Preparing Cerebral Endothelial Tubes from Mouse Parenchymal Arterioles

Abstract

Source: Hakim, M. A., et al., Isolation and Functional Analysis of Arteriolar Endothelium of Mouse Brain Parenchyma. J. Vis. Exp. (2022)

This video demonstrates a method for isolating the endothelial tubes from mouse cerebral parenchyma. Through a combination of enzymatic digestion and mechanical dissociation, the process yields an intact tube with endothelial cells.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Solutions and drugs

1. Physiological salt solution (PSS)
   1. Prepare a minimum of 1 L of PSS using 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 mM glucose.
   2. Prepare necessary solutions lacking CaCl₂ (zero Ca²⁺ PSS) for dissection of cerebral arterioles and isolation of endothelial tube.
      ....

Results

Figure 1: Dissection and isolation of parenchymal arterioles. (A) Isolated mouse brain with intact Circle of Willis. (B) A dissected rectangular piece of brain tissue containing the MCA (white arrow). (C) Isolation of pia from the upper part of the tis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bath Chiller (Isotemp 500LCU)	ThermoFisher Scientific	13874647
Borosilicate glass capillaries (Pinning)	Warner Instruments	G150T-6
Borosilicate glass capillaries (Sharp Electrodes)	Warner Instruments	GC100F-10
Borosilicate glass capillaries (Trituration)	World Precision Instruments (WPI), Sarasota, FL, USA	1B100-4
BSA: Bovine Serum Albumin	Sigma	A7906
CaCl2: Calcium Chloride	Sigma	223506
Collagenase (Type H Blend)	Sigma	C8051
Data Acquision Digitizer	Molecular Devices, Sunnyvale, CA, USA	Digidata 1550A
Dissection Dish (Glass Petri with Charcoal Sylgard bottom)	Living Systems Instrumentation, St. Albans City, VT, USA	DD-90-S-BLK
Dithioerythritol	Sigma	D8255
DMSO: Dimethyl Sulfoxide	Sigma	D8418
Elastase (porcine pancreas)	Sigma	E7885
Flow Control Valve	Warner Instruments	FR-50
Fluorescence system interface, ARC lamp & power supply, hyperswitch and PMT	Molecular Devices, Sunnyvale, CA, USA	IonOptix Systems
Forceps (Fine-tipped, sharpened)	FST	Dumont #5 & Dumont #55
Glucose	Sigma-Aldrich (St. Louis, MO, USA)	G7021
HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)	Sigma	H4034
KCl: Potassium Chloride	Sigma	P9541
MgCl2: Magnesium Chloride	Sigma	M2670
Micromanipulator	Siskiyou	MX10
Micropipette puller (digital)	Sutter Instruments, Novato, CA, USA	P-97 or P-1000
Microscope (Nikon-inverted)	Nikon Instruments Inc, Melville, NY, USA	Ts2
Microscope (Nikon-inverted)	Nikon Instruments Inc	Eclipse TS100
Microscope Stage (Aluminum)	Siskiyou, Grants Pass, OR, USA	8090P
Microsyringe Pump Controller	World Precision Instruments (WPI), Sarasota, FL, USA	SYS-MICRO4
NaCl: Sodium Chloride	Sigma	S7653
Papain	Sigma	P4762
Phase contrast objectives	Nikon Instruments Inc	(Ph1 DL; 10X & 20X)
Scissors (3 mm & 7 mm blades)	Fine Science Tools (or FST), Foster City, CA, USA	Moria MC52 & 15000-00
Scissors (Vannas style; 9.5 mm & 3 mm blades)	World Precision Instruments	555640S, 14364
Temperature Controller (Dual Channel)	Warner Instruments	TC-344B or C
Valve Control System	Warner Instruments	VC-6