This study investigates the effects of ischemia-reperfusion injury on brain tissue using a rat model. The methodology involves assessing brain damage through staining techniques that differentiate viable cells from necrotic cells.
During brain ischemia, impaired blood flow to the brain reduces oxygen and nutrient supply to the tissue, causing brain damage. After brief ischemia, as the blood flow to the brain gets restored, reperfusion with increased oxygen supply results in reactive ion species overproduction, leading to oxidative tissue damage, known as ischemia-reperfusion injury.
To assess brain damage following ischemia-reperfusion injury in a rat model, first, harvest the rat brain. Rinse in a chilled buffer to remove debris and blood. Section the brain to obtain coronal slices of desired thicknesses. Carefully transfer the slices into a buffer-containing tray - with anterior surfaces facing up - for subsequent staining.
Add solution of warm triphenyl tetrazolium chloride, TTC, a water-soluble redox dye, over the brain slices. Incubate under dark conditions, as TTC is light-sensitive. TTC enters cells within the tissue section.
In living cells, active mitochondrial succinate dehydrogenase enzyme in the