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 samples at 4 &#176;C within 36 h of collection until ready for processing. Alternatively, collect stool samples using a commercially available tool for easy and extended DNA stability at ambient temperature, especially for at-home use.</li>
	<li>Sanitize a biosafety-level fume hood with a 10% bleach solution, or other Environmental Protection Agency-approved disinfectant.</li>
	<li>Remove the stool from cool storage and bring into the fume hood; use a disposable spatula to make ~1 g aliquots and store in a -80 &#176;C freezer until completely ready for processing.</li>
	<li>Discard all disposable items in biohazard trash. Disinfect all surfaces (hood and any surfaces touched by the processor) and items being removed from the hood.</li>
</ol>
2. Aseptic collection of mouse fecal samples
NOTE: Use aseptic techniques, including sterilized instruments.
<ol>
	<li>Euthanize the mouse by CO2 asphyxiation. Spray the chest and sides of the mouse with 70% ethanol and carefully open the skin and peritoneal cavity to expose the gastrointestinal tract.</li>
	<li>Isolate the cecum and use sterile surgical scissors to cut it in half. Briefly, expose the cecum and cut 0.5 cm proximally from the ileum and 0.5 cm distally at its junction with the colon. Transfer the isolated cecum onto a sterile Petri dish.</li>
	<li>Use a sterile spatula to transfer cecal content into sterile tubes, and store aliquots in a -80 &#176;C freezer19. 
	&#8203;NOTE: Since the majority of bacteria in the gut are anaerobes, exposure to oxygen may damage or kill the organisms during the isolation procedure in a room atmosphere. Thus, fecal samples should be isolated in anaerobic chambers to maintain the viability of the bacteria.</li>
</ol>
3. Fecal matter transplantation
<ol>
	<li>Resuspend fresh or previously frozen fecal pellets in sterile saline in a 1:20 (w:v) proportion and vortex until homogenized.</li>
	<li>Pass the homogenate through a 30 &#181;m pore nylon filter to remove large particulate matter. Centrifuge at 79 &#215; g for 5 min and collect the supernatant to use for transplantation.</li>
	<li>Oral gavage 100 &#181;L of the slurry per germ-free recipient mouse for 3 consecutive days, followed by gavage every 3 days for 2 weeks. Use conventional mice to study mechanisms of the gut microbiota if they have been first treated with antibiotics to eliminate the recipient&#39;s own endemic microbiota. For example, administer ceftriaxone (400 mg/kg) daily to the recipient mice for 5 consecutive days by oral gavage before gavage of the fecal slurry. 
	NOTE: Studies suggest that a minimum of 2 weeks of this treatment is necessary to elicit cardiovascular changes, including blood pressure20.</li>
	<li>Ensure that germ-free recipient mice are single-housed in gnotobiotic film isolators and fed with sterile food and water.</li>
</ol>
4. Systolic blood pressure measurements
NOTE: Gnotobiotic mice that received FMT from conventionally housed 3-month-old C57Bl/6 mice were implanted with osmotic minipumps (Alzet, model 2002) for infusion of low-dose angiotensin II (140 ng/kg/min) for 2 weeks. Blood pressure was monitored weekly via tail cuff. The protocol for implanting osmotic minipumps has been previously reported21. Tail-cuff was performed as briefly summarized below. A noninvasive method of measuring blood pressure, such as tail cuff, is suitable for FMT studies in gnotobiotic mice. The detailed steps on how to perform tail cuff have been described previously22.
<ol>
	<li>Briefly, retrieve the mice from the gnotobiotic isolators and preheat the tail-cuff machine platform and mouse holder.</li>
	<li>Place the conscious mice in restraints on the heated platform and collect at least three rounds of systolic pressure measurements using tail cuff plethysmography. Perform the following steps below on 3 consecutive days prior to the proper measurement days, to train mice to being restrained in order to reduce stress.
	<ol>
		<li>Gently place the mouse in the preheated holder and leave the tail outside. Carefully tape down the top without pinching it, so as not to stress the mouse.</li>
		<li>Allow the mouse to rest in the holder; place on the platform for 3-5 min covered by a sheet to acclimate.</li>
	</ol>
	</li>
	<li>Average the measurements from all the rounds for an average systolic pressure for each animal.</li>
</ol>
5. Assessment of FMT to cardiovascular changes
<ol>
	<li>Following blood pressure measurement, euthanize the mice and aseptically collect cecal contents, as described in section 2 .</li>
	<li>Harvest the intestines and other tissues, including the heart, aorta, liver, mesenteric arteries, and kidneys, to examine the role of the gut microbiota in cardiometabolic health. To harvest tissues, locate the tissue in the mouse and use scissors to excise them.</li>
