Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Abstract

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca²⁺ [Ca²⁺]i increases and subsequent activation of small- and intermediate conductance Ca²⁺-activated K⁺ (SKCa/IKCa) channels. Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca²⁺]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 °C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial "tubes" are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca²⁺]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Figure 1:. Isolation of cerebral arteries from the mouse brain.

(A) Brain isolated with intact arteries (white arrow indicates the posterior cerebral artery) in dessection solution. (B) Magnified view of posterior cerebral artery (white arrow; ~3x vs. Panel A). (C) Isolated posterior cerebral arteries secured with stainless steel pins in specimen dish. (D) Posterior cerebral arteries following removal of connective tissues. Inset shows cut segments of arteries; Scale bar = 500 µm. Please click here to view a larger version of this figure.

Figure 2:. Preparation of cerebral endothelial tube.

(A) Equipment used for triturating the partially digested arterial segments; a = micromanipulator, b = chamber for preparing tubes, c = aluminum stage, d = microsyringe, e = microscope, f = microsyringe pump controller. (B) Arterial segments (white arrows) in solution for enzymatic digestion. (C) Endothelial tubes prepared by trituration; a = trituration pipette, b = intact endothelial tubes. Inset shows the trituration process to remove adventitia and smooth muscle cells; Scale bar = 100 µm. (D) An intact endothelial tubes secured on the glass cover slip with pinning pipette; a = pinning pipette, b = intact endothelial tubes with diameter of ~100 µm. Please click here to view a larger version of this figure.

Figure 3:. Equipment for superfusion and simultaneous [Ca²⁺]_i and V_m measurements.

(A) Equipment for superfusion of the endothelial tube and temperature control, a = high intensity ARC lamp power supply, b = fluorescence system interface, c = temperature controller, d = six-reservoirs of superfusion system, e = fiber optic light sources, f = valve controller, g = hyperswitch of [Ca²⁺]_i photometry system. (B) Superfusion chamber apparatus; a and b = headstages, c = ground electrode, d = inline heater, e = superfusion tubing, f = vacuum suction, g = superfusion chamber, h = aluminum stage holding the chamber. (C) Experimental platform on a vibration isolation table; a = cell framing adaptor with camera, b = microscope light, c = microscope, d = aluminum stage, e = micromanipulator for headstage control, f = vibration isolation table. Please click here to view a larger version of this figure.

Figure 4:. Equipment for operating electrophysiology and data recording.

a = oscilloscope, b = function generator, c = audible baseline monitor, d = 50/60 Hz noise filter, e and f = electrometers, g = data acquisition system. Please click here to view a larger version of this figure.

Figure 5:. Simultaneous [Ca²⁺]_i and V_m recordings.

(A) Differential Interference Contrast image (400x) of a mouse cerebral arterial endothelial tube adjusted for experiment in photometric window using a 40x objective. A sharp electrode is placed into a cell as shown at the top of the image (white arrow). (B) Raw traces for simultaneous measurements of F₃₄₀/F₃₈₀ ratio (top) and V_m (bottom) in response to ATP (100 µM). Please click here to view a larger version of this figure.