5), 1324-1332 (2019).</li><li>Kootte, R. 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=Improvement+of+insulin+sensitivity+after+lean+donor+feces+in+metabolic+syndrome+is+driven+by+baseline+intestinal+microbiota+composition.">Improvement of insulin sensitivity after lean donor feces in metabolic syndrome is driven by baseline intestinal microbiota composition.</a> Cell Metabolism. 26 (4), 611-619 (2017).</li><li>Li, 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=Gut+microbiota+dysbiosis+contributes+to+the+development+of+hypertension.">Gut microbiota dysbiosis contributes to the development of hypertension.</a> Microbiome. 5 (1), 14 (2017).</li><li>Shi, 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=Restructuring+the+gut+microbiota+by+intermittent+fasting+lowers+blood+pressure.">Restructuring the gut microbiota by intermittent fasting lowers blood pressure.</a> Circulation Research. 128 (9), 1240-1254 (2021).</li><li>Zhong, H. 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=Washed+microbiota+transplantation+lowers+blood+pressure+in+patients+with+hypertension.">Washed microbiota transplantation lowers blood pressure in patients with hypertension.</a> Frontiers in Cellular and Infection Microbiology. 11, 679624 (2021).</li><li>Ferguson, J. F., 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=High+dietary+salt-induced+dendritic+cell+activation+underlies+microbial+dysbiosis-associated+hypertension.">High dietary salt-induced dendritic cell activation underlies microbial dysbiosis-associated hypertension.</a> JCI Insight. 5 (13), 126241 (2019).</li><li>Yu, E. W., 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=Fecal+microbiota+transplantation+for+the+improvement+of+metabolism+in+obesity:+The+FMT-TRIM+double-blind+placebo-controlled+pilot+trial.">Fecal microbiota transplantation for the improvement of metabolism in obesity: The FMT-TRIM double-blind placebo-controlled pilot trial.</a> PLoS Medicine. 17 (3), 1003051 (2020).</li><li>Leong, K. S. W., 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=Effects+of+fecal+microbiome+transfer+in+adolescents+with+obesity:+the+gut+bugs+randomized+controlled+trial.">Effects of fecal microbiome transfer in adolescents with obesity: the gut bugs randomized controlled trial.</a> JAMA Network Open. 3 (12), 2030415 (2020).</li><li>Zhang, Z., 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=Impact+of+fecal+microbiota+transplantation+on+obesity+and+metabolic+syndrome-a+systematic+review.">Impact of fecal microbiota transplantation on obesity and metabolic syndrome-a systematic review.</a> Nutrients. 11 (10), 2291 (2019).</li><li>Laubitz, D., 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=Dynamics+of+gut+microbiota+recovery+after+antibiotic+exposure+in+young+and+old+mice+(a+pilot+study).">Dynamics of gut microbiota recovery after antibiotic exposure in young and old mice (a pilot study).</a> Microorganisms. 9 (3), 647 (2021).</li><li>Xiao, L., 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=High-fat+feeding+rather+than+obesity+drives+taxonomical+and+functional+changes+in+the+gut+microbiota+in+mice.">High-fat feeding rather than obesity drives taxonomical and functional changes in the gut microbiota in mice.</a> Microbiome. 5 (1), 43 (2017).</li><li>Brunt, V. 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=Suppression+of+the+gut+microbiome+ameliorates+age-related+arterial+dysfunction+and+oxidative+stress+in+mice.">Suppression of the gut microbiome ameliorates age-related arterial dysfunction and oxidative stress in mice.</a> The Journal of Physiology. 597 (9), 2361-2378 (2019).</li><li>Choo, J. M., Rogers, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbiota+transplantation+for+colonization+of+germ-free+mice.">Gut microbiota transplantation for colonization of germ-free mice.</a> STAR Protocols. 2 (3), 100610 (2021).</li><li>Kim, T. T., 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=Fecal+transplant+from+resveratrol-fed+donors+improves+glycaemia+and+cardiovascular+features+of+the+metabolic+syndrome+in+mice.">Fecal transplant from resveratrol-fed donors improves glycaemia and cardiovascular features of the metabolic syndrome in mice.</a> American Journal of Physiology. Endocrinology and Metabolism. 