Name: sequential blood collection from inferior vena cava followed by portal vein to evaluate gut microbial metabolites in mice
Uploaded: 2024-06-21T14:00:00.000+00:00
Duration: 4 min 9 s
Description: Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous ...

AP is funded by an R01 award from the NIH/NHLBI (1R01HL146753). DM is funded by a T32 fellowship from the NIH and by a Trainee/Staff Pilot Awards from the UCSF Benioff Center for Microbiome Medicine.

All steps in this non-survival procedure were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California San Francisco. The mouse gender and strain used were male C57BL/6J mice (weighing 25-35 g and aged 12-15 weeks). Female and/or other standard mouse strains can also be used.
1. Anesthesia
<ol>
	<li>Wipe the bottom half of the mouse abdomen with 70% ethanol swabs. Administer anesthesia by giving an intraperitoneal injection of tribromoethanol (250-400 mg/kg). Ketamine/xylazine or isoflurane anesthesia can also be used. Apply ophthalmic ointment to the eyes to prevent corneal damage.</li>
	<li>Inject buprenorphine (0.05-0.1 mg/kg) intraperitoneally for analgesia. 
	NOTE: Buprenorphine is required because of the pain associated with the laparotomy procedure.&#160;</li>
</ol>
2. Laparotomy
<ol>
	<li>Place the anesthetized mouse on the surgical surface (with a heating pad underneath) in the supine position. Fasten all mouse limbs on the surgical board with tape. Confirm that the appropriate depth of anesthesia has been achieved by toe&#160;pinch without pedal withdrawal reflex.</li>
	<li>Lift the skin and make a longitudinal incision with dissecting&#160;scissors along the midline from the umbilicus to the xiphoid process and caudally towards the base of the abdomen (external genital organ).</li>
	<li>Make a similar incision into the peritoneum with dissecting scissors without perforating or damaging the intestines, diaphragm, or liver.</li>
	<li>Expand the surgical field by&#160;making a full-length transverse incision&#160;(extending the width of the abdomen) with scissors on both sides of the umbilicus.</li>
	<li>Move the large and small intestines to the surgeon&#39;s right side using a clean gauze soaked in warm PBS to expose the IVC below.</li>
</ol>
3. Ligature preparation for PV
<ol>
	<li>Gently manipulate the intestinal segments within the surgical field with a cotton swab to locate the PV at the root of the mesentery.</li>
	<li>Using a dissecting microscope to visualize the vessels and other abdominal&#160;structures, carefully pass a 7-0 prolene suture behind the PV near the liver hilum (between the duodenal vein and splenic vein)19 so that it is ready for ligation in step 5 (Figure 1). 
	NOTE: Using a microscope from this step onwards is not mandatory but significantly improves the&#160;overall procedure&#39;s success rate.</li>
</ol>
4. Blood sample collection from the IVC
<ol>
	<li>Insert the 28G &#189;-inch needle of a heparinized 1 cc insulin syringe into the IVC. Collect 0.2-0.3 mL of blood sample. 
	NOTE: If too much blood is removed in this step, it will result in&#160;lower volumes of PV blood&#160;collected in step 5.</li>
	<li>Leave the needle inside the IVC so as to avoid the bleeding that would occur with removal.</li>
</ol>
5. Blood sample collection from the PV
<ol>
	<li>Gently tie the 7-0 prolene ligature around the PV; the PV should expand and dilate as it backfills. Insert the 28G &#189;-inch needle of another heparinized 1cc insulin syringe into the PV.</li>
	<li>Aspirate slowly to collect up to 0.3-0.5 mL of blood from the PV. The PV may collapse but it should gradually re-expand with blood refilling from the mesenteric venous system.</li>
</ol>
6. Sample storage
<ol>
	<li>Remove the needles from the IVC and PV after blood collection.</li>
	<li>Transfer the blood samples from syringes to 1.5 mL heparinized&#160;eppendorf&#160;tubes and place&#160;them on ice for later processing into plasma for storage and analysis as needed.</li>
	<li>Euthanize the mouse using anesthetic overdose followed by cervical dislocation (per UCSF IACUC approved guidelines).</li>
</ol>

Improved Blood Sampling Technique to Assess Gut Microbial Metabolites in Mice

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The authors declare that they have no competing financial interests.