	<li>Run a metagenomic sequencing analysis on fecal samples/cecal contents collected from the donor and recipient mice to confirm engraftment of the gut microbiota after FMT23. The first proof of successful colonization of microbiota is confirming that the donor and recipient&#39;s microbiota are similar.</li>
	<li>Use the Swiss-roll technique (see section 6) on the harvested intestinal tissue, coupled with immunostaining and histology to examine morphological and cellular expression changes24.</li>
</ol>
6. Making gut intestine Swiss-rolls
<ol>
	<li>Day 1
	<ol>
		<li>In a properly euthanized mouse sprayed with 70% ethanol, dissect the mouse gut from the anal side (fixed in retroperitoneum) to the stomach side. Place the entirety of the isolated gastrointestinal tract in a Petri dish containing phosphate-buffered saline (PBS). Gently hold the proximal end from the stomach end and remove the surrounding fat and connective tissue by hand.</li>
		<li>Isolate the small intestine (cephalad from the appendix) and make a Z-type zigzag with each length. Then, cut to obtain the duodenum, jejunum, and ileum successively, as previously described25. Isolate the colon by cutting the section of the intestine below the cecum.</li>
		<li>Cut the duodenum, jejunum, ileum, and colon.</li>
		<li>Flush and wash the gut inside using PBS with a syringe and a needle with a ball tip, so as not to tear the intestine.</li>
		<li>Put the gut on filter paper. Label the paper with the name of the section (e.g., duodenum) and then &#39;P&#39; at the right top corner for proximal or &#39;D&#39; for distal at the right bottom corner.</li>
		<li>Cut the gut longitudinally with ball-tip scissors. Open the gut on the filter paper. Wash with more PBS as needed.</li>
		<li>Sandwich the gut between two filter papers. Staple the filter papers at four points/corners near the gut.</li>
		<li>Soak in 10% formalin neutral buffer solution (4.0 g of sodium phosphate, monobasic, 6.5 g of sodium phosphate, dibasic, 100 mL of 37% formaldehyde, 900 mL of distilled water). Shake using a platform rocker at 5 rpm at room temperature overnight.</li>
	</ol>
	</li>
	<li>Day 2
	<ol>
		<li>Prepare 2% agarose in distilled water and heat with a stir bar in a beaker covered with aluminum foil.</li>
		<li>Retrieve the tissues; strip the upper filter paper. 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 G needle or two, as needed.</li>
		<li>Aspirate 1 mL 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.</li>
		<li>Allow the agarose to cool and solidify. Use a razor blade to trim the extra agarose around the tissue section.</li>
		<li>Put the gut sections in tissue processing/embedding cassettes (bigger than the regular ones to accommodate the increased height due to agarose). Soak in 70% ethanol at 4 &#176;C.</li>
		<li>Prepare paraffin-embedded tissue slides and proceed to immunostaining, as outlined below.</li>
	</ol>
	</li>
</ol>
7. Immunostaining of the gut intestinal tract
<ol>
	<li>Deparaffinization
	<ol>
		<li>Pass through the following baths with slides in a rack: xylene for 3 min, fresh xylene again for 3 min, xylene with 100% ethanol (1:1) for 3 min, 95% ethanol for 3 min, 70% ethanol for 3 min, and 50% ethanol for 3 min.</li>
		<li>Rinse gently with cold running tap water. Store in a bath of tap water.</li>
	</ol>
	</li>
	<li>Antigen retrieval
	<ol>
		<li>Following deparaffinization, boil the slides in a rack in a bath of antigen retrieval buffer (0.01M trisodium citrate dihydrate at pH 6 and 0.05% Tween-20) at 100 &#176;C for 20 min.</li>
		<li>Run under cold tap water.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Remove the slides from the bath and place the tissue face up in a slide box with wet laboratory wipes/paper towels in the bottom. Draw an outline around the tissue with a hydrophobic marker pen.</li>
		<li>Drop Tris-buffered saline (TBS) + 0.025% Triton X-100 onto the tissue and incubate for 5 min. Repeat this step.</li>
		<li>Block with TBS + 10% fetal bovine serum (FBS) + 1% bovine serum albumin (BSA) for 2 h at room temperature. Turn the slides on their side and remove the blocking buffer on a laboratory wipe.</li>
		<li>Add primary antibody solution and incubate at 4 &#176;C for at least 2 h or overnight. Wash gently with TBS + 0.025% Triton X-100 by gently pipetting ~200 &#181;L over the section.</li>
		<li>Add secondary antibody solution and incubate for 1 h at room temperature. Rinse the slides 3 x 5 min with TBS by rinsing with a pipette, as in step 7.3.4.</li>
		<li>Mount with mounting medium and a coverslip.</li>
	</ol>
	</li>
</ol>

