Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

Summary

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Abstract

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

Introduction

The pial circulation on the surface of the brain has been the object of much study, partly because of its experimental accessibility. However, the topology of the cerebral vasculature creates distinct regions. In contrast to the robust pial network rich in anastomoses with substantial capacity for redirecting the blood flow, the intracerebral parenchymal arterioles (PAs) present limited collateral supply, each of them perfusing a discrete volume of nervous tissue^1,2. This creates a bottleneck effect on the blood flow which, combined with unique physiological features^3,

Protocol

All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Colorado, Anschutz Medical Campus and were performed according to the guidelines from the National Institutes of Health.

1. Solutions

1. Use MOPS-buffered saline for the dissection and to keep samples at 4 °C before their utilization. Do not gas the solution. Prepare MOPS buffered saline with following composition: 135 mM NaCl, 5 mM KCl, 1 mM KH₂PO₄, 1 mM MgSO₄, 2.5 mM CaCl₂, 5 mM glucose, 3 mM MOPS, 0.02 mM EDTA, 2 mM pyruvate, 10 mg/mL bovine serum albumin, pH 7.3 at 4 ....

Results

Endothelial small-conductance (SK) and intermediate-conductance (IK) Ca²⁺-sensitive K⁺ channels exert a dilatory influence on the diameter of PAs. Bath application of 1 µM NS309, a synthetic IK and SK channel agonist, caused near maximal dilation (Figure 2A,B). However, capillary endothelial cells lack IK and SK channels and did not hyperpolarize in response to NS309²⁰. As a result, stimulating ca.......

Discussion

The pressurized HiCaPA (hippocampal capillary-parenchymal arteriole) preparation described in the present manuscript is an extension of our well-established procedure to isolate, pressurize, and study parenchymal arterioles²⁹. We recently reported that Kir2.1 channels in brain capillary endothelial cells sense increases in [K⁺]_o associated with neural activation, and generate an ascending hyperpolarizing signal that dilates upstream arterioles²⁰. Revea.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22µm Syringe Filters	CELLTREAT Scientific Products	229751
12-0 Nylon (12cm) Black	Microsurgery Instruments, Inc	S12-0 NYLON
Automatic Temperature Controller	Warner Instruments	TC-324B
Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm	Sutter Instruments	B120-69-10
Bovine serum albumin	Sigma-Aldrich	A7030
CaCl2 dihydrate	Sigma-Aldrich	C3881
D-(+)-Glucose	Sigma-Aldrich	G5767
Dissection Scope	Olympus	SZ11
ECOLINE VC-MS/CA 4-12 — complete Pump with Drive and MS/CA 4-12 pump-head	Ismatec	ISM 1090
EGTA	Sigma-Aldrich	E4378
Fine Scissors - Sharp	Fine Science Tools	14063-09
Inline Water Heater	Warner Instruments	SH-27B
Integra™ Miltex™Tissue Forceps	Fisher Scientific	12-460-117
KCl	Sigma-Aldrich	P9333
KH2PO4	Sigma-Aldrich	P5379
Magnesium sulfate heptahydrate	Sigma-Aldrich	M1880
MgCl Anhydrous	Sigma-Aldrich	M8266
Micromanipulator	Narishige	MN-153
ML 133 hydrochloride	Tocris	4549
MOPS	Sigma-Aldrich	M1254
NaCl	Sigma-Aldrich	S9625
NaH2PO4	Sigma-Aldrich	S9638
NaHCO3	Sigma-Aldrich	S8875
NS309	Tocris	3895
Picospritzer III - Intracellular Microinjection Dispense Systems, 2-channel	Parker Hannifin	052-0500-900
Pressure Servo Controller with Peristaltic Pump	Living Systems Instrumentation	PS-200
Sodium pyruvate	Sigma-Aldrich	P3662
Super Fine Forceps	Fine Science Tools	11252-20
Surgical Scissors - Sharp-Blunt	Fine Science Tools	14001-13
Vertical Micropipette Puller	Narishige	PP-83