Measuring the Infarct Zone and Brain Edema in a Rat Brain Following Middle Cerebral Artery Occlusion

Take a rat subjected to middle cerebral artery occlusion, restricting blood flow to the right brain hemisphere.

Reduced blood supply deprives brain cells of oxygen, disrupts water regulation, causes cell swelling or edema, and leads to blood-brain barrier disruption.

Damaged cells eventually die, forming an infarct zone.

Inject a blue dye into the rat's tail vein to detect BBB disruption.

Harvest the brain, slice it with razor blades, and incubate the slices in a redox dye solution.

In living cells, the dye reacts with mitochondrial dehydrogenases and undergoes reduction, turning viable tissue red.

Infarcted areas, lacking dehydrogenase activity, remain white.

Scan the slices to obtain high-resolution images.

Using image analysis software, apply a blue filter to eliminate the blue dye color, converting the images to grayscale.

Apply thresholding to distinguish healthy black tissue from infarcted white tissue.

Calculate the infarct zone and brain edema as a percentage of the unaffected contralateral hemisphere.

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 1,600-by-1,600 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 OK. 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.