315 (4), 511-519 (2018).</li><li>Lu, 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=Subcutaneous+angiotensin+II+infusion+using+osmotic+pumps+induces+aortic+aneurysms+in+mice.">Subcutaneous angiotensin II infusion using osmotic pumps induces aortic aneurysms in mice.</a> Journal of Visualized Experiments. (103), e53191 (2015).</li><li>Wang, Y., Thatcher, S. E., Cassis, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+blood+pressure+using+a+noninvasive+tail+cuff+method+in+mice.">Measuring blood pressure using a noninvasive tail cuff method in mice.</a> Methods in Molecular Biology. 1614, 69-73 (2017).</li><li>Ishimwe, J. 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=The+gut+microbiota+and+short-chain+fatty+acids+profile+in+postural+orthostatic+tachycardia+syndrome.">The gut microbiota and short-chain fatty acids profile in postural orthostatic tachycardia syndrome.</a> Frontiers in Physiology. 13, 879012 (2022).</li><li>Bialkowska, A. B., Ghaleb, A. M., Nandan, M. O., Yang, V. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1,3-Butanediol+attenuates+hypertension+and+suppresses+kidney+injury+in+female+rats.">1,3-Butanediol attenuates hypertension and suppresses kidney injury in female rats.</a> American Journal of Physiology. Renal Physiology. 319 (1), 106-114 (2020).</li><li>Bokoliya, S. C., Dorsett, Y., Panier, H., Zhou, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedures+for+fecal+microbiota+transplantation+in+murine+microbiome+studies.">Procedures for fecal microbiota transplantation in murine microbiome studies.</a> Frontiers in Cellular and Infection Microbiology. 11, 711055 (2021).</li><li>Van Beusecum, J. P., Xiao, L., Barbaro, N. R., Patrick, D. M., Kirabo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+adoptive+transfer+of+high+salt+treated+antigen-presenting+dendritic+cells.">Isolation and adoptive transfer of high salt treated antigen-presenting dendritic cells.</a> Journal of Visualized Experiments. (145), e59124 (2019).</li><li>Harrison, D. G., Marvar, P. J., Titze, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+inflammatory+cells+in+hypertension.">Vascular inflammatory cells in hypertension.</a> Frontiers in Physiology. 3, 128 (2012).</li><li>Sylvester, M. 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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=Depletion+of+murine+intestinal+microbiota:+effects+on+gut+mucosa+and+epithelial+gene+expression.">Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression.</a> PLoS One. 6 (3), 17996 (2011).</li><li>Le Roy, T., 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=Comparative+evaluation+of+microbiota+engraftment+following+fecal+microbiota+transfer+in+mice+models:+age,+kinetic+and+microbial+status+matter.">Comparative evaluation of microbiota engraftment following fecal microbiota transfer in mice models: age, kinetic and microbial status matter.</a> Frontiers in Microbiology. 9, 3289 (2019).</li><li>Sun, 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=Fecal+microbiota+transplantation+alleviated+Alzheimer's+disease-like+pathogenesis+in+APP/PS1+transgenic+mice.">Fecal microbiota transplantation alleviated Alzheimer's disease-like pathogenesis in APP/PS1 transgenic mice.</a> Translation Psychiatry. 9 (1), 189 (2019).</li><li>Kim, 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=Critical+role+for+the+microbiota+in+CX(3)CR1(+)+intestinal+mononuclear+phagocyte+regulation+of+intestinal+T+cell+responses.">Critical role for the microbiota in CX(3)CR1(+) intestinal mononuclear phagocyte regulation of intestinal T cell responses.</a> Immunity. 49 (3), 151-163 (2018).</li><li>Hintze, K. 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A valuable approach to studying the causal role of gut microbiota in cardiovascular and metabolic disease is to transfer the total microbiota or select species of interest into germ-free mice. Here, we describe protocols to collect fecal samples from humans and conventionally housed mice into germ-free mice to study the role of gut microbiota in hypertensive disorders.
In mice, we use aseptically collected cecal contents processed in an aerobic chamber, and in humans, collect feces. FMT can be...

