Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Summary

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Abstract

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O₂). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O₂ (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

Introduction

As the leading cause of adult disability and the fourth leading cause of death, stroke is one of the most debilitating disease states facing the adult population of the United States.¹ Animal models of stroke allow for experimental investigation of new methods of reducing ischemic injury and improving post-stroke recovery. One novel avenue for such translational research is preconditioning. Preconditioning is the intentional use of a non-damaging stimulus to reduce damage from a subsequent, and more severe, injury.² Hypoxic preconditioning has been shown to produce pleiotropic changes in the brain that provide protection against stroke in both

Protocol

NOTE: This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern Medical Center which abides by the National Institutes for Health (NIH) policy for experimental animal usage.

1. Repetitive Hypoxic Preconditioning

1. Custom design four flowmeters on gas regulators and attach to standard 15 L induction chambers with PVC tubes to allow compressed gas from the oxygen (O₂) tanks to flow into the chambers via an inlet port. See Equipment and Materials for more details about custom design.
2. Divide mice into 2 groups: Repetitive Hypoxic Preconditioning (RHP) Group, which rec....

Results

This study included 25 male Swiss Webster mice that were 10 weeks of age at the start of randomization into RHP (n = 10) or 21% O₂ (n = 15) groups. Two weeks after the final RHP exposure, surgical procedures were performed, with groups blinded and counterbalanced between days. Following tMCAo, 1 mouse died during post-operative recovery and 1 mouse was excluded from the study because it did not meet the reperfusion CBF criterion. Both excluded mice were from the 21% O₂ group. In accordance with ARRI.......

Discussion

A single exposure to systemic hypoxia (i.e., 2 hr of 11% O₂) in mice “transiently” protects the brain from tMCAo,²⁹ meaning the epigenetic response to the hypoxic preconditioning challenge is short-lasting, and the baseline phenotype is restored within days. Repetitive presentations of the hypoxic preconditioning stimulus dramatically extend the duration of the neuroprotective phenotype.⁶ Many studies have shown that the frequency, magnitude, and duration of the repet.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Flowmeters, regulators	VetEquip, Inc	Specialty order	Four flowmeters are attached to 6.0 mm flexible PVC tubing which connects to the inlet port on each induction chamber with a plastic female connector. These flowmeters are bolted to a 6.5" x 1" x 1" metal bar. This metal bar is bolted to a MI-246-P pressure gauge with a DISS outlet. This pressure gauge and flowmeter equipment can be attached to each new gas cylinder with a wrench.
21% O2 tank	AirGas	OX USP200
11% O2 tank	AirGas	Specialty order
8% O2 tank	AirGas	Specialty order
15 L induction chambers	VetEquip	941454
Moor Laber Dopper Flow	Moor Instruments	moorVMS-LDF1-HP	0.8 mm diameter probe
High Intensity Illuminator	Nikon	NI-150
Zoom Stereo Microscope	NIkon	SMZ800	Other surgical microscopes may be used.
Kent Scientific Right Temperature CODA	Kent Scientific Corporation	Discontinued	Recommended replacement is PhysioSuite with RightTemp Temperature Monitoring and Homeothermic Control (Kent Scientific, #PS-RT).
Hovabator Incubator	Stromberg's	2362-E	Our model is the 2362N. 2362E is a later model and includes an electronic thermostat.
V010 Anesthesia system	VetEquip	901807	Includes: ten foot high-pressure oxygen hose, frame, flowmeter, oxygen flush assembly, vaporizer, breathing circuit, chamber, nosecones, waste gas evacuation tubing and two VapoGuard filters
250 ml isoflurane	Butler Schein	NDC-11695
D-6 Vet Trim Animal Cordless Trimmer	Andis	#23905	Replacement blades are available from Andis (#23995)
Betadine	Fisher Scientific	19-898-867
Q-tips	Multiple sellers	Catalog number not available
Gauze Pads	Fisher Scientific	67622
Surgical drape	Fisher Scientific	GM300
Silk Sutures	Look/Div Surgical Specialties	SP115
Nylon Sutures	Look/Div Surgical Specialties	SP185
Durmont #5 forceps (2)	Fine Science Tools	11251-35	Angled 45°
Surgical Scissors	Fine Science Tools	14028-10
3 mm Vannas	Kent Scientific Corporation	INS600177	Straight blade
Hartman Hemostats	Fine Scientific Tools	13002-10
Occluding filaments	Washington University	Specialty order	Filaments are silicone coated at Washington Univeristy and provided to UTSW facilities for a fee.
Evans Blue	Sigma Aldritch	E2129-10G
Filter Paper	Sigma Aldritch	WHA1001150	150 mm, circles, Grade 1
Weigh Boats	Fisher Scientific	02-202-101	2.5" diameter
0.9% Sodium Chloride Injection USP	Baxter Pharmaceutics	2B1321
0.3 cc insulin syringe with 29 gauge needle	Becton Dickinson Labware	309301
Flat bottom restrainer	Braintree Scientific	FB M	2.0" diameter
TTC	Sigma	T8877
10x PBS, pH 7.4	Fisher Scientific	BP399-20
Water Bath	Multiple sellers	Catalog number not available	Scintillation tubes with TTC may be manually held under running warm water as an alternative to the water bath.
Styrofoam board	Multiple sellers	Catalog number not available
Large Syringe Kit	PumpSystems Inc	P-SYRKIT-LG
Perfusion Pump	PumpSystems Inc	NE-300
60 cc syringe	Fisher Scientific	NC9203256
27 gauge winged infusion set	Kawasumi Laboratories, Inc	D3K1-25G 1
20 ml scintillation vial	Fisher Scientific	50-367-126
Stainless steel spatula	Fisher Scientific	14-373-25A
Alto acrylic 1.0 mm mouse brain, coronal	CellPoint Scientific	Catalog number not available
0.21 mm stainless steel blades, 25 pk	CellPoint Scientific	Catalog number not available	Reusable cryostat blades are an inexpensive alternative.
4% paraformaldehyde	Santa Cruz Biotechnology	SC-281692
Superfrost microscope slides	Fisher Scientific	12-550-15
HP Scanjet G4050	Multiple sellers	Catalog number not available	Other commercial scanners are suitable for this step in the protocol.
ImageJ	National Institute of Health	Catalog number not available
Analytical Balance	Mettler Toledo	XSE 205U
Precision Compact Oven	Thermo Scientific	PR305225M
1.7 ml microcentrifuge tubes (Eppendorfs)	Denville Scientific	C2170
Formamide	Fisher Scientific	BP228-100
96-well plates	Fisher Scientific	07-200-9
Epoch Microplate Spectrophotometer	BioTek	Catalog number not available