Summary
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.
Abstract
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.
Introduction
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.
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Protocol
Vanderbilt University'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
- Collect a stool sample, using a sterile container if the subject is in the clinic. Refrigerate the stool samples at 4 °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.
- Sanitize a biosafety-level fume hood with a 10% bleach solution, or other Environmental Protection Agency-approved disinfectant.
- 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 °C freezer until completely ready for processing.
- 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.
2. Aseptic collection of mouse fecal samples
NOTE: Use aseptic techniques, including sterilized instruments.
- 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.
- 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.
- Use a sterile spatula to transfer cecal content into sterile tubes, and store aliquots in a -80 °C freezer19.
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.
3. Fecal matter transplantation
- Resuspend fresh or previously frozen fecal pellets in sterile saline in a 1:20 (w:v) proportion and vortex until homogenized.
- Pass the homogenate through a 30 µm pore nylon filter to remove large particulate matter. Centrifuge at 79 × g for 5 min and collect the supernatant to use for transplantation.
- Oral gavage 100 µL 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'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. - Ensure that germ-free recipient mice are single-housed in gnotobiotic film isolators and fed with sterile food and water.
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.
- Briefly, retrieve the mice from the gnotobiotic isolators and preheat the tail-cuff machine platform and mouse holder.
- 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.
- 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.
- Allow the mouse to rest in the holder; place on the platform for 3-5 min covered by a sheet to acclimate.
- Average the measurements from all the rounds for an average systolic pressure for each animal.
5. Assessment of FMT to cardiovascular changes
- Following blood pressure measurement, euthanize the mice and aseptically collect cecal contents, as described in section 2 .
- 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.
- 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's microbiota are similar.
- 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.
6. Making gut intestine Swiss-rolls
- Day 1
- 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.
- 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.
- Cut the duodenum, jejunum, ileum, and colon.
- 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.
- Put the gut on filter paper. Label the paper with the name of the section (e.g., duodenum) and then 'P' at the right top corner for proximal or 'D' for distal at the right bottom corner.
- Cut the gut longitudinally with ball-tip scissors. Open the gut on the filter paper. Wash with more PBS as needed.
- Sandwich the gut between two filter papers. Staple the filter papers at four points/corners near the gut.
- 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.
- Day 2
- Prepare 2% agarose in distilled water and heat with a stir bar in a beaker covered with aluminum foil.
- 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.
- 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.
- Allow the agarose to cool and solidify. Use a razor blade to trim the extra agarose around the tissue section.
- 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 °C.
- Prepare paraffin-embedded tissue slides and proceed to immunostaining, as outlined below.
7. Immunostaining of the gut intestinal tract
- Deparaffinization
- 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.
- Rinse gently with cold running tap water. Store in a bath of tap water.
- Antigen retrieval
- 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 °C for 20 min.
- Run under cold tap water.
- Staining
- 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.
- Drop Tris-buffered saline (TBS) + 0.025% Triton X-100 onto the tissue and incubate for 5 min. Repeat this step.
- 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.
- Add primary antibody solution and incubate at 4 °C for at least 2 h or overnight. Wash gently with TBS + 0.025% Triton X-100 by gently pipetting ~200 µL over the section.
- 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.
- Mount with mounting medium and a coverslip.
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Representative Results
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 step in choosing the microbiota is ensuring that the disease phenotype of interest is present in the donor and is associated with dysbiotic changes. For example, a high-salt diet is strongly linked to dysbiosis and cardiovascular dysfunction. This study used a mouse donor that was fed an 8% NaCl diet. Changes in the gut microbiota in response to the high salt included a decrease in bacterial biodiversity (Figure 2A), which clustered separately from the normal salt microbiota (Figure 2B). The Firmicutes/Bacteroidetes ratio was also increased (Figure 2C), suggesting high salt-induced microbiota changes (modified from Ferguson et al.12).
To determine the role of high salt-induced dysbiosis in predisposition to hypertension, FMT from high salt-fed mice to germ-free mice was performed and blood pressure responses to a low subpressor dose of angiotensin II (Ang II) assessed. C57BL/6 male mice at 3 months of age were used in this study. Recipient mice were implanted with osmotic minipumps to administer a continuous low dose of Ang II (140 ng/kg/) for 2 weeks in mice. Mice that received FMT from high salt-fed donors exhibited a significant increase in blood pressure with Ang II treatment compared to normal salt microbiota recipients (Figure 3). This finding indicates that FMT primed the recipient mice to develop hypertension12. Details on the tail-cuff protocol in rodents have been reported previously12,26.
