This work was supported, in part, by grants from the National Institutes of Health (R01 HL152683 and R21 AI126097 to Q. Li) and by American Heart Association Grant-in-Aid 17GRNT33460395 (to Q. Li) (heart.org).

All procedures performed on animals were approved by the Institutional Animal Care and Use Committee of the University of Texas Medical Branch at Galveston (Protocol # 1512071A).
1. Collection of fresh fecal pellets
<ol>
	<li>Prepare sterile paper towels, blunt-end forceps, and 50 mL conical tubes.
	<ol>
		<li>Place some paper towels and forceps in separate autoclave bags and autoclave them at 180 &#176;C in dry heat for 30 min. Use sterile conical tubes as well. Weigh the c...

<ol>
	<li>D'Argenio, V., Salvatore, 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+role+of+the+gut+microbiome+in+the+healthy+adult+status.">The role of the gut microbiome in the healthy adult status.</a> Clinica Chimica Acta. 451, 97-102 (2015).</li><li>Baquero, F., Nombela, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiome+as+a+human+organ.">The microbiome as a human organ.</a> Clinical Microbiology and Infection. 18, 2-4 (2012).</li><li>Hawrelak, J. A., Myers, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+causes+of+intestinal+dysbiosis:+a+review.">The causes of intestinal dysbiosis: a review.</a> Alternative Medcine Review. 9 (2), 180-197 (2004).</li><li>Carding, S., Verbeke, K., Vipond, D. T., Corfe, B. M., Owen, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysbiosis+of+the+gut+microbiota+in+disease.">Dysbiosis of the gut microbiota in disease.</a> Microbial Ecology in Health and Disease. 26 (1), 26191 (2015).</li><li>Ma, H. Q., Yu, T. T., Zhao, X. J., Zhang, Y., Zhang, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fecal+microbial+dysbiosis+in+Chinese+patients+with+inflammatory+bowel+disease.">Fecal microbial dysbiosis in Chinese patients with inflammatory bowel disease.</a> World Journal of Gastroenterology. 24 (13), 1464-1477 (2018).</li><li>Chu, 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=Specific+changes+of+enteric+mycobiota+and+virome+in+inflammatory+bowel+disease.">Specific changes of enteric mycobiota and virome in inflammatory bowel disease.</a> Journal of Digestive Diseases. 19 (1), 2-7 (2018).</li><li>Manichanh, C., Borruel, N., Casellas, F., Guarner, 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+gut+microbiota+in+IBD.">The gut microbiota in IBD.</a> Nature reviews Gastroenterology and Hepatology. 9 (10), 599-608 (2012).</li><li>Podolsky, D. 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S. <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:+Current+status+and+challenges+in+China.">Fecal microbiota transplantation: Current status and challenges in China.</a> JGH Open: An Open Access Journal of Gastroenterology and Hepatology. 2 (4), 114-116 (2018).</li><li>Markley, J. C., Carson, R. P., Holzer, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomembranous+enterocolitis:+A+clinico+pathologic+study+of+fourteen+cases+with+a+common+etiologic+factor.">Pseudomembranous enterocolitis: A clinico pathologic study of fourteen cases with a common etiologic factor.</a> AMA Archives of Surgery. 77 (3), 452-461 (1958).</li><li>Wilcox, M. 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J., Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+inflammatory+bowel+diseases.">Experimental models of inflammatory bowel diseases.</a> Cellular and Molecular Gastroenterology and Hepatology. 1 (2), 154-170 (2015).</li><li>Sikka, 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=Interleukin+10+knockout+frail+mice+develop+cardiac+and+vascular+dysfunction+with+increased+age.">Interleukin 10 knockout frail mice develop cardiac and vascular dysfunction with increased age.</a> Experimental Gerontology. 48 (2), 128-135 (2013).</li><li>Fecal microbiota transplantation-standardization study group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanjing+consensus+on+methodology+of+washed+microbiota+transplantation.">Nanjing consensus on methodology of washed microbiota transplantation.</a> Chinese Medical Journal (Engl). 133 (19), 2330-2332 (2020).</li><li>Kodani, 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=Flexible+colonoscopy+in+mice+to+evaluate+the+severity+of+colitis+and+colorectal+tumors+using+a+validated+endoscopic+scoring+system).">Flexible colonoscopy in mice to evaluate the severity of colitis and colorectal tumors using a validated endoscopic scoring system).</a> Journal of Visualized Experiments: JoVE. 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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=Suppression+of+tumorigenesis+in+the+Apc(min)+mouse:+down-regulation+of+beta-catenin+signaling+by+a+combination+of+tea+plus+sulindac.">Suppression of tumorigenesis in the Apc(min) mouse: down-regulation of beta-catenin signaling by a combination of tea plus sulindac.</a> Carcinogenesis. 24 (2), 263-267 (2003).</li><li>Kline, K. 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=Neonatal+injury+increases+gut+permeability+by+epigenetically+suppressing+E-Cadherin+in+adulthood.">Neonatal injury increases gut permeability by epigenetically suppressing E-Cadherin in adulthood.</a> The Journal of Immunology. 204 (4), 980-989 (2020).</li><li>DeGruttola, A. K., Low, D., Mizoguchi, A., Mizoguchi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+understanding+of+dysbiosis+in+disease+in+human+and+animal+models.">Current understanding of dysbiosis in disease in human and animal models.