In the present protocol, we present a modified middle cerebral artery occlusion technique that measures ischemic brain injury to determinate an infarct zone, brain edema, and BBB permeability in the same set of rodent brains. In terms of ethical and economic costs, the main advantage of this technique is that only one brain sample is required for all three measurements. This assessment of the main parameters of ischemic brain injury will help to comprehensively investigate pathophysiology of ischemic stroke.
To begin, remove the rat brain, and cut 2 millimeter thick horizontal sections with a 0.009-inch stainless steel, uncoated, single edge razor blade. Place the sections in 0.005%TTC and incubate them for 30 minutes at 37 degrees Celsius, then place the brain tissue on the microscope slides and perform optical scanning of the six brain slices with a resolution of 1600 x 1600 DPI. Use a photo editor to add a blue filter to the images.
After opening the software, go to image, adjustments, and channel mixer. Select blue as the output channel and check monochrome, then click okay. Save the image in the JPEG file format.
Use the polygon selection tool in ImageJ version 1.37 to select each hemisphere of each of the six brain slices and save it as a separate image file. To calculate the infarct volume and brain edema, use a macro to convert the pixels in the image to white or black, based on a threshold. Then, close all files and use a second macro to compare the number of white and black pixels to determine the infarct volume and brain edema.
Copy the raw data to a spreadsheet. When calculating infarct volume, correct for tissue swelling by using the ratios of ipsilateral and contralateral cerebral hemispheres. Express the brain edema areas as a percentage of the standard areas of the unaffected contralateral hemisphere.
Determine blood brain barrier disruption 24 hours after occlusion. Divide the right and left hemispheres into six slices and put each slice into a microcentrifuge tube. Homogenize each slice of the brain tissue in trichloroacetic acid, using 4 milliliters of 50%trichloroacetic acid per gram of brain tissue.
Centrifuge the samples at 10, 000 times G for 20 minutes, then transfer the supernatant into a fresh tube and dilute it 1:3 with 96%ethanol. Perform luminescence spectrophotometry with a plate reader. An independent sample t-test indicated that 19 rats that underwent permanent middle cerebral artery occlusion, or MCAO, demonstrated a significant increase in brain infarct volume compared to the 16 sham operated rats.
The rats that underwent permanent MCAO also demonstrated a significant increase in the extent of brain edema and blood brain barrier breakdown after 24 hours, compared to the sham operated animals. When attempting this procedure, it is important to ensure that chemical concentration comply with the protocol. This protocol makes it possible to evaluate all three parameters of ischemic damage in one sample of the brain.
