Neuronal Adaptation to Repetitive Hypoxic Preconditioning in Mice

Abstract

Source: Poinsatte, K., et al., Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice. J. Vis. Exp. (2015)

This video demonstrates the method of repetitive hypoxic preconditioning (RHP) by exposing a mouse to controlled hypoxia cycles before inducing an ischemic stroke. The experiment evaluates its effects on blood-brain barrier stability, neuronal adaptation, and inflammation.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Repetitive Hypoxic Preconditioning

1. Custom design four flowmeters on gas regulators and attach them to standard 15 L induction chambers with polyvinyl chloride (PVC) tubes to allow compressed gas from the oxygen (O₂) tanks to flow into the chambers via an inlet port.
2. Divide mice into 2 groups: The repetitive hypoxic preconditioning (RHP) Group, which receives exposures to 8% and 11% O₂, and the Control Group, which receives exposures to 21% O₂ (room air) concurrently.
3. Remove the top filter lid of each cage and place the cage, with food and water bottles intact, in the chambers connected to their respective O₂ tanks. Close and secure the lid of the chamber.
   1. Open the main gas valve for the tanks and set the initial flow for each induction chamber to 2 L per minute (LPM) for the first 5 minutes of exposure. Reduce the flow rate to 1 LPM for the remainder of the exposure.
   2. At the end of the exposure, reduce the flow to 0 LPM and close the tanks' gas valves.
   3. Open the chamber lids and replace the filter top lid on each cage. Place the cages in standard housing until the next hypoxic exposure.
4. Spray down each induction chamber with NPD (Steris) or an equivalent disinfectant/deodorizer after each use.
5. Expose both 21% and RHP mice at the same time of day over the course of two weeks.

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