</a> Inflammatory Bowel Diseases. 22 (5), 1137-1150 (2016).</li><li>Chevalier, 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=Effect+of+gut+microbiota+on+depressive-like+behaviors+in+mice+is+mediated+by+the+endocannabinoid+system.">Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system.</a> Nature Communications. 11 (1), 6363 (2020).</li><li>Kao, 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=Effect+of+oral+capsule-+vs+colonoscopy-delivered+fecal+microbiota+transplantation+on+recurrent+clostridium+difficile+infection:+A+randomized+clinical+trial.">Effect of oral capsule- vs colonoscopy-delivered fecal microbiota transplantation on recurrent clostridium difficile infection: A randomized clinical trial.</a> JAMA. 318 (20), 1985-1993 (2017).</li><li>Cammarota, 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=International+consensus+conference+on+stool+banking+for+faecal+microbiota+transplantation+in+clinical+practice.">International consensus conference on stool banking for faecal microbiota transplantation in clinical practice.</a> Gut. 68 (12), 2111-2121 (2019).</li><li>Vemuri, R., 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+microgenderome+revealed:+sex+differences+in+bidirectional+interactions+between+the+microbiota,+hormones,+immunity+and+disease+susceptibility.">The microgenderome revealed: sex differences in bidirectional interactions between the microbiota, hormones, immunity and disease susceptibility.</a> Seminar Immunopathology. 41 (2), 265-275 (2019).</li><li>Wilkinson, N. M., Chen, H. -. C., Lechner, M. G., Su, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+immunity.">Sex differences in immunity.</a> Annual Review of Immunology. , (2022).</li><li>Lee, C. 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=Frozen+vs+fresh+fecal+microbiota+transplantation+and+clinical+resolution+of+diarrhea+in+patients+with+recurrent+clostridium+difficile+infection:+A+randomized+clinical+trial.">Frozen vs fresh fecal microbiota transplantation and clinical resolution of diarrhea in patients with recurrent clostridium difficile infection: A randomized clinical trial.</a> JAMA. 315 (2), 142-149 (2016).</li><li>Hamilton, M. J., Weingarden, A. R., Sadowsky, M. J., Khoruts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+frozen+preparation+for+transplantation+of+fecal+microbiota+for+recurrent+clostridium+difficile+infection.">Standardized frozen preparation for transplantation of fecal microbiota for recurrent clostridium difficile infection.</a> American Journal of Gastroenterology. 107 (5), 761-767 (2012).</li><li>Tang, G., Yin, W., Liu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+frozen+fecal+microbiota+transplantation+as+effective+as+fresh+fecal+microbiota+transplantation+in+patients+with+recurrent+or+refractory+Clostridium+difficile+infection:+A+meta-analysis.">Is frozen fecal microbiota transplantation as effective as fresh fecal microbiota transplantation in patients with recurrent or refractory Clostridium difficile infection: A meta-analysis.</a> Diagnostic Microbiology and Infectious Disease. 88 (4), 322-329 (2017).</li><li>Cui, B., 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+through+mid-gut+for+refractory+Crohn's+disease:+safety,+feasibility,+and+efficacy+trial+results.">Fecal microbiota transplantation through mid-gut for refractory Crohn's disease: safety, feasibility, and efficacy trial results.</a> Journal of Gastroenterology and Hepatology. 30 (1), 51-58 (2015).</li><li>Li, P., 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=Timing+for+the+second+fecal+microbiota+transplantation+to+maintain+the+long-term+benefit+from+the+first+treatment+for+Crohn's+disease.">Timing for the second fecal microbiota transplantation to maintain the long-term benefit from the first treatment for Crohn's disease.</a> Applied Microbiology and Biotechnology. 103 (1), 349-360 (2019).</li><li>Moayyedi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+fecal+microbiota+transplantation+in+patients+with+inflammatory+bowel+disease.">Update on fecal microbiota transplantation in patients with inflammatory bowel disease.</a> Gastroenterology and Hepatology. 14 (5), 319 (2018).</li><li>Saha, S., Mara, K., Pardi, D. S., Khanna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+safety+of+fecal+microbiota+transplantation+for+recurrent+clostridioides+difficile+infection.">Long-term safety of fecal microbiota transplantation for recurrent clostridioides difficile infection.</a> Gastroenterology. 160 (6), 1961-1969 (2021).</li><li>Perler, B. K., 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=Long-term+efficacy+and+safety+of+fecal+microbiota+transplantation+for+treatment+of+recurrent+clostridioides+difficile+infection.">Long-term efficacy and safety of fecal microbiota transplantation for treatment of recurrent clostridioides difficile infection.</a> Journal of Clinical Gastroenterology. 54 (8), 701-706 (2020).</li><li>Safety alert regarding use of fecal microbiota for transplantation and risk of serious adverse events likely due to transmission of pathogenic organisms. FDA Available from: <a target="_blank" href="https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-regarding-use-fecal-microbiota-tramsplantation-and-risk-serious-adverse-events-likely">https://www.fda.gov/vaccines-blood-biologics/safety-availability-biologics/safety-alert-regarding-use-fecal-microbiota-tramsplantation-and-risk-serious-adverse-events-likely</a> (2020)</li></ol>