Cardiometabolic disorders, including heart disease and stroke, are the leading global causes of death1. Physical inactivity, poor nutrition, advancing age, and genetics modulate the pathophysiology of these disorders. Accumulating evidence supports the concept that gut microbiota affect cardiovascular and metabolic disorders, including type 2 diabetes2, obesity3, and hypertension4, which may hold a key to the development of new therapeutic approaches for these diseases.
The exact mechanisms by which the microbiota cause diseases are still unknown, and current studies are highly variable, in part due to methodological differences. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. FMT is widely used in animal studies to induce or suppress a phenotype. For example, caloric intake and glucose metabolism can be modulated by transferring fecal matter from a sick donor to a healthy recipient5,6. In humans, FMT has been shown to be a safe treatment option for patients with recurrent Clostridium difficile infection7. Evidence supporting its use in cardiovascular disease management is emerging; for instance, FMT from lean to metabolic syndrome patients improves insulin sensitivity8. Gut dysbiosis is also associated with high blood pressure in both human and rodent studies9,10,11. FMT from mice fed a high salt diet into germ-free mice predisposes the recipients to inflammation and hypertension12.
Despite the high rate of FMT success in rodents, translational challenges remain. Clinical trials using FMT to treat obesity and metabolic syndrome indicate minimal to no effects on these disorders13,14,15. Thus, more studies are needed to identify additional therapeutic avenues targeting the gut microbiota for the treatment of cardiometabolic disorders. Most of the available evidence on the gut microbiota and cardiovascular disease is associative. The described protocol discusses how to utilize a combination of FMT and the Swiss-rolling technique to show both an association between disease and gut microbiota and directly assess the integrity of all parts of the gut intestine16,17,18.
The overall goal of this method is to provide guidance for studying the effects of the gut microbiome in experimental cardiovascular disease. This protocol provides more details and key considerations in the experimental design to promote physiological translation and increase the rigor and reproducibility of the findings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alexa Fluor 488 Tyamide SuperBoost</td>
			<td>ThermoFisher</td>
			<td>B40932</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerobic chamber</td>
			<td>COY</td>
			<td>7150220</td>
			<td></td>
		</tr>
		<tr>
			<td>Apolipoprotein AI</td>
			<td>Novus Biologicals</td>
			<td>NBP2-52979</td>
			<td></td>
		</tr>
		<tr>
			<td>Artery Scissors - Ball Tip</td>
			<td>Fine Science Tools</td>
			<td>14086-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach solution</td>
			<td>Fisher Scientific</td>
			<td>14-412-53</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Fisher Scientific</td>
			<td>B14</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 antibody</td>
			<td>ThermoFisher</td>
			<td>&#160;14-0032-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD68 monoclonal antibody</td>
			<td>ThermoFisher</td>
			<td>14-0681-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Fisher Scientific</td>
			<td>75-004-221</td>
			<td></td>
		</tr>
		<tr>
			<td>CODA high throughput monitor</td>
			<td>Kent Scientic Corporation</td>
			<td>CODA-HT8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Fisher Scientific</td>
			<td>10-500-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable graduate transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>137119AM</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable syringes</td>
			<td>Fisher Scientific</td>
			<td>14-823-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>AA33361M1</td>
			<td></td>
		</tr>
		<tr>
			<td>Feeding Needle</td>
			<td>Fine Science Tools</td>
			<td>18061-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (30 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>NC0922459</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper sheet</td>
			<td>Fisher Scientific</td>
			<td>09-802</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin (10%)</td>
			<td>Fisher Scientific</td>
			<td>23-730-581</td>
			<td></td>
		</tr>
		<tr>
			<td>High salt diet</td>
			<td>Teklad</td>
			<td>TD.03142</td>
			<td></td>
		</tr>
		<tr>
			<td>OMNIgene.GUT</td>
			<td>DNAgenotek</td>
			<td>OM-200+ACP102</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmotic mini-pumps</td>
			<td>Alzet</td>
			<td>&#160;MODEL 2002</td>
			<td></td>
		</tr>
		<tr>
			<td>PAP Pen</td>
			<td>Millipore Sigma</td>
			<td>Z377821-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Fisher Scientific</td>
			<td>AS4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Fisher Scientific</td>
			<td>21-236-18C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Fisher Scientific</td>
			<td>14-388-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Serile Phosphate-buffered saline</td>
			<td>Fisher Scientific</td>
			<td>AAJ61196AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Smart spatula</td>
			<td>Fisher Scientific</td>
			<td>NC0133733</td>
			<td></td>
		</tr>
		<tr>
			<td>Stool collection device</td>
			<td>Fisher Scientific</td>
			<td>50-203-7255</td>
			<td></td>
		</tr>
		<tr>
			<td>TBS Buffer</td>
			<td>Fisher Scientific</td>
			<td>R017R.0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Millipore Sigma</td>
			<td> 
			9036-19-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Varimix platform rocker</td>
			<td>Fisher Scientific</td>
			<td>09047113Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Fisher Scientific</td>
			<td>02-215-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>1330-20-7, 100-41-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

