Characterization of a surgical model of lymphatic insufficiency in the rat mesentery
Authors: Stolarz Amanda J , Garner Brittney R , Fletcher Terry W , Sarimollaoglu Mustafa , Galanzha Ekaterina , Zharov Vladimir , Rusch Nancy J 
Published: November 2023

The mesenteric lymphatic system clears fluid, lipids, plasma proteins, and immune cells from the intestinal interstitial space, thereby contributing to intestinal homeostasis. Conversely, insufficient lymphatic clearance can result in accumulation of immune cells in the mucosa and submucosa of the gastrointestinal (GI) wall and predispose to immune cell‐mediated tissue injury leading to chronic GI inflammation and lymphedema development. The goal of this study was to establish a model of intestinal lymphadenectomy to begin to understand the functional response of the GI lymph vessels (LVs) to localized lymphatic insufficiency and ultimately devise strategies to restore lymph flow. We modified the standard rat model of severe, generalized mesenteric lymphedema, which involves radical surgical resection of the entire mesenteric lymph chain and ducts. Instead, we ligated a single proximal LV draining one mesenteric arcade to more closely recapitulate surgical removal of sentinel lymph nodes. Rats were anesthetized and one loop of mesentery was exposed on a heated microscope stage. High‐speed optical imaging in vivo was performed on a chosen LV pre‐operatively and then performed again on 2 or 7 days after LV ligation, enabling each LV to serve as its own control. Using this strategy, our initial results indicate that lymph flow is reduced by 48% (n=2) on Day 2 in a LV distal to the ligated site, but lymph flow shows a compensatory increase of 120% (n=2) at sites remote from the ligation. Additional evidence of lymphedema formation distal to the ligated site is the 150% increase in tissue hydration (wet/dry weight ratio) on Day 7 compared to Day 0 and Day 2, an effect not observed for the remote site. Isolated rat mesenteric LVs were cannulated, pressurized (4–5 mm Hg) and equilibrated in physiological salt solution (37°C) to monitor rhythmic contractions (n=2). Edge‐detection software recorded changes in external diameter. On Day 2, LVs isolated from the ligated region exhibited reduced contraction amplitude and area under the curve (AUC), resulting in reductions of ~40% (amplitude) and ~77% (AUC) compared to LVs isolated from the remote site. These reductions were accompanied by an increase in contraction frequency to ~198% when compared to LVs from the remote site. On Day 7, rhythmic contractions of LVs from the ligated region appeared to recover to ~74% (amplitude) and ~94% (AUC) compared to the remote site; however, contraction frequency was reduced to ~62% compared to LVs from the remote site. Notably, the contraction and flow abnormalities observed on Day 2 were detected prior to a measurable increase of tissue wet/dry ratio, suggesting LV contractile dysfunction may precede lymphedema. Thus, this model of mesenteric lymphatic insufficiency may be a useful preclinical tool to identify mechanisms of lymphatic dysfunction after surgical lymphadenectomy and evaluate anti‐lymphedema interventions to restore flow.Support or Funding InformationFunded by Rho Chi‐AFPE Graduate Fellowship, PhRMA Predoctoral Fellowship, UAMS Pilot Grant, and NIH R21CA131164.