Background

The endothelial barrier regulates the passage of ions, water, and proteins across blood vessels. The vasculature is heterogeneous, with veins being generally more permeable than arteries (Crone and Christensen, 1981; Durán et al., 2008; Hilfenhaus et al., 2018). Several diseases, including sepsis, stroke, and hypertension are exacerbated by a failure in endothelial barrier function (Wang et al., 2002; Granger et al., 2010; Rosenberg, 2012; Schoknecht and Shalev, 2012). Thus, it is critically important to understand the molecular regulation of vascular function in general, and of the endothelium in particular.

Ussing chambers enable the most detailed analysis of the electrophysiological properties of barrier-forming cells (Clarke, 2009; Herrmann and Turner, 2016). Originally developed to measure sodium permeability across frog skin (Ussing and Zerahn, 1951; Lindemann, 2001), Ussing chambers have been used to measure ion permeability in systems ranging from epithelial cells cultured on Transwell permeable supports (Molina et al., 2015) to intact tissues, such as the intestine (Laukoetter et al., 2007). The vast majority of Ussing chamber measurements have been performed on epithelial cells. By contrast, Ussing chamber analysis of cultured endothelial cells is much more difficult, although there are some examples where the approach was used to measure endothelial ion channel activity (Wigham et al., 2000; Hatou et al., 2010). The barrier function of cultured endothelial cells is generally characterized with other techniques, including cells cultured on Transwells with the EndOhm/ Millicell-ERS system, measuring flux with fluorescent tracers, and/or cells cultured on gold electrodes using electric cell-substrate impedance sensing (ECIS) (Dubrovskyi et al., 2013; Adil and Somanath, 2021; Maier-Begandt et al., 2021).

Here, we describe a method for Ussing chamber analysis of the electrophysiological properties of semi-intact blood vessels, which have been dissected and longitudinally cut. This method can be applied to veins and arteries, and avoids difficulties encountered when measuring intact perfused blood vessels (Crone and Christensen, 1981). Several parameters can be measured, including transendothelial electrical resistance (TEER), short circuit current (Isc), and determination of relative ion permeability. Each of these measurements reflects both transcellular and paracellular ion transport, where transcellular ion transport is largely mediated by plasma membrane ion channels and the paracellular component is mediated through tight junctions (Anderson and van Itallie, 2009; Clarke, 2009). Together, the transcellular and paracellular pathways regulate tissue barrier function, a parameter that can be impacted by several pathophysiological conditions, such as sepsis (Joffre et al., 2020).

The ability to analyze an intact vascular barrier enables electrophysiological analysis of the endothelium in a native context that complements and validates measurements done with cultured cells or isolated perfused vessels. Native tissues also have better tissue integrity than endothelial cells in culture, and are more amenable to Ussing chamber analysis. Here, we describe techniques needed for electrophysiological analysis of intact vessels, and how these were applied to analyze the effect of a proinflammatory cytokine called TNFα on vein barrier function and ion permselectivity.