This paper describes an innovative in vivo method for near simultaneous collection of blood samples sequentially&#160; from the IVC and PV in the same experimental mouse. This method is useful for measuring the levels of gut microbiota-generated products, such as SCFAs, that transit through the portal circulation. The average maximal volume of blood that can be collected during a terminal procedure in mice (weighing 25-30 g) is approximately 1 mL/mouse, which in turn represents 50% of the total circulating blood...

Short-chain fatty acids (SCFA) are a major class of metabolites produced by the gut microbiota. Their critical roles in the interaction between the gut microbiome and other distant organs1 have been supported by research describing&#160;how they modulate inflammation, signal through dedicated receptors, and serve as substrates in&#160;cellular metabolism2,3,4,5. Recent work&#160;from our group has proposed that SCFAs are key in vivo inflammatory regulators of lung immune tone in vivo via the gut-lung axis6,7. Additional reports have described their functional influence on metabolism via the gut-brain axis8,9. Overall, the influence of SCFAs on host physiology and pathology is under active and intense investigation by numerous research groups spanning a wide range of disease processes.
Acetate (C2), propionate (C3), and butyrate (C4) are the primary SCFAs and are generated by gut microbiota through the fermentation of ingestible dietary fiber in the cecum and large intestine. All three SCFAs&#160;can also be obtained directly from the diet, and only&#160;acetate may also be produced by mammalian cells. SCFAs are absorbed in the colon and are partially utilized by intestinal epithelial cells (as an energy source, for local tissue immune modulation and to support gut&#160;barrier maintenance). They are also transported into the portal vein via the mesenteric venous system10. Butyrate is mainly consumed by intestinal epithelia, propionate by the liver11,12,&#160;and acetate has been reported to act on muscle and adipose tissues after entering the peripheral circulation13,14.
A comprehensive assessment of SCFA production, absorption, and functional activity requires knowledge of SCFA levels within the colonic lumen, in the portal circulation and peripheral blood. This can be accomplished by blood collection from portal vein (PV) and systemic circulation simultaneously or sequentially in the same animal. Since SCFAs are volatile15, measuring their levels in expelled fecal pellets may&#160;not accurately reflect levels within the colon. Furthermore, compared to measurements from colonic contents, the level of SCFAs present in the PV may more accurately reflect the net sum of the steady-state levels absorbed by the host versus the uneven levels produced by the gut microbiome&#160;throughout the length of the colon11. These PV SCFA levels may thus be more relevant and appropriate for studying the effects of SCFAs on host physiology and pathology beyond the local effects within the intestine.
To perform the coordinated and near simultaneous collection of PV and systemic circulating blood, the diaphragm should remain intact so as to maintain normal blood circulation and support spontaneous breathing. Therefore, the inferior vena cava (IVC) presents an ideal site to obtain systemic circulation blood while collecting PV blood. This IVC blood can also be used for other purposes, such as measuring circulating cytokines to evaluate systemic inflammation.
Currently, only a few methods for collecting blood from both the systemic circulation and PV have been reported in larger rodents16,17. Conventional methods, which require cannulation of vessels in rats, are technically difficult to perform in mice. In addition, the maximum amount of blood collected by these methods is usually no more than 0.3 mL18.
In this paper, we present a novel method that simplifies the process of dual blood collection&#160;from mouse IVC followed by PV in the same animal. The unique feature of the method is the ligation of the PV near the hepatic hilum just prior to PV blood sampling. This approach can expand the dimensions of the PV, thereby significantly improving the success rate as well as increasing the maximum collectable blood volume up to 0.5 mL.