Dysbiotic gut microbiota contribute to disease partly due to an inflamed and leaky gut wall. Thus, examining the intestinal wall by immunohistochemistry can be used to interrogate changes in specific gut areas in any disease state even beyond FMT. Figure 4 demonstrates that we can perform immunohistochemistry on the ileum using a Swiss-roll technique and various stain markers, such as hematoxylin and eosin (H&E), Masson's trichrome, and immune cells markers such as anti-CD3 and anti-CD68., as previously described 12, and apolipoprotein AI (AI), which had accumulated in the ileum of proteinuric mice (Figure 4).
Figure 1: Diagram summarizing the protocol design. Fecal samples collected from a human subject or conventional mouse are used for transplantation into germ-free mice. Abbreviations: FMT = fecal microbiota transplantation; BP = blood pressure. Please click here to view a larger version of this figure.
Figure 2: Mice on a high-salt diet exhibit gut microbiota dysbiosis. (A) Estimation of species biodiversity in cecal content obtained from mice on normal salt (black) and high salt (red) diets. (B) Nonmetric multidimensional scaling shows that the bacteria from normal salt and high salt diet mice form separate clusters. (C) High salt is associated with an increased Firmicutes/Bacteroidetes ratio. (***p < 0.0001, using two-tailed unpaired Student's t-tests). This figure is adapted from Ferguson et al.12. Abbreviations: NMDS = nonmetric multidimensional scaling; NS = normal salt; HS = high salt. Please click here to view a larger version of this figure.
Figure 3: FMT 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. This figure is adapted from Ferguson et al.12. Abbreviations: FMT = fecal microbiota transplantation; BP = blood pressure; Ang II = angiotensin II; NS = normal salt; HS = high salt. Please click here to view a larger version of this figure.
Figure 4: Immunohistochemistry image illustrating how to assess disease marker changes in the gut. Representative images showing apolipoprotein AI stain in ilea obtained from mice that had hyperlipidemia without (A) or with (B) proteinuria. Magnification: 5x at a 200 µm scale (A,B, left); 10x at a 100 µm scale (A,B, right). Please click here to view a larger version of this figure.
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Discussion
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 performed immediately while the sample is still fresh or snap-frozen in liquid nitrogen and kept at -80 °C until ready to be used. If the sample cannot be immediately frozen, such as in the case of humans collecting their samples at home, it is important to use a preservative for nucleic acids such as ethanol. Most bacteria in the gut are anaerobes; when preparing the fecal samples for transplantation, it must be done quickly to avoid exposing bacteria to aerobic environments, as this may reduce the efficacy due to a decreased amount of anaerobes in the mixture27. For a longitudinal study, metagenomic sequencing of the samples should be performed together to avoid batch effects. Samples for FMT can come from one or multiple donors pooled together and then distributed to multiple recipients across the experimental groups.
It is extremely crucial that the success of FMT is confirmed experimentally prior to exploring mechanistic implications. Fecal or cecal contents can be analyzed by metagenomics and similarities assessed between donors and recipients. The expected results would be similar microbial diversity through alpha diversity indices, principal coordinates analysis, and functional profiles. It is only then that the conclusion that the gut microbiota plays an important role in disease etiology can be inferred. This step is even more important if the study utilizes conventionally-housed mice that were pretreated with antibiotics to deplete their endemic microbiota. This is because if this step was not successful, the surviving recipients' bacteria can influence disease outcomes. However, successful FMT, especially in preclinical studies, does not always meaningly result in clinical translation. Thus, additional confirmatory steps are necessary to implicate direct gut-related mechanisms in disease pathophysiology, including changes in gut morphology using the Swiss-roll technique described here.
There is an established direct role of immune cell activation in the development of hypertension, demonstrated through adoptive transfer studies28,29,30. Although germ-free mice are the gold standard in determining a causal role of the microbiota in disease, the model may be limited in studying inflammatory mechanisms of cardiometabolic diseases. Germ-free mice have an undeveloped immune system, which dampens their translational relevance in studying the interplay between gut microbiota and inflammation. Alternatively, this protocol can be modified to transplant fecal samples into conventional mice, following the depletion of their microbiota using antibiotics or bowel cleansing with polyethylene glycol31,32. FMT into conventionally-housed mice pretreated with antibiotics eliminates the need for the strictly-controlled and expensive environment that is required to maintain germ-free mice. Current evidence demonstrates that the choice of antibiotics, whether one or a combination, and treatment protocol vary among studies33,34,35. The choice of antibiotics will determine the types of bacteria that will be depleted, due to, variations in mechanisms of action, and consequently affect the phenotype. Thus, the experimental design should take into account the types of bacteria known to mediate the phenotype, and the broad-spectrum antibiotics, mode of administration, and regimen should be selected accordingly.