As an innovative investigational treatment, FMT has become a hot topic in the treatment of various disorders in recent years since dysbiosis of the commensal microbiota is implicated in the pathogenesis of multiple human diseases, including IBD, obesity, diabetes mellitus, autism, heart disease, and cancer26. Although the mechanism has not been determined, it is believed that FMT works by building a new biological flora and preventing the loss of residual bacteria. The method presented herein adop...

The human intestinal micro-ecosystem is extremely complex, with more than 1000 species of bacteria in the intestine of a healthy person1. The intestinal flora is involved in maintaining the normal physiological functions of the intestine and the immune response and has an inseparable relationship with the human body. Accumulating evidence suggests that the intestinal microbiome constitutes the last human organ, which is part of the human body, not just a group of parasites2. A &#39;healthy&#39; symbiotic relationship between the gut microbiota, their metabolites, and the host immune system established in early life is critical to maintaining gut homeostasis. In some abnormal conditions such as chronic inflammation, changes in the internal and external environment of the body seriously disrupt the gut homeostasis, resulting in a persistent imbalance of the gut&#39;s microbial community, named dysbiosis3. In fact, exposure to multiple environmental factors, including diet, drugs, and pathogens, can lead to changes in the microbiota.
Dysbiosis is associated with the pathogenesis of a variety of intestinal diseases, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and pseudomembranous enteritis, as well as a growing list of extra-intestinal disorders, including cardiovascular disease, obesity, and allergy4. Microbiota profiling revealed that patients with IBD have a dramatic decrease in bacterial diversity, as well as marked alterations in the populations of some specific bacterial strains5,6. These studies demonstrated less Lachnospiraceae and Bacteroidetes but more Proteobacteria and Actinobacteria in IBD patients. It is believed that the pathogenesis of IBD is related to various pathogenic factors, including abnormal intestinal flora, dysregulated immune response, environmental challenges, and genetic variants7.Abundant evidence suggests that intestinal bacteria play a role in the initiation and application phases of IBD8,9, indicating that correcting gut dysbiosis may represent a novel approach for the therapy and/or maintenance treatment of IBD.
The prototype of fecal microbiota transplantation (FMT) began in ancient China10. In 1958, Dr. Eiseman and his colleagues successfully treated four cases of severe pseudomembranous enteritis with fecal matter from healthy donors via enema, opening a new chapter in modern Western medicine using human feces to treat human diseases11. Clostridium difficile infection (CDI) has been found to be the main cause of pseudomembranous enteritis12 and FMT is highly effective in the treatment of CDI. In the last eight years, FMT has become a standard-of-care therapy for the treatment of recurrent CDI13, prompting further studies investigating the role of FMT in other disorders, such as IBD. Over the past twenty years, numerous case reports and cohort studies have documented the use of FMT in patients with IBD14. A meta-analysis, including 12 trials, showed that 62% of patients with Crohn&#39;s disease (CD) achieved clinical remission after FMT, and 69% of CD patients had clinical response15. Despite these encouraging findings, the role of FMT in the management of IBD remains uncertain, and the mechanisms by which FMT ameliorates intestinal inflammation are poorly understood. Further investigation is necessary before FMT can join the current armamentarium of treatment options for IBD in the clinics.
