Name: Author Spotlight: Gut Microbiome-Lung Tissue Communication Through Short-Chain Fatty Acid Analysis
Uploaded: 2024-06-21T14:00:00
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 tribr...

Improved Blood Sampling Technique to Assess Gut Microbial Metabolites in Mice

<ol>
	<li>vander Hee, B., Wells, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+regulation+of+host+physiology+by+short-chain+fatty+acids.">Microbial regulation of host physiology by short-chain fatty acids.</a> Trends Microbiol. 29 (8), 700-712 (2021).</li><li>Koh, A., De Vadder, F., Kovatcheva-Datchary, P., B&#228;ckhed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+dietary+fiber+to+host+physiology:+Short-chain+fatty+acids+as+key+bacterial+metabolites.">From dietary fiber to host physiology: Short-chain fatty acids as key bacterial metabolites.</a> Cell. 165 (6), 1332-1345 (2016).</li><li>Vinolo, M. A. R., Rodrigues, H. G., Nachbar, R. T., Curi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+inflammation+by+short+chain+fatty+acids.">Regulation of inflammation by short chain fatty acids.</a> Nutrients. 3 (10), 858-876 (2011).</li><li>Layden, B. T., Angueira, A. R., Brodsky, M., Durai, V., Lowe, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+chain+fatty+acids+and+their+receptors:+new+metabolic+targets.">Short chain fatty acids and their receptors: new metabolic targets.</a> Transl Res. 161 (3), 131-140 (2013).</li><li>den Besten, G., 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=The+role+of+short-chain+fatty+acids+in+the+interplay+between+diet,+gut+microbiota,+and+host+energy+metabolism.">The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism.</a> J Lipid Res. 54 (9), 2325-2340 (2013).</li><li>Tian, 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=Elevated+gut+microbiome-derived+propionate+levels+are+associated+with+reduced+sterile+lung+inflammation+and+bacterial+immunity+in+mice.">Elevated gut microbiome-derived propionate levels are associated with reduced sterile lung inflammation and bacterial immunity in mice.</a> Front Microbiol. 10, 159 (2019).</li><li>Liu, Q., Tian, X., Maruyama, D., Arjomandi, M., Prakash, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+immune+tone+via+gut-lung+axis:+Gut-derived+LPS+and+short-chain+fatty+acids&#8217;+immunometabolic+regulation+of+lung+IL-1&#946;,+FFAR2,+and+FFAR3+expression.">Lung immune tone via gut-lung axis: Gut-derived LPS and short-chain fatty acids&#8217; immunometabolic regulation of lung IL-1&#946;, FFAR2, and FFAR3 expression.</a> Am J Physiol Lung Cell Mol Physiol. 321 (1), L65-L78 (2021).</li><li>De Vadder, F., 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=Microbiota-generated+metabolites+promote+metabolic+benefits+via+gut-brain+neural+circuits.">Microbiota-generated metabolites promote metabolic benefits via gut-brain neural circuits.</a> Cell. 156 (1-2), 84-96 (2014).</li><li>Dalile, B., Van Oudenhove, L., Vervliet, B., Verbeke, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+short-chain+fatty+acids+in+microbiota&#8211;gut&#8211;brain+communication.">The role of short-chain fatty acids in microbiota&#8211;gut&#8211;brain communication.</a> Nat Rev Gastroenterol Hepatol. 16 (8), 461-478 (2019).</li><li>Sun, M., Wu, W., Liu, Z., Cong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota+metabolite+short+chain+fatty+acids,+GPCR,+and+inflammatory+bowel+diseases.">Microbiota metabolite short chain fatty acids, GPCR, and inflammatory bowel diseases.</a> J Gastroenterol. 52 (1), 1-8 (2017).</li><li>Cummings, J. H., Pomare, E. W., Branch, H. W. J., Naylor, C. P. E., MacFarlane, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+chain+fatty+acids+in+human+large+intestine,+portal,+hepatic+and+venous+blood.">Short chain fatty acids in human large intestine, portal, hepatic and venous blood.</a> Gut. 28 (10), 1221-1227 (1987).</li><li>Macfarlane, G. T., Gibson, G. R., Cummings, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fermentation+reactions+in+different+regions+of+the+human+colon.">Comparison of fermentation reactions in different regions of the human colon.</a> J Appl Bacteriol. 72 (1), 57-64 (1992).</li><li>Neis, E. P., 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=Distal+versus+proximal+intestinal+short-chain+fatty+acid+release+in+man.">Distal versus proximal intestinal short-chain fatty acid release in man.</a> Gut. 68 (4), 764-765 (2019).</li><li>Yamashita, H., 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=Effects+of+acetate+on+lipid+metabolism+in+muscles+and+adipose+tissues+of+type+2+diabetic+Otsuka+Long-Evans+Tokushima+Fatty+(OLETF)+Rats.">Effects of acetate on lipid metabolism in muscles and adipose tissues of type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) Rats.</a> Biosci Biotechnol Biochem. 73 (3), 570-576 (2009).