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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=Broad+scope+method+for+creating+humanized+animal+models+for+animal+health+and+disease+research+through+antibiotic+treatment+and+human+fecal+transfer.">Broad scope method for creating humanized animal models for animal health and disease research through antibiotic treatment and human fecal transfer.</a> Gut Microbes. 5 (2), 183-191 (2014).</li><li>Wilde, 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=Tail-cuff+technique+and+its+influence+on+central+blood+pressure+in+the+mouse.">Tail-cuff technique and its influence on central blood pressure in the mouse.</a> Journal of the American Heart Association. 6 (6), 005204 (2017).</li><li>Liu, X., 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-fiber+diet+mitigates+maternal+obesity-induced+cognitive+and+social+dysfunction+in+the+offspring+via+gut-brain+axis.">High-fiber diet mitigates maternal obesity-induced cognitive and social dysfunction in the offspring via gut-brain axis.</a> Cell Metabolism. 33 (5), 923-938 (2021).</li></ol>

No conflicts of interest, financial or otherwise, are declared by the authors.

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><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>&amp;nbsp;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 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>&amp;nbsp;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>&lt;br/&gt; 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 µL) 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 key ...

Watch this Scientific Journal Video about Murine Fecal Isolation and Microbiota Transplantation at JoVE.com

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 ...

murine-fecal-isolation-and-microbiota-transplantation

Research

JoVE Journal

Immunology and Infection

3.9K Views.  Vanderbilt University. 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. 

Video: Murine Fecal Isolation and Microbiota Transplantation

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Isolation, Processing and Analysis of Murine Gingival Cells

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique and important tissue to study immune mechanisms because it is involved in host immune response against oral biofilm that might cause periodontal diseases. Furthermore, the close proximity of the gingiva to alveolar bone tissue enables also studying bone remodeling under inflammatory conditions. Our method yields large amount of immune cells that allows analysis of even rare cell populations such as Langerhans cells and T regulatory cells as we demonstrated previously 1. Employing mice to study local immune responses involved in alveolar bone loss during periodontal diseases is advantageous because of the availability of various immunological and experimental tools. Nevertheless, due to their small size and the relatively inconvenient access to the murine gingiva, many studies avoided examination of this critical tissue. The method described in this work could facilitate gingival analysis, which hopefully will increase our understating on the oral immune system and its role during periodontal diseases.

This study describes an efficient technique to isolate and process gingival tissues from the mouse oral cavity in order to produce a single-cell culture. The resulting cells can be further used for flow cytometry analysis and molecular studies.

We have developed a technique to precisely isolate and process murine gingival tissue for flow cytometry and molecular studies. The gingiva is a unique ...

A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be used to model diseases that are otherwise difficult or impossible to study in humans or other animal models. The gut microbiome can have a profound impact on human health and disease. However, the murine gut microbiome is very different than the one found in humans. &#160;There is a need for improved pre-clinical hu-mice models that have an engrafted human gut microbiome. Therefore, we created double hu-mice that feature both a human immune system and stable human-like gut microbiome. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are one of the best animals for humanization due to their high level of immunodeficiency. However, germ-free NSG mice, and various other important germ-free mice models are not currently commercially available. Further, many research settings do not have access to gnotobiotic facilities, and working under gnotobiotic conditions can often be expensive and time consuming. Importantly, germ-free mice have several immune deficiencies that exist even after the engraftment of microbes. Therefore, we developed a protocol that does not require germ-free animals or gnotobiotic facilities. To generate double hu-mice, NSG mice were treated with radiation prior to surgery to create bone-marrow, liver, thymus-humanized (hu-BLT) mice. The mice were then treated with broad spectrum antibiotics to deplete the pre-existing murine gut microbiome. After antibiotic treatment, the mice were given fecal transplants with healthy human donor samples via oral gavage. Double hu-BLT mice had unique 16S rRNA gene profiles based on the individual human donor sample that was transplanted. Importantly, the transplanted human-like microbiome was stable in the double hu-BLT mice for the duration of the study up to 14.5 weeks post-transplant.

We describe a novel method for generating double humanized BLT-mice that feature a functional human immune system and a stable engrafted human-like gut microbiome. This protocol can be followed without the need for germ-free mice or gnotobiotic facilities.

Humanized mice (hu-mice) that feature a functional human immune system have fundamentally changed the study of human pathogens and disease. They can be ...

Biochemistry

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

Fecal Microbiota Transplantation (FMT) is a safe and highly effective treatment for recurrent and refractory C. difficile infection (CDI). Various methods of FMT administration have been reported in the literature including nasogastric tube, upper endoscopy, enema and colonoscopy. FMT via colonoscopy yields excellent cure rates and is also well tolerated. We have found that patients find this an acceptable and tolerable mode of delivery. At our Center, we have initiated a fecal transplant program for patients with recurrent or refractory CDI. We have developed a protocol using an iterative process of revision and have performed 24 fecal transplants on 22 patients with success rates comparable to the current published literature. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool maximizes therapeutic success. Here we detail each step of the FMT protocol that can be carried out at any endoscopy center with a high degree of safety and success.

