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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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 conical tubes and write down their weight on the tubes.</li>
	</ol>
	</li>
	<li>Turn on the biosafety cabinet in the animal room.</li>
	<li>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.</li>
	<li>Place some sterile paper towels on the bottom of the cage and place the metal rack back on top of the cage.</li>
	<li>Identify age-matched fecal donors and place the mouse cage in the biosafety cabinet. Open the cage and gently grab a donor mouse (C57BL/6J) by the tail and place it on the metal rack on top of the clean cage.</li>
	<li>Place the cage cover on top of the rack and wait for the animal(s) to defecate. 
	NOTE: Put a few mouse littermates on the rack simultaneously.</li>
	<li>Collect the fecal pellets and put them in a sterile 50 mL conical tube. Pool the pellets by sex. Do not mix fecal pellets collected from males and females.</li>
	<li>Weight the tube again and calculate the weight of the fecal pellets.</li>
</ol>
2. Preparation of fecal suspension
<ol>
	<li>Prepare a sterile solution (10% glycerol in normal saline).</li>
	<li>Add 10 mL of 10% glycerol/normal saline to the conical tube for each gram of fecal pellets. 
	NOTE: Increase the solution volume to 20 mL if necessary. This study used 1 mL of solution for each pellet (5-10 mg) as well.</li>
	<li>Homogenize the mixture at a low speed with a benchtop homogenizer or a blender inside a fume hood to resuspend the feces (3 X 30 s).</li>
	<li>Filter the fecal suspension through 2 layers of sterile cotton gauze (10.2 cm x 10.2 cm). Store the filtrate temporarily in a refrigerator for up to 6 h or package it into sterile cryogenic vials and store it in a -80 &#176;C freezer.</li>
	<li>Thoroughly clean the homogenizer or blender following a standard procedure.</li>
</ol>
3. Administration of fecal suspension by oral gavage
<ol>
	<li>Thaw the frozen fecal suspension on ice if using frozen samples. Mix the thawed fecal suspension by vortexing.</li>
	<li>Transfer the fresh or thawed fecal suspension to 1 mL syringes. 
	NOTE: Each mouse will receive a total of 200 &#181;L of fecal suspension, and each IL-10-/-&#160;mouse in the control group will get 200 &#181;L of 10% glycerol/normal saline.</li>
	<li>Weigh the mice and choose the right gavage needle size and maximum dosage volume. 
	NOTE: For mice with a bodyweight between 20-25 grams, use a 20 G 3.81 cm curved gavage needle with a 2.25 mm ball. Please check gavageneedle.com for more information.</li>
	<li>Test the gavage needle by measuring the length from the tip of the mouse&#39;s nose to the xiphoid process (bottom of the sternum). Fill the syringe with 10% glycerol/saline or fecal suspension and remove air bubbles inside the syringe and the needle.&#160;&#160;&#160;&#160; 
	NOTE: If the needle is longer than the length, put a mark on the needle shaft/tubing at the level of the nose. Do not pass the needle/tubing through the animal past that point to prevent gastric perforation.</li>
	<li>Place one mouse cage in the biosafety cabinet, remove the plastic cage cover, and leave the metal rack in place.</li>
	<li>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. This way, the forelegs are stretched out to the side, which will prevent the front feet from pushing the needle out. Gently extend the animal&#39;s head backward and hold the head in place with one hand.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Practice mouse handling until the experimenter has full confidence before proceeding to the experiment.</li>
	<li>Place the gavage needle on top of the tongue inside the mouth. Gently advance along the upper palate until the needle reaches the esophagus. Pass the needle smoothly in one motion. Do not force the needle if any resistance is felt. Take the needle out and try again.</li>
	<li>Once the needle is properly placed and verified, slowly administer the material by pushing the syringe attached to the needle. Do not rotate the needle or push the needle forward, which may rupture the esophagus. After dosing, gently pull the needle out.</li>
	<li>Return the mouse to its home cage. Monitor the animal for 5-10 min by looking for signs of labored breathing or distress. Monitor the mice again between 12-24 h after the FMT.</li>
</ol>

<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>C57BL/6J mice</td>
			<td>Jackson Lab</td>
			<td>664</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>Glycerol</td>
			<td>MilliporeSigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-10 knockout mice</td>
			<td>Jackson Lab</td>
			<td>4366</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>

evaluating the effects of fecal microbiota transplantation in an interleukin-10 deficient mouse

Source: Xiao, Y. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63350">Therapeutic Evaluation of Fecal Microbiota Transplantation in an Interleukin 10-Deficient Mouse Model.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a procedure of fecal microbiota transplantation in an interleukin-10 deficient mouse with colon inflammation. Colonized fecal microbes in the gut release secondary metabolites, eliciting anti-inflammatory cytokine release and reducing colon inflammation.

Evaluating the Effects of Fecal Microbiota Transplantation in an Interleukin-10 Deficient Mouse

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Begin with a restrained mouse lacking interleukin-10, an anti-inflammatory cytokine, which results in colon inflammation.
Take a syringe filled with a fecal suspension rich in diverse gut microbes, from a healthy mouse.
Next, attach an oral tube to the syringe.
Insert this tube into the mouth, guiding it downward to the stomach of the mouse.
Now, administer the fecal suspension directly into the stomach, allowing it to gradually reach the colon.
Once in the colon, the fecal microbes multiply and colonize, reestablishing the gut microbes.
The colonized microbes then release various secondary metabolites.
These metabolites help in mucin production, which repairs the colon epithelium and restores the gut integrity.
Within the colon epithelium, the fecal microbes and their metabolites interact with immune cells.
This interaction causes the immune cells to release anti-inflammatory cytokines, leading to a reduction in colon inflammation in the interleukin-10 deficient mouse.

evaluating-effects-fecal-microbiota-transplantation-an-interleukin-10

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

44 Views. Źródło: Xiao, Y. et al., Terapeutyczna ocena przeszczepu mikrobioty kałowej w modelu myszy z niedoborem interleukiny 10.J. Vis. Exp. (2022) Ten film pokazuje procedurę przeszczepu mikrobioty kałowej u myszy z niedoborem interleukiny-10 z zapaleniem jelita grubego. Skolonizowane drobnoustroje kałowe w jelitach uwalniają metabolity wtórne, wywołując uwalnianie cytokin przeciwzapalnych i zmniejszając stan zapalny jelita grubego. 

Video: Ocena skutków przeszczepu mikrobioty kałowej u myszy z niedoborem interleukiny-10 - Concept

Fecal (micro) RNA Isolation

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs from fecal samples of animal and human subjects. The aim is to isolate total RNA with high purity and quantity for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The advantages of this optimized protocol in the miRNA isolation are capabilities of isolating highly purified RNA products with additional washing steps described, increased quantity of RNA obtained with an improved method in the resuspension of sample, and important tips of decontamination. One limitation is the inability to process and purify larger sample of more than 200 mg as these sample sizes would cause a difficulty in the clear formation of the interphase. Consequently, the large sample size may contaminate the aqueous phase to be extracted as described in the protocol with organic matters that affect the quality of RNA isolated in the end. However, RNA isolates from a sample of up to 200 mg are sufficient for most of downstream analyses.

Fecal micro RNA Isolation

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs ...

JoVE Journal

Immunology and Infection

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

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 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 ...

Evaluating the Effects of Fecal Microbiota Transplantation in an Interleukin-10 Deficient Mouse

Transkrypcja

Evaluating the Effects of Fecal Microbiota Transplantation in an Interleukin-10 Deficient Mouse