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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	<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>

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 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).

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

943 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

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Use of a Monocyte Monolayer Assay to Evaluate Fc&#947; Receptor-mediated Phagocytosis

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express Fc&#947;Rs (e.g., Fc&#947;RI, Fc&#947;RIIA, Fc&#947;RIIB, and Fc&#947;RIIIA) that are involved in immune responses. The MMA exploits the mechanism of Fc&#947;R-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate Fc&#947;Rs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out Fc&#947;R-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo Fc&#947;R-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo- or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of Fc&#947;R-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

The monocyte monolayer assay (MMA) is an in vitro assay that utilizes isolated primary monocytes obtained from mammalian peripheral whole blood to evaluate Fc&#947; receptor (Fc&#947;R)-mediated phagocytosis.

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Evaluation of Blood Lactate and Plasma Insulin During High-intensity Exercise by Antecubital Vein Catheterization

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different exercise modalities. During some exercise modalities (e.g., high-intensity interval exercise), blood metabolites and hormonal levels change in short periods of time. In the present study, we describe a method to catheterize the antecubital vein, which allows the collection of several blood samples during exercise. Insulin and venous lactate concentrations were measured during high-intensity exercise by the application of the described method. The exercise consisted of three 30 s bouts of high-intensity exercise separated by 4 min of recovery. After the last recovery period, a Wingate test was performed. Blood samples from the antecubital vein were obtained before and after each 30 s bout and before and after the Wingate test. As a result, it was possible to evaluate the plasma insulin and venous blood lactate variations during the exercise.

Here we present a protocol to obtain several blood samples from the antecubital vein during high-intensity interval training. This protocol may be useful for the measurement of blood metabolites and endocrinal markers during exercise.

The measurement of metabolic and endocrinal markers during physical activity is of relevance to understanding the physiological implications of different ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...