<table><tbody><tr><td>2,2,2 Tribromoethanol, 97% (Avertin)</td><td>Sigma Aldrich&amp;nbsp;</td><td>T48402-25G</td><td>Anesthetic agent&amp;nbsp;</td></tr><tr><td>Buprenorphine Hydrochloride Injection 0.3 mg/mL</td><td>PAR Pharmaceutical&amp;nbsp;</td><td>NDC 42023-179-05</td><td>Analgesic agent</td></tr><tr><td>Dressing Forceps</td><td>Miltex&amp;nbsp;</td><td>6-100</td><td>Dissection&amp;nbsp;</td></tr><tr><td>Graefe Forceps</td><td>Roboz</td><td>RS-5136</td><td>Dissection&amp;nbsp;</td></tr><tr><td>Hepatin sodium 1000 USP units/mL</td><td>Hikma</td><td>NDC 0641-0391-12</td><td>Blood sample syringes/tubes heparinization</td></tr><tr><td>Prolene 7-0</td><td>Ethicon</td><td>8696G</td><td>Portal vein ligature</td></tr><tr><td>Scissors</td><td>F.S.T</td><td>14058-11</td><td>Dissection&amp;nbsp;</td></tr><tr><td>Student Halsted-Mosquito Hemostats</td><td>F.S.T&amp;nbsp;</td><td>91308-12</td><td>Dissection&amp;nbsp;</td></tr><tr><td>Surgical tape&amp;nbsp;</td><td>3M Transpore</td><td>1527-1</td><td>Mouse limbs fixation</td></tr><tr><td>U-100 Insulin Syringe 28G1/2</td><td>EXEL</td><td>26027</td><td>Blood sample collection&amp;nbsp;</td></tr></tbody></table>

sequential blood collection from inferior vena cava followed by portal vein to evaluate gut microbial metabolites in mice

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous distant organ systems. Short-chain fatty acids (SCFA) are one class of metabolites produced by gut microbes during the fermentation of indigestible dietary fiber. They are now recognized&#160;as important contributors to how the gut microbiome influences extra-intestinal organ systems via the gut-lung, gut-brain, and other gut-organ axes throughout the host. SCFAs are absorbed from the colon, through intestinal tissue,&#160;into the portal vein (PV). They then pass through the liver, and are consumed in various organs such as the brain, muscle, adipose tissue, and lungs. SCFAs are most easily measured in the expelled fecal material however, more accurate measurements have been obtained from intra-colonic fecal contents. Here we propose that sampling PV and systemic circulating plasma of a single subject may be preferable for studying the&#160;absorption, transport, and systemic levels of SCFAs in mice. We present a new technique for efficient blood sampling from the PV and inferior vena cava (IVC) that allows for the collection of relatively large volumes of blood from the portal and systemic circulations. This is accomplished by ligating the PV, thereby allowing&#160;for the dilation or enlargement of the PV as it backfills from the mesenteric veins that drain into it. Using this method, we were able to improve the rate of successful collection as well as the total amount of blood collected (up to 0.3 mL from IVC and 0.5 mL from PV).

Using the method described above, we can collect blood samples from the IVC and PV sequentially in the same mouse with a success&#160;rate of more than 95%. The average volumes of blood samples collected are 0.25 mL for the IVC and 0.35 mL for the PV.
Using gas chromatography-mass spectrometry (GC-MS), we measured the concentration of SCFAs in feces, PV blood, and IVC blood and were thus able to trace the absorption and transit of acetate (C2), propionate (C3), and butyrate (C4) from the colon...

Author Spotlight: Gut Microbiome-Lung Tissue Communication Through Short-Chain Fatty Acid Analysis

The protocol demonstrates a method to collect blood from portal veins and inferior vena cava from mice sequentially to evaluate the production and absorption of gut microbial metabolites.