murine fecal isolation and microbiota transplantation

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal microbiota transplantation (FMT) is a valuable approach to delineating a direct role of the total microbiota or isolated species in disease pathophysiology. It is a safe treatment option for patients with recurrent Clostridium difficile infection. Preclinical studies demonstrate that manipulating the gut microbiota is a useful tool to study the mechanistic link between dysbiosis and disease. Fecal microbiota transplantation may help elucidate novel gut microbiota-targeted therapeutics for the management and treatment of cardiometabolic disease. Despite a high success rate in rodents, there remains translational changes associated with the transplantation. The goal here is to provide guidance in studying the effects of gut microbiome in experimental cardiovascular disease. In this study, a detailed protocol for the collection, handling, processing, and transplantation of fecal microbiota in murine studies is described. The collection and processing steps are described for both human and rodent donors. Lastly, we describe using a combination of the Swiss-rolling and immunostaining techniques to assess gut-specific morphology and integrity changes in cardiovascular disease and related gut microbiota mechanisms.

The steps described above are summarized in Figure 1. Mouse cecal contents or human feces are resuspended in sterile saline to prepare a slurry to give to germ-free mice (100 &#181;L) by gavage, first for 3 consecutive days, then once every 3 days. At the end of the protocol, blood pressure is measured by the tail-cuff method, mice are euthanized, and tissues are harvested for assessment of changes in the gut microbiota and cardiovascular and metabolic changes.
A ...

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Murine Fecal Isolation and Microbiota Transplantation

The goal here is to outline a protocol to investigate the mechanisms of dysbiosis in cardiovascular disease. This paper discusses how to aseptically collect and transplant murine fecal samples, isolate intestines, and use the "Swiss-roll" method, followed by immunostaining techniques to interrogate changes in the gastrointestinal tract.

Gut microbiota dysbiosis plays a role in the pathophysiology of cardiovascular and metabolic disorders, but the mechanisms are not well understood. Fecal ...