</li><li>R&#237;os-Covi&#225;n, D., 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=Intestinal+short+chain+fatty+acids+and+their+ink+with+diet+and+human+health.">Intestinal short chain fatty acids and their ink with diet and human health.</a> Front Microbiol. 7, 185 (2016).</li><li>Jaworska, K., 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=An+in+vivo+method+for+evaluating+the+gut-blood+barrier+and+liver+metabolism+of+microbiota+products.">An in vivo method for evaluating the gut-blood barrier and liver metabolism of microbiota products.</a> J Vis Exp. (140), e58456 (2018).</li><li>Ashcroft, S. P., 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=Protocol+to+assess+arteriovenous+differences+across+the+liver+and+hindlimb+muscles+in+mice+following+treadmill+exercise.">Protocol to assess arteriovenous differences across the liver and hindlimb muscles in mice following treadmill exercise.</a> STAR Protoc. 4 (1), 101985 (2023).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> J Pharmacol Pharmacother. 1 (2), 87-93 (2010).</li><li>Cheever, A. W., Warren, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+vein+ligation+in+mice:+Portal+hypertension,+collateral+circulation,+and+blood+flow.">Portal vein ligation in mice: Portal hypertension, collateral circulation, and blood flow.</a> J Appl Physiol. 18, 405-407 (1963).</li><li>Bloemen, J. G., 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=Short+chain+fatty+acids+exchange+across+the+gut+and+liver+in+humans+measured+at+surgery.">Short chain fatty acids exchange across the gut and liver in humans measured at surgery.</a> Clin Nutr. 28 (6), 657-661 (2009).</li><li>Hoyles, L., 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=Microbiome&#8211;host+systems+interactions:+Protective+effects+of+propionate+upon+the+blood&#8211;brain+barrier.">Microbiome&#8211;host systems interactions: Protective effects of propionate upon the blood&#8211;brain barrier.</a> Microbiome. 6 (1), 55 (2018).</li><li>Shimizu, 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=Propionate-producing+bacteria+in+the+intestine+may+associate+with+skewed+responses+of+IL10-producing+regulatory+T+cells+in+patients+with+relapsing+polychondritis.">Propionate-producing bacteria in the intestine may associate with skewed responses of IL10-producing regulatory T cells in patients with relapsing polychondritis.</a> PLoS One. 13 (9), e0203657 (2018).</li><li>Wang, 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=Propionate+protects+against+lipopolysaccharide-induced+mastitis+in+mice+by+restoring+blood&#8211;milk+barrier+disruption+and+suppressing+inflammatory+response.">Propionate protects against lipopolysaccharide-induced mastitis in mice by restoring blood&#8211;milk barrier disruption and suppressing inflammatory response.</a> Front Immunol. 8, 1108 (2017).</li><li>Lucas, S., 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=Short-chain+fatty+acids+regulate+systemic+bone+mass+and+protect+from+pathological+bone+loss.">Short-chain fatty acids regulate systemic bone mass and protect from pathological bone loss.</a> Nat Commun. 9 (1), 55 (2018).</li><li>Xie, C., Wei, W., Zhang, T., Dirsch, O., Dahmen, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+systemic+and+hepatic+hemodynamic+parameters+in+mice.">Monitoring of systemic and hepatic hemodynamic parameters in mice.</a> J Vis Exp. (92), e51955 (2014).</li><li>S&#228;nger, C., 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=Intrahepatic+vascular+anatomy+in+rats+and+mice+-+Variations+and+surgical+implications.">Intrahepatic vascular anatomy in rats and mice - Variations and surgical implications.</a> PLoS One. 10 (11), e0141798 (2015).</li><li>Phillips, B. J., Mirzaie, M., Turco, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+Vein+Injuries:+A+Review.">Portal Vein Injuries: A Review.</a> J Emerg Trauma. 2 (2), 4 (2017).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2,2,2 Tribromoethanol, 97% (Avertin)</td>
			<td>Sigma Aldrich&#160;</td>
			<td>T48402-25G</td>
			<td>Anesthetic agent&#160;</td>
		</tr>
		<tr>
			<td>Buprenorphine Hydrochloride Injection 0.3 mg/mL</td>
			<td>PAR Pharmaceutical&#160;</td>
			<td>NDC 42023-179-05</td>
			<td>Analgesic agent</td>
		</tr>
		<tr>
			<td>Dressing Forceps</td>
			<td>Miltex&#160;</td>
			<td>6-100</td>
			<td>Dissection&#160;</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Roboz</td>
			<td>RS-5136</td>
			<td>Dissection&#160;</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&#160;</td>
		</tr>
		<tr>
			<td>Student Halsted-Mosquito Hemostats</td>
			<td>F.S.T&#160;</td>
			<td>91308-12</td>
			<td>Dissection&#160;</td>
		</tr>
		<tr>
			<td>Surgical tape&#160;</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&#160;</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).