In this protocol, we applied FMT on IL-10-/- mice, which develop colitis spontaneously after weaning and have served as a gold standard to mirror the multifactorial nature of IBD16,17,18.&#160;IL-10&#8722;/&#8722; mice have been extensively used to dissect IBD etiology because they present similar molecular and histological features to IBD patients, and, like patients, the disease can be ameliorated with anti-TNF&#945; therapy16. Aging IL10&#8722;/&#8722; mice (&#62;9 months of age) have an increased heart size and impaired cardiac function compared to age-matched wild-type mice19, making it an excellent model for studying colitis-induced heart diseases. However, other murine models of colitis, such as the dextran sodium sulfate model and the T-cell-induced colitis model, can be used as well. We administered fecal suspension via oral gavage, proven to be an effective and better route than enema in humans20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>BD Syringe, 1 mL</td>
			<td>Fisher Scientific</td>
			<td>14-829-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end forceps</td>
			<td>Knipex</td>
			<td>926443</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain natriuretic peptide EIA kit</td>
			<td>Sigma</td>
			<td>RAB0386</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Lab</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tubes</td>
			<td>ThermoFisher</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved feeding Needles</td>
			<td>Kent Scientific</td>
			<td>FNC-20-1.5-2</td>
			<td></td>
		</tr>
		<tr>
			<td>GLH-115 homogenizer</td>
			<td>Omni International</td>
			<td>GLH-115</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>MilliporeSigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-10 knockout mice</td>
			<td>Jackson Lab</td>
			<td>004366</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Critical care</td>
			<td>NDC66794-017-10</td>
			<td></td>
		</tr>
		<tr>
			<td>USP normal saline</td>
			<td>Grainger</td>
			<td>6280</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>Euthanex Corp.</td>
			<td>EZ-108SA</td>
			<td></td>
		</tr>
	</tbody>
</table>

therapeutic evaluation of fecal microbiota transplantation in an interleukin 10-deficient mouse model

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted considerable attention. Accumulating evidence suggests that dysbiotic microbiota plays an active role in triggering or worsening the inflammatory process in IBD and that fecal microbiota transplantation (FMT) is an attractive therapeutic strategy since transferring a healthy microbiota to IBD patient could restore the appropriate host-microbiota communication. However, the molecular mechanisms are unclear, and the efficacy of FMT has not been very well established. Thus, further studies in animal models of IBD are necessary. In this method, we applied FMT from wild-type C57BL/6J mice to IL-10 deficient mice, a widely used mouse model of colitis. The study elaborates on collecting fecal pellets from the donor mice, making the fecal solution/suspension, administering the fecal solution, and monitoring the disease. We found that FMT significantly mitigated the cardiac impairment in IL-10 knockout mice, underlining its therapeutic potential for IBD management.

We performed healthy donor FMT 3 times (once a month for 3 months) on 2-month old C57BL/6J wild type (WT) and IL-10 knockout mice. Age-matched C57BL/6J mice (age difference should be &#60;2 months) served as the fecal donors and fresh fecal pellets were used each time. EIA assays revealed that BNP was markedly elevated in the plasma of IL-10-deficiency mice and that healthy donor FMT significantly mitigated the increase in BNP levels (Figure 1A, n = 5, p &#60; 0.05). Echocardiograph...

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Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model

The interaction of genetic susceptibility, mucosal immunity, and intestinal microecological environment is involved in the pathogenesis of inflammatory bowel disease (IBD). In this study, we applied fecal microbiota transplantation to IL-10 deficient mice and investigated its impact on colonic inflammation and heart function.

With the development of microecology in recent years, the relationship between intestinal bacteria and inflammatory bowel disease (IBD) has attracted ...

