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

The overall goal of the following experiment is to quantify neuroprotection following repetitive hypoxia preconditioning or RHP by simultaneously measuring infarct volume and blood brainin barrier integrity. This is achieved through subjecting mice to nine stochastic hypoxia exposures that vary in both duration and intensity to provide neurovascular protection from stroke. Stroke is then induced in the mouse and followed with Evan's blue injection.

Evan's blue selectively binds albumin and allows for the quantification of blood brainin barrier disruption Subsequent to sacrifice of the animal after injection. The brain is removed and stained with TTC to quantify the infarct volume. The results show neuroprotective benefits of repetitive hypoxia preconditioning on mice after ischemic stroke.

The main advantage of this technique over existing methods like single exposure hypoxic preconditioning, is that RHP produces neuroprotection for at least eight Weeks in mice. This dual quantification method can answer key questions in stroke research, such as how to simultaneously measure infarct volumes in blood-brain barrier disruption, Though this method can be used to provide insights into ischemic stroke, RHP can also be applied to other CNS based and systemic pro-inflammatory disease states. To begin divide mice into two groups.

The RHP group, which receives exposures of 8%and 11%oxygen, and the control group, which receives exposures of 21%oxygen concurrently, remove the top filter lid of each cage and place the cage with food and water bottles intact into the chambers connected to their respective oxygen tanks. Close and secure the lid of the chamber. Open the main gas valve for the tanks and set the initial flow for each induction chamber to two liters per minute or LPM.

After five minutes of exposure, reduce the flow rate to one LPM for the remainder of the exposure. At the end of the exposure, reduce the flow to zero LPM and close the gas valve. For the tanks.

Open the chamber lids and replace the filter top lid on each cage. Place the cages in standard housing until the next hypoxic exposure. Spray down each induction chamber with a disinfectant and deodorizer after each use.

Expose both RHP and control mice at the same time of day over the course of the two weeks of the protocol. Then perform a transient middle cerebral artery occlusion or A-T-M-C-A-O as described in the text. EB injection should be performed 22 hours after reperfusion and should circulate in the bloodstream for two hours prior to sacrifice NTTC staining.

Prepare the amount of EB needed for injection. Then draw the desired amount of room temperature into a 0.3 cc insulin syringe. With a 29 gauge needle, restrain the mouse using a flat bottom restrainer.

Hold the tail so that the lateral vein is uppermost. Lateral veins are located on either side of the center line of the tail. Hold the tip of the tail to keep the mouse steady for injection.

Insert the needle into the vein approximately three to five millimeters. Being careful not to perforate the vein, inject all of the dye over the course of one minute. If the solution is going into the vein, there should be minimal resistance as pressure is applied to the syringe.

Confirm successful systemic venous administration of EB by an immediate color change in the tail limbs and eyes of the mouse. Remove the needle from the tail and gently apply pressure using clean gauze in order to stop the bleeding. Begin timing.

When the mouse's skin turns blue, allow the EB to circulate for two hours to penetrate the weakened blood brain barrier. Perfusion and TTC staining should occur 24 hours after reperfusion at 24 hours after T-M-C-A-O and two hours after EB administration sacrifice the animal with an isof fluorine overdose in a small induction chamber. Perfusion should begin immediately following sacrifice in order to minimize autolysis, which begins in the absence of oxygen following death.

Quickly secure the animal on a styrofoam platform with forearms pinned through the paws. Cut a lateral incision through the abdominal wall from the midline just below the rib cage. Carefully cut through the diaphragm to expose the heart.

Start the perfusion pump at a flow rate of five milliliters per minute with a 60 cc syringe filled with ice cold 0.01 molar phosphate buffered saline or PBS connected to a 27 gauge winged infusion needle. Place the tip of the needle about 0.5 centimeters into the left ventricle of the heart and cut the right atrium Progressively diluted venous blood should flow out of the atrium during the perfusion until venous blood appears. Colorless trans cardio e perfused 30 milliliters of 0.01 molar PBS through the heart.

Next, add five milliliters of TTC solution into transparent 20 scintillation bottles. Immediately following perfusion, decapitate the animals and dissect out the brains using small scissors and a spatula if necessary. Check that the hemisphere contralateral to the occluded middle cerebral artery appears pale without noticeable EB leakage or edema.

Next poor PBS into an acrylic brain matrix designed to make 1.0 millimeter thick coronal sections of a mouse brain. Place the brain dorsal side up into the matrix and immediately pour PBS over the brain. Keep the brain moist.

Remove the olfactory bulbs by inserting a stainless deal, 0.21 millimeter thick blade into the second slot from the rostral side of the matrix. Then remove the cerebellum by inserting a blade in the fourth slot from the coddle side of the matrix. Insert a blade into the middle slot of the remaining slots in the matrix.

Then insert the remaining blades evenly bisecting the remaining tissue to ensure the most even distribution of tissue during the slicing. Once all blades have been inserted, add one to two drops of PBS to the brain to moisten it. Next, remove the blades one at a time from the matrix beginning with the rostral region.

Keep the first seven slices for TTC analysis. After T-M-C-A-O, use a small spatula to carefully transfer the slices from the blade to the TTC filled vial. After all sections are in the vial, cap it and place it in a warm water bath until the sections turn pink.

Gently turn the bottle in the bath if necessary to avoid section overlap, which could lead to uneven staining. Then dispose of the TTC and pour a 4%paraform aldehyde solution into the vial to cover the brain sections. This terminates the TTC chemical reaction.

Immediately arrange the sections on a clean one by three inch glass slide and orient the sections from rostral to coddle when the sections are arranged on the slide. Scan the slide. Using a standard scanner, set the resolution at a minimum of 600 DPI for image analysis.

Be sure to include the name of the animal and a metric ruler in the scanned image. Finally, flip over the slide and scan the backside to ensure all data is collected. RHP reduces infarct volumes by 46%compared to control mice, but has no effect on hemispheric swelling derived from the TTC data in the same animals.

RHP reduces blood-brain barrier disruption as defined by Evans Blue leak normalized to the contralateral hemisphere representative TTC stains from both RHP treated and 21%oxygen exposed control. Mice are shown with corresponding infarct volumes and Evans blue leak listed below each sample here. Ipsilateral is the ischemic hemisphere of the brain and contralateral is the unaffected side of the brain.

While attempting this procedure, it is important to remember to inject Evan's blue 22 hours after fusion and allow it to circulate for two hours before animal sacrifice after Its development. This technique paved the way for investigators in the field of preclinical stroke research to explore mechanisms of endogenous protection from stroke in Mice. After watching this video, you should have a clear understanding of how to measure the neuroprotective effect of RHP through the dual quantification of blood-brain barrier integrity and infarct volumes.