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 metabolism2,3,4,5. 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 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 can also be obtained directly from the diet, and only 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 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, 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 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 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 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.
Protocol
Representative Results
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…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
Referencias
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