Fecal Microbiota Transplantation via Colonoscopy for Recurrent C. difficile Infection

Fecal Microbiota Transplantation via colonoscopy is a safe and effective treatment for recurrent and refractory C.difficile infection. A systematic approach to patient and donor screening, preparation of stool, and delivery of the stool during the colonoscopy will maximize therapeutic success.

Fecal Microbiota Transplantation (FMT) is a safe and highly effective treatment for recurrent and refractory C. difficile infection (CDI). Various methods ...

Medicine

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.

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 ...

Preparation and Maintenance of Bioexclusion IsoPositive Cage Experiment for Human Fecal Transplantation into Germ-Free Mice

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of the specific role of individuals, defined or complex groups of microorganisms in host response. Traditionally bred and reared in flexible-film or semi-rigid isolators, germ-free mouse husbandry and experimental manipulation are costly and require numerous trained staff and a large space footprint in animal housing facilities. The IsoPositive caging system allows for experimental manipulation of germ-free mice in individual, hermetically-sealed, positive-pressure isolator cages (isocages), reducing cost and enabling greater flexibility in experimental manipulations. 
Here, a protocol is described for transferring germ-free mice from breeding isolators to isocages and subsequent fecal transfer from human donor stool into mice to create stable long-term gut "humanized" mice for future studies. The materials and preparation needed for the utilization of the isocage system are described, including the use of chlorine-dioxide sterilant chemical sterilant to clean cages, supplies, equipment, and personal protective equipment. The methods for confirming the germ-free status of transferred mice and how to determine contamination in the caging system are discussed. The procedure for husbandry, including bedding, food, and water supply, is further discussed. The protocol for human fecal slurry preparation and gavage into germ-free mice to create gut "humanized" mice, along with stool collection to monitor the microbial community composition of these mice, are described. An experiment illustrates that two weeks post-human fecal transplant allows for stable colonization of donor microbiota in the murine hosts, enabling subsequent experimental usage. Furthermore, the collection of humanized mouse feces in viability preservation media, enabling use in further functional experiments, is described. Overall, these methods allow for the safe and effective establishment of humanized mouse communities in experimental gnotobiotic cages for further manipulation.

This protocol describes the best practices for germ-free mouse transfer to and housing in experimental single-cage isolators (isocages) while maintaining sterile conditions. Methods for fecal transplant into germ-free mice and the collection of viable bacteria from these gut "humanized" mice for further applications are discussed.

Germ-free mice are an important investigation tool for understanding the contribution of microorganisms in host health and disease, enabling assessment of ...

Fecal Microbiota Transplantation and Detection of Prevalence of IgA-Coated Bacteria in the Gut

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the biological function of intestinal bacteria as a whole or at the species level. Several different FMT methods have been published. Here, we present an FMT protocol that successfully depletes gut microbiota in a matter of days, followed by transplantation of fecal microbiota from fresh or frozen donor intestinal contents to conventional mice. Real time-PCR is applied to test the efficacy of bacterial depletion. Sequencing of the 16S ribosomal RNA (rRNA) is then applied to test the relative abundance and identity of gut microbiota in recipient mice. We also present a flow cytometry-based detection method of immunoglobulin A (IgA)-coated bacteria in the gut.

We established an efficient way to deplete intestinal bacteria in three days, and subsequently transplant fecal microbiota via gavage of fecal fluid prepared from fresh or frozen intestinal contents in mice. We also present an optimized method to detect the frequency of IgA-coated bacteria in the gut.

Gut microbiota exert pleiotropic roles in human health and disease. Fecal microbiota transplantation (FMT) is an effective method to investigate the ...

Biology

Generating a Double Humanized BLT (Bone-Marrow, Liver, Thymus) Mouse Model

Source: Daharsh, L. et al., <a href="https://app.jove.com/v/59773">A Double Humanized BLT-mice Model Featuring a Stable Human-Like Gut Microbiome and Human Immune System.</a>&#160;J. Vis. Exp. (2019)
This video demonstrates the method for generating a double humanized bone BLT (marrow, liver, and thymus) mouse with a functional human immune system and a stable engrafted human-like gut microbiome.

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

JoVE Encyclopedia of Experiments

Immunology

Immunotherapy

Model Development and Disease Study

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

Source: Xiao, Y. et al., <a href="https://app.jove.com/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.

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

Murine Fecal Isolation and Microbiota Transplantation

Summary

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