Murine Mesenteric Lymphadenectomy for Selective Disruption of Lymphatic Communication with Region-Specific Gut

Our research focuses on T-cell responses to neoantigens and peripheral tissues, and these responses are heavily influenced by priming, and so we primarily focus on where these priming events occur. Currently, the only widely used options to block the trafficking between lymph nodes and tissues are systemic modalities like S1P receptor agonists and the integrin blockade. But this broad inhibition prevents us from studying the contribution of specific local lymph nodes.

Our protocol provides an in-depth description of highly selective lymphadenectomies at each of the four main mesenteric lymph node stations, and since these lymph nodes are quite specialized in the gut segment that they drain, this well-tolerated survival procedure allows us to disrupt specific segments of the gut lymph axis while leaving the rest intact. After confirming appropriate anesthetic depth via a hind foot pinch, use sterile surgical scissors to make a 0.5 to 1 centimeter midline skin incision in the anesthetized mouse. Using sterile forceps, dissect the skin away from the abdominal wall and identify the linea alba.

Then make another 0.5 to 1 centimeter incision through the linea alba to access the peritoneal cavity. Using a blunt-tip gavage needle, instill one milliliter of body-temperature sterile saline into the peritoneal cavity. Moisten the sterile drapes to prepare for bowel positioning.

To resect the first lymph node station, orient the exteriorized bowel onto moistened drapes with the cecum positioned inferiorly, proximal colon superiorly, and terminal ileum toward the surgeon. Identify the mesenteric lymph node chain that runs longitudinally along the colon. Using angled fine-tipped forceps, bluntly dissect the first lymph node station away from the ileocolic vessels by placing the closed forceps between these structures and gently opening them.

To reduce the theoretical risk of collateral flow to nearby nodes, perform an optional suture ligation with single interrupted sutures along the lymph node chain, one millimeter distal and one millimeter proximal to the first lymph node station using monofilament nylon suture and fine-tipped forceps. Gently retract the lymph node station caudally, then using fine-point scissors, excise the target lymph node station from the cephalad to caudal direction. Observe the distal branches of the ileocolic vessels, ileal vessels, and jejunal vessels to confirm that the blood supply remains intact.

To resect the second lymph node station, gently eviscerate the small intestine and orient the exteriorized bowel onto moistened drapes with the cecum positioned inferiorly, proximal colon superiorly, and small intestine toward the surgeon's right. Next, identify the second lymph node station which runs longitudinally along the colon and begins at the confluence of ileal and jejunal vessels. Using angled fine-tipped forceps, bluntly dissect the second lymph node station away from the colonic vessels.

To reduce the theoretical risk of collateral flow to nearby nodes, perform an optional suture ligation with single interrupted sutures along the lymph node chain, one millimeter distal and one millimeter proximal to the second lymph node station using monofilament nylon suture and fine-tipped forceps. Gently retract the lymph node station caudally, then using fine-point scissors, excise the second lymph node station from the cephalad to caudal direction. Inspect the operative field to ensure hemostasis.

To resect the fourth lymph node station, gently eviscerate the entire small intestine up to the posterior tethering point. Orient the bowel onto moistened drapes with the cecum inferiorly, proximal colon superiorly, and small intestine toward the surgeon's right. Identify the fourth lymph node station which resides just medial to the distal colon and lateral to the third station.

Using angled fine-tipped forceps or extra-fine point scissors, bluntly dissect the fourth lymph node station away from the colonic vessels and surrounding adipose tissue. To reduce the theoretical risk of collateral flow to nearby nodes, perform an optional suture ligation with single interrupted sutures at each identifiable lymph node, usually one millimeter from the node using monofilament nylon suture and fine-tipped forceps. Then gently retract the fourth lymph node station cephalad.

Using fine or extra-fine point scissors, excise the lymph node stations from caudal to cephalad. Repeat the procedure for the second node in the fourth lymph node station. Carefully dissect the node, perform suture ligation if required, and resect it.

Inspect the operative field to confirm hemostasis. After returning the abdominal contents to the peritoneal cavity, instill one milliliter of body-temperature sterile saline into the abdominal cavity to compensate for evaporative fluid loss. Close the abdomen in two layers:first the abdominal wall, then the skin using a 6-0 monofilament polypropylene suture in a running pattern.

Tissue extracted using the mesenteric lymphadenectomy procedure consistently resulted in samples with low adipose content, as evidenced by immediate sinking in PBS. Flow cytometry analysis confirmed successful lymph node extraction, with over 95%of cells in mesenteric lymphadenectomy-derived samples identified as CD45 positive, compared with only 7%in visceral adipose tissue controls.