Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

Abstract

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g. endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g. nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

Advanced age protects microvascular endothelium from aberrant Ca2+ influx and cell death induced by hydrogen peroxide

“…Intact endothelial tubes were isolated from the SEA as described (Socha et al . , ; Socha & Segal, ). Briefly, the abdominal musculature was transferred to a dissection chamber at 4°C and pinned onto a transparent silicone rubber block (Sylgard 184; Dow‐Corning, Midland, MI, USA).…”

Electrical dynamics of isolated cerebral and skeletal muscle endothelial tubes: Differential roles of G‐protein‐coupled receptors and K+ channels

“…The general isolation procedure for the SEA has been previously described in detail [see for video illustration]. Briefly, a ventral midline incision was made from the sternum to the pubis.…”

“…For PCAs, enzyme solution contained the following: 0.31 mg·mL −1 papain (P4762, Sigma), 0.5 mg·mL −1 dithioerythritol (D8255, Sigma), 0.75 mg·mL −1 collagenase (C8051, Sigma), and 0.13 mg·mL −1 elastase (E7885, Sigma). As previously described, the SEA enzyme cocktail included 0.62 mg·mL −1 papain, 1.0 mg·mL −1 dithioerythritol, and 1.5 mg·mL −1 collagenase. Following partial digestion, each arterial segment was gently triturated to remove adventitia and smooth muscle cells using borosilicate glass capillary tubes [1B100‐4, World Precision Instruments (WPI), Sarasota, FL, USA] with an internal tip diameter of 80‐120 μm.…”

Gene expression profiles of ion channels and receptors in mouse resistance arteries: Effects of cell type, vascular bed, and age

“…Cells were sampled while visualizing dispersed vessels at 200× magnification to ensure pure, cell type–specific samples. The endothelium was discerned as intact “tubes” surrounded by dispersed SMCs that typically adopted a “C” shape on the bottom of the dish. For each vessel type, ~5000 ECs and ~500 SMCs were collected per mouse by aspiration; a fresh micropipette was used for each cell type.…”