Isolation and Cannulation of Cerebral Parenchymal Arterioles

Summary

This manuscript describes a simple and reproducible protocol for isolation of intracerebral arterioles (a group of blood vessels encompassing parenchymal arterioles, penetrating arterioles and pre-capillary arterioles) from mice, to be used in pressure myography, immunofluorescence, biochemistry, and molecular studies.

Abstract

Intracerebral parenchymal arterioles (PAs), which include parenchymal arterioles, penetrating arterioles and pre-capillary arterioles, are high resistance blood vessels branching out from pial arteries and arterioles and diving into the brain parenchyma. Individual PA perfuse a discrete cylindrical territory of the parenchyma and the neurons contained within. These arterioles are a central player in the regulation of cerebral blood flow both globally (cerebrovascular autoregulation) and locally (functional hyperemia). PAs are part of the neurovascular unit, a structure that matches regional blood flow to metabolic activity within the brain and also includes neurons, interneurons, and astrocytes. Perfusion through PAs is directly linked to the activity of neurons in that particular territory and increases in neuronal metabolism lead to an augmentation in local perfusion caused by dilation of the feed PA. Regulation of PAs differs from that of better-characterized pial arteries. Pressure-induced vasoconstriction is greater in PAs and vasodilatory mechanisms vary. In addition, PAs do not receive extrinsic innervation from perivascular nerves — innervation is intrinsic and indirect in nature through contact with astrocytic endfeet. Thus, data regarding contractile regulation accumulated by studies using pial arteries does not directly translate to understanding PA function. Further, it remains undetermined how pathological states, such as hypertension and diabetes, affect PA structure and reactivity. This knowledge gap is in part a consequence of the technical difficulties pertaining to PA isolation and cannulation. In this manuscript we present a protocol for isolation and cannulation of rodent PAs. Further, we show examples of experiments that can be performed with these arterioles, including agonist-induced constriction and myogenic reactivity. Although the focus of this manuscript is on PA cannulation and pressure myography, isolated PAs can also be used for biochemical, biophysical, molecular, and imaging studies.

Introduction

The cerebral circulation is uniquely organized to support the metabolic demands of central neurons, cells that have limited energy stores and are consequently highly sensitive to changes in oxygen pressure and supply of necessary nutrients. As particular neuronal subpopulations becomes active when specific tasks are performed, the vasculature promotes a highly localized increase in perfusion to prevent local hypoxia and depletion of nutrients ¹. This is a form of functional hyperemia known as neurovascular coupling, and is dependent on the proper operation of the neurovascular unit, composed of active neurons, astrocytes, and cerebral arteries

Protocol

1. Cannula and Chamber Preparation

1. Insert clean borosilicate glass capillaries (outer diameter: 1.2 mm; internal diameter: 0.69 mm; 10 mm in length) into the grooves of a pipette puller with a platinum filament (diameter: 100 µm).
2. Using appropriate settings, pull the capillary to generate a cannula with a long and thin tip (Figure 2) using a micropipette puller. The settings used are: Heat - 700, Pull - 100, Velocity - 50, Time - 10.
3. Insert cannula into the holder of pressure myograph chamber. Align the cannulas appropriately.
4. Carefully break the tips of the cannulas by using a forceps under the dissectin....

Results

Figure 5A shows a representative constriction of mouse PAs to 60 mM KCl aCSF to evaluate the integrity of the preparation. PAs should constrict between 15 - 30% in the presence of 60 mM KCl. If the constriction is below 15%, discard the PA and cannulate another one, as it suggests that the arteriole was damaged during the isolation and cannulation process.

Figure 5B illustrates PA constriction t.......

Discussion

Cerebral parenchymal arterioles are high resistance arterioles with few anastomoses and branches that perfuse distinct neuronal populations. These specialized blood vessels are central players in cerebrovascular autoregulation and neurovascular coupling through astrocyte-mediated vasodilation ¹. The importance of these specialized blood vessels in cerebral vascular disease has been known for approximately 50 years, when the pioneering work of Dr. Miller Fisher described structural alterations in parenchymal ar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Artificial Cerebrospinal Fluid
NaCl	Fisher Scientific	S-640
KCl	Fisher Scientific	P217
MgCl Anhydrous	Sigma-Aldrich	M-8266
NaHCO3	Fisher Scientific	S233
NaH2PO4	Sigma-Aldrich	S9638
D-(+)-Glucose	Sigma-Aldrich	G2870
CaCl2	Sigma-Aldrich	C4901
Bovine Serum Albumin	Sigma-Aldrich	A9647
Isolation/Cannulation
Stereo Microscope	Olympus	SZX7
Super Fine Forceps	Fine Science Tools	11252-00
Vannas Spring Scissors	Fine Science Tools	15000-00
Wiretrol 50 μl	VWR Scientific	5-000-1050
0.2 μm Sterile Syringe Filter	VWR Scientific	28145-477
Micropipette Puller	Sutter Instruments	P-97
Borosilicate Glass O.D.: 1.2 mm, I.D.: 0.68 mm	Sutter Instruments	B120-69-10
Dark Green Nylon Thread	Living Systems Instrumentation	THR-G
Linear Alignment Single Vessel Chamber	Living Systems Instrumentation	CH-1-LIN
Pressure Servo Controller with Peristaltic Pump	Living Systems Instrumentation	PS-200
Video Dimension Analyzer	Living Systems Instrumentation	VDA-10
Four Channel Recorder with LabScribe 3 Recording and Analysis Software	Living Systems Instrumentation	DAQ-IWORX-404
Heating Unit	Warner Instruments	64-0102
Automatic Temperature Controller	Warner Instruments	TC-324B