A Surgical Approach to Induce Local Reversal of Blood Flow to Assess Plasticity of Segmented Endothelium in the Mouse Ear

Abstract

I aim to test the hypothesis that arteriolar endothelial cells will behave as venular endothelial cells and mediate leukocyte rolling following local reversal of blood flow. In live models, this hypothesized reprogramming of endothelial cells has not yet been studied, as techniques are limited to identify endothelial cell phenotypes in vivo. Venular endothelial cells play an essential role in regulating leukocyte infiltration of tissues and thereby mediate leukocyte-dependent diseases in humans (autoimmune disease, arthritis, graft vs. host disease, inflammatory bowel disease, ischemia/reperfusion injury, to name a few). The Von Andrian Lab has developed a technique by use of conventional intravital microscopy that allows identification of venular segments of microvasculature in live animals. In brief, the technique involves the preparation of a tissue in an anesthetized animal so that its microvasculature can be studied by brightfield or fluorescence microscopy. We apply methods that allow us to identify subsets of circulating blood cells in microvessels; as these cells display characteristic interactions with microvascular endothelial cells, we can analyze film developed via intravital microscopy to distinguish vasculature where leukocyte rolling occurs. With these insights, we can map arteriolar and venular segments of microvessels in specific sites of live animals. In an attempt to induce endothelial cell reprogramming in vivo, I have executed a novel surgical approach with the intent of reversing blood flow in the mouse ear. In male BALB/c mice aged 10-12 weeks, I have used a heparinized, polyethylene catheter to cannulate the left common carotid artery and thereafter the left external jugular vein; the catheter thus serves as a fistula, completing a vascular shunt redirecting oxygenated blood from the left common carotid artery into the external jugular vein. A secondary shunt has also been designed and practiced, forming a double arteriovenous anastomosis to increase negative pressure in the ear and further promote complete reversal of flow in the vasculature of the ear. I have analyzed and mapped the microvasculature of the left ear by conducting epifluorescence intravital microscopy at numerous time points prior to and following surgery to rigorously test my hypothesis that arteriolar endothelial cells can shift phenotypes to mediate leukocyte rolling. The mouse ear requires minimal, non-invasive preparation for analysis by conventional intravital microscopy and thus serves as an excellent site for repeated monitoring of microvascular structures. Following successful blood flow reversal, mice will be kept alive for postoperative observation for up to 30 days, allowing enough time for microvasculature to adapt and respond to the local change in the direction of blood flow, as I hypothesize that this process may require an extensive time window.