Three clinical experiments suggested that fecal microbiota transplantation carries the therapeutic potential for a number of human diseases. Further mechanistic studies will help bring FMT from bench to bedside. This method adopts oral gavage for the delivery route, allowing more precise volume delivery and the fast absorption.It requires only moderate technical skills. Demonstrating the procedure will be Xioying Zhong, a senior research associate from our laboratory. Begin by preparing sterile paper towels, blunt end forceps, and 50 milliliter conical tubes.Place some paper towels and forceps in separate autoclave bags and autoclave them at 180 degrees Celsius in dry heat for 30 minutes. Weigh the sterile conical tubes and write down their weight on the tubes. Turn on the biosafety cabinet in the animal room.Take an autoclaved clean mouse cage without any bedding and place it in the biosafety cabinet. Remove the cover and the food rack and place them inside the cabinet. Place some sterile paper towels on the bottom of the cage, and place the metal rack back on top of the cage.Identify age matched fecal donors and place the mouse cage in the biosafety cabinet. Open the cage and gently grab a donor mouse by the tail and place it on the metal rack on top of the clean cage. Place the cage cover on top of the rack and wait for the animal to defecate.Collect the fecal pellets and put them in a sterile 50 milliliter conical tube. Pull the pellets by sex. Weigh the tube again and calculate the weight of the fecal pellets.Prepare a sterile solution by adding 10%glycerol solution to normal saline. Add 10 milliliters of this solution to the conical tube for each gram of fecal pellets. Homogenize this mixture at a low speed three times for 30 seconds using a benchtop homogenizer inside a fume hood to resuspend the feces.Filter the fecal suspension through two layers of sterile cotton gauze. Store the filtrate temporarily in a refrigerator for up to six hours, or package it into sterile cryogenic vials and store it in a minus 80 degree freezer. Thoroughly clean the homogenizer or blender following a standard procedure.Thaw the frozen fecal suspension on ice if using frozen samples. Mix the thawed fecal suspension by vortexing. Transfer the fresh or thaw fecal suspension to one milliliter syringes.Weigh the mice and choose the right gavage needle size and maximum dosage volume. Test the gavage needle by measuring the length from the tip of the mouse's nose to the bottom of the sternum. Fill the syringe with 10%glycerol in saline or fecal suspension and remove air bubbles inside the syringe and the needle.Place one mouse cage in the biosafety cabinet. Remove the plastic cage cover and leave the metal rack in place. Open the metal rack with one hand.Grab one mouse by the tail and put it on the metal rack. Hold the mouse by the tail with one hand and use the thumb and middle fingers of another hand to restrain the animal by grasping the skin over the shoulders. Gently extend the animal's head backward and hold the head in place with one hand.Place the gavage needle on top of the tongue inside the mouth. Gently advance along the upper pallet until the needle reaches the esophagus. Pass the needle smoothly in one motion.Once the needle is properly placed and verified, slowly administer the material by pushing the syringe attached to the needle. After dosing, gently pull the needle out. Mark the mouse and return it to its home cage.Monitor the animal for five to 10 minutes by looking for signs of labored breathing or distress. Monitor the mice again between 12 to 24 hours after the fecal microbiota transplantation. Enzyme immunoassay revealed that B type natriuretic peptide was markedly elevated in the plasma of interleukin 10 deficient mice.A healthy donor fecal microbiota transplantation significantly mitigated the increase in B type natriuretic peptide levels. Echocardiography detected a significant decrease in the left ventricle ejection fraction in the interleukin 10 deficient mice compared to the wild type mice. The decrease was significantly abrogated by fecal microbiota transplantation.These findings suggest that healthy donor fecal microbiota transplantation mitigated colitis induced cardiac impairment. When attempting the protocol, choosing the right gavage needle is essential. Flexible plastic feeding tubes could be a better choice for beginners.It is also important to practice animal handling before gavage.

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Research

JoVE Journal

Immunology and Infection

2.5K Views.  University of Texas Medical Branch at Galveston. Three clinical experiments suggested that fecal microbiota transplantation carries the therapeutic potential for a number of human diseases. Further mechanistic studies will help bring FMT from bench to bedside. This method adopts oral gavage for the delivery route, allowing more precise volume delivery and the fast absorption.It requires only moderate technical skills. Demonstrating the procedure will be Xioying Zhong, a senior research associate from our laboratory. Begin by preparin...