Sequential Blood Collection from Inferior Vena Cava Followed by Portal Vein to Evaluate Gut Microbial Metabolites in Mice

Gut microbial products are known to act both locally within the intestine and get absorbed into circulation, where their effects can extend to numerous ...

sequential-blood-collection-from-inferior-vena-cava-followed-portal

Research

JoVE Journal

Immunology and Infection

1.1K Views.  University of California San Francisco and San Francisco General Hospital. The protocol demonstrates a method to collect blood from portal veins and inferior vena cava from mice sequentially to evaluate the production and absorption of gut microbial metabolites.

Video: Author Spotlight: Gut Microbiome-Lung Tissue Communication Through Short-Chain Fatty Acid Analysis

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

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

Behavior

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Medicine

Exploring Metabolite Exchange in Brown Adipose Tissue

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering thermogenesis. To achieve this, BAT utilizes a diverse menu of circulating nutrients to support its high metabolic demand. Additionally, BAT secretes metabolite-derived bioactive factors that can serve as either metabolic fuels or signaling molecules, facilitating BAT-mediated intratissue and/or intertissue communication. This suggests that BAT actively participates in systemic metabolite exchange, an interesting feature that is beginning to be explored. Here, we introduce a protocol for in vivo mouse-level optimized BAT arteriovenous metabolomics. The protocol focuses on relevant methods for thermogenic stimulations and an arteriovenous blood sampling technique using Sulzer's vein, which selectively drains interscapular BAT-derived venous blood and systemic arterial blood. Next, a gas chromatography-based metabolomics protocol using those blood samples is demonstrated. The use of this technique should expand the understanding of BAT-regulated metabolite exchange at the inter-organ level by measuring the net uptake and release of metabolites by BAT.

Author Spotlight: Illuminating New Avenues for Adipose Tissue Metabolism and Disease Prevention 

In this protocol, methods relevant for BAT-optimized arteriovenous metabolomics using GC-MS in a mouse model are outlined. These methods allow for the acquisition of valuable insights into BAT-mediated metabolite exchange at the organismal level.

Brown adipose tissue (BAT) plays a crucial role in regulating metabolic homeostasis through a unique energy expenditure process known as non-shivering ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that are not only driven by genetic factors, but also environmental factors that can trigger the onset and/or worsen the disease course. A previously published study on the dynamics of the gut microbiota in lupus-prone MRL/lpr female mice showed how changes of the gut microbiota can alter disease progression. Here, a protocol is described for extracting representative samples from the gut microbiota for studies of autoimmunity. Microbiota samples are collected from the anus and processed, from which the DNA is extracted using a phenol-chloroform method and purified by alcohol precipitation. After PCR is performed, purified amplicons are sequenced using a Next Generation Sequencing platform at Argonne National Laboratory. Finally, the 16S ribosomal RNA gene sequencing data is analyzed. As an example, data obtained from gut microbiota comparisons of MRL/lpr mice with or without CX3CR1 are shown. Results showed significant differences in genera containing pathogenic bacteria such as those in the phylum Proteobacteria, as well as the genus Bifidobacterium, which is considered part of the healthy commensal microbiota. In summary, this simple, cost-effective DNA isolation method is reliable and can help the investigation of gut microbiota changes associated with autoimmune diseases.

This protocol provides a simple, cost-effective DNA isolation method for the analysis of murine gut microbiota alterations during the development of autoimmune disease.

Gut microbiota has an important role in educating the immune system. This relationship is extremely important for understanding autoimmune diseases that ...

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

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 ...

Sequential Blood Collection from Inferior Vena Cava Followed by Portal Vein to Evaluate Gut Microbial Metabolites in Mice

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Chapters in this video

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

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

Arteriovenous Metabolomics to Measure In Vivo Metabolite Exchange in Brown Adipose Tissue

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

Analysis of Fecal Microbiota Dynamics in Lupus-Prone Mice Using a Simple, Cost-Effective DNA Isolation Method

Blood Collection Through Subclavian Vein Puncture in Mice

Murine Fecal Isolation and Microbiota Transplantation