Here we have described a protocol to guide others in investigating the mechanisms of dysbiosis in disease, from collecting fecal samples to using the Swiss roll method to study changes in the gut. To begin, spray the chest and sides of the euthanized mouse with 70%ethanol, and carefully open the skin and peritoneal cavity to expose the gastrointestinal tract. Isolate the cecum, and use sterile surgical scissors to cut it in half.Briefly expose the cecum, and cut 0.5 centimeters proximally from the ileum and 0.5 centimeters distally at its junction with the colon. Transfer the isolated cecum onto a sterile Petri dish. Use a sterile spatula to transfer the cecal content into sterile tubes, and store the aliquots in a 80 degrees Celsius freezer.Re-suspend fresh or previously frozen fecal pellets in sterile saline in a 1 to 20 proportion, and vortex until homogenized. Pass the homogenate through a 30-micrometer-pore nylon filter to remove large particulate matter. Centrifuge at 79G for five minutes, and collect the supernatant to use for transplantation.Oral gavage 100 microliters of the slurry per germ-free recipient mouse for three consecutive days, followed by gavage every three days for two weeks. Gently place the mouse in restraints on the preheated mouse holder, and leave the tail outside. Carefully tape down the top without pinching it so as not to stress the mouse.Allow the mouse to rest in the holder, and place it on the tail cuff machine platform for three to five minutes, covered by a sheet to acclimate. Collect at least three rounds of systolic pressure measurements using tail cuff plethysmography, and average the measurements from all the rounds for an average systolic pressure for each animal. Aseptically collect cecal contents from the euthanized mouse.To harvest the intestines and other tissues, locate the tissue in the mouse, and excise them with scissors to examine the role of the gut microbiota in cardiometabolic health. On day one, dissect the mouse gut from the anal side to the stomach side. And place the entirety of the isolated gastrointestinal tract in a Petri dish containing PBS.Gently pull the proximal end from the stomach end, and remove the surrounding fat and connective tissue by hand. Isolate the small intestine, and make a Z-type zigzag with each length. Then, cut to obtain the duodenum, jejunum, and ileum.Isolate the colon by cutting the section of the intestine below the cecum. Cut the duodenum, jejunum, ileum, and colon. Use a syringe and a needle with a ball tip to carefully flush and wash the gut inside with PBS without tearing the intestine.Place the gut on filter paper, and label the paper with the name of the section and then P at the top left corner for proximal or D for distal at the bottom left corner. Cut the gut longitudinally with ball-tip scissors. Open the gut on the filter paper, and wash with more PBS as needed.Sandwich the gut between two filter papers, and staple the filter papers at four points or corners near the gut. Soak in 10%formalin neutral buffer solution, and shake using a platform rocker at five rpm overnight at room temperature. On day two, heat the prepared 2%agarose in distilled water with a stir bar in a beaker covered with aluminum foil.To retrieve the tissues, strip the upper filter paper, and roll the gut from the proximal side so that the proximal side goes inside first, and roll inward so that the lumen is inside on the slide as well. Pin with a 30-gauge needle or two as needed. Aspirate one milliliter of agarose using disposable graduate transfer pipettes, and pour the agarose on a rolled gut section on a flat surface while avoiding air bubbles in the tissues.After the agarose cools down and solidifies, use a razor blade to trim the extra agarose around the tissue section. Finally, place the gut sections in tissue processing or embedding cassettes, and soak them in 70%ethanol at four degrees Celsius. The estimation of species biodiversity in cecal content obtained from mice on normal salt diets and high-salt diets is shown here.Changes in the gut microbiota in response to the high salt included a decrease in bacterial biodiversity. Non-metric multidimensional scaling shows that the bacteria from normal salt and high-salt-diet mice form separate clusters. High salt is associated with an increased Firmicutes to Bacteroidetes ratio.Fecal microbiota transplantation from high-salt-fed mice predisposes germ-free mice to angiotensin II-induced hypertension. Transferring high-salt-induced dysbiotic gut microbiota was associated with significantly increased systolic pressure in germ-free mice compared to mice that received normal salt gut microbiota. The representative images show apolipoprotein A-I stain in ilia obtained from mice that had hyperlipidemia without or with proteinuria.This protocol aligns steps to achieve successful FMT in key experimental considerations for both human and rodent donors.

Research

JoVE Journal

Immunology and Infection

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Murine Full-thickness Skin Transplantation

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An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

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