This protocol has its limitations. Germ-free mice are kept in gnotobiotic facilities, which makes experimental procedures related to studying cardiovascular diseases challenging. For example, radiotelemetry is the gold standard method to measure blood pressure, but involves very invasive surgical procedures. This is not practical for immune, naïve germ-free mice. Thus, a noninvasive tail-cuff is used for this purpose, to avoid microbial contamination12. Typically, to obtain relatively accurate measurements using tail-cuff plethysmography, animals are trained and acclimated to the platform before systolic pressure measurements. In germ-free FMT studies, researchers should consider collecting and averaging multiple measurements36.
These challenges may present in other endpoints beyond blood pressure. For example, studies exploring behavioral aspects of cardiovascular diseases often require frequent handling and external exposure. A study exploring the effects of a high-fiber diet in maternal obesity-induced cognitive and social dysfunction demonstrated successful FMT in conventional mice that were pretreated with antibiotics37. Studies exploring longitudinal cardiovascular responses may consider FMT in conventionally housed mice.
In addition to measuring blood pressure, animals can be euthanized and tissues harvested for further examination. Notably, cecal contents must be collected to be evaluated for successful engraftment of the transplanted microbiota. This detailed protocol will guide researchers in studying the mechanism of the gut microbiome from FMT to tissue levels, and is applicable to functional studies. This is significant because there is a tremendous amount of methodological and currently available literature. For mechanistic purposes, plasma, intestines, kidneys, hearts, and other tissues can be harvested to investigate the pathways involved. The causal effects of the gut microbiota in disease are connected to the metabolites they produce and their release into the system due to a leaky gut. The health of the entire gut can be assessed through histological and immunostaining approaches. The Swiss-roll technique has been used to demonstrate the effect of disease FMT in mice12,24.
The contribution of the gut microbiota in cardiovascular diseases such as hypertension remains associative. Here, we presented methodological approaches to collect, process, and transplant fecal matter from human subjects or conventional mice to germ-free mice. The protocol summarizes additional experimental practices and parameters to investigate in delineating a cause and/or effect of the gut microbiota in cardiometabolic health. Studies should be replicated to ensure the reproducibility and rigor of the desired phenotype.
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Disclosures
No conflicts of interest, financial or otherwise, are declared by the authors.
Acknowledgments
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.
Materials
| Name | Company | Catalog Number | Comments |
| Alexa Fluor 488 Tyamide SuperBoost | ThermoFisher | B40932 | |
| Anaerobic chamber | COY | 7150220 | |
| Apolipoprotein AI | Novus Biologicals | NBP2-52979 | |
| Artery Scissors - Ball Tip | Fine Science Tools | 14086-09 | |
| Bleach solution | Fisher Scientific | 14-412-53 | |
| Bovine Serum Albumin | Fisher Scientific | B14 | |
| CD3 antibody | ThermoFisher | 14-0032-82 | |
| CD68 monoclonal antibody | ThermoFisher | 14-0681-82 | |
| Centrifuge | Fisher Scientific | 75-004-221 | |
| CODA high throughput monitor | Kent Scientic Corporation | CODA-HT8 | |
| Cryogenic vials | Fisher Scientific | 10-500-26 | |
| Disposable graduate transfer pipettes | Fisher Scientific | 137119AM | |
| Disposable syringes | Fisher Scientific | 14-823-2A | |
| Ethanol | Fisher Scientific | AA33361M1 | |
| Feeding Needle | Fine Science Tools | 18061-38 | |
| Filter paper sheet | Fisher Scientific | 09-802 | |
| Formalin (10%) | Fisher Scientific | 23-730-581 | |
| High salt diet | Teklad | TD.03142 | |
| OMNIgene.GUT | DNAgenotek | OM-200+ACP102 | |
| Osmotic mini-pumps | Alzet | MODEL 2002 | |
| PAP Pen | Millipore Sigma | Z377821-1EA | |
| Petri dish | Fisher Scientific | AS4050 | |
| Pipette tips | Fisher Scientific | 21-236-18C | |
| Pipettes | Fisher Scientific | 14-388-100 | |
| Serile Phosphate-buffered saline | Fisher Scientific | AAJ61196AP | |
| Smart spatula | Fisher Scientific | NC0133733 | |
| Stool collection device | Fisher Scientific | 50-203-7255 | |
| TBS Buffer | Fisher Scientific | R017R.0000 | |
| Triton X-100 | Millipore Sigma | 9036-19-5 |
|
| Varimix platform rocker | Fisher Scientific | 09047113Q | |
| Vortex mixer | Fisher Scientific | 02-215-41 | |
| Xylene | Fisher Scientific | 1330-20-7, 100-41-4 |
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