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. 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 proline 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 proline 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.