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

Summary

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.

Abstract

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 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, 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 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 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).

Introduction

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 how they modulate inflammation, signal through dedicated receptors, and serve as substrates in cellular metabolism^2,3,4,5. Recent work from our group has proposed that SCFAs are key in vivo inflammatory regulators of lung immune tone in vivo via the gut-lung....

Protocol

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

1. Wipe the bottom half of the mouse abdomen with 70% ethanol swabs. Administer anesthesia by giving an intraperitoneal injection of tribr.......

Discussion

This paper describes an innovative in vivo method for near simultaneous collection of blood samples sequentially  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.......

Materials

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