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

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

The scope of our lab research is to understand the communication between the gut microbiome and lung tissues, and how the components of the gut microbiome may directly influence lung immune and inflammatory responses. We focus on the effects of specific metabolites, namely, short-chain fatty acids. Short-chain fatty acids such as, acetate, propionate, and butyrate have been implicated as having roles in influencing diverse disease processes in experimental mice.These include lipid metabolism in type 2 diabetes, polychondritis, pathological bone loss, allergic asthma, and blood-brain barrier permeability. The measurement of short-chain fatty acid concentrations is most convenient in fecal pellets expelled from the mice. However, these fecal short-chain fatty acid levels do not reliably correspond to what is present in vivo, in the colon, and more importantly, what is transmitted into the host circulation.We believe that the levels of short-chain fatty acids and other metabolites measured within the portal blood will more accurately reflect the steady state in vivo concentrations compared to those measured from fecal samples. Ligating the portal vein near the hepatic hilum can expand the dimensions of the portal vein, and thereby significantly improve the success rate, as well as increase the maximum blood volume that can be collected.

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...

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.

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

Sequential Collection of Blood from the Portal Veins and Inferior Vena Cava of Mice

After anesthetizing the mouse, place it in a supine position on a heated surgical surface. Apply eye ointment and perform toe pad pinches to ensure the animal is adequately anesthetized. Lift the skin of the abdomen.With a pair of scissors, make a longitudinal incision at the midline from the umbilicus to the xiphoid process and caudally towards the abdomen's base. Make a similar incision into the peritoneum without perforating or damaging the intestines, diaphragm, or liver. Next, make a full length transverse incision on both sides of the umbilicus to expand the surgical field.Then use clean gauze soaked in warm PBS to move the large and small intestines to the right and expose the IVC below. With a cotton swab, gently manipulate the intestinal segments within the surgical field to locate the portal vein at the root of the mesentery. Then use a dissecting microscope to carefully pass a 7-0 prolene suture behind the portal vein near the hepatic hilum.Next, insert a 28 gauge half inch needle of a heparinized one cubic centimeter insulin syringe into the IVC. Collect 0.2 to 0.3 milliliters of the blood sample. Leave the needle inside the IVC to avoid the bleeding that would occur with removal.Now, gently tie the 7-0 prolene ligature around the portal vein. Insert the 28 gauge half inch needle of another heparinized insulin syringe into the portal vein. Aspirate to slowly collect up to 0.5 milliliters of blood from the portal vein.Once blood collection is complete, remove the needles from the IVC and the portal vein. Transfer the blood samples from syringes to 1.5 milliliter heparinized tubes. Place the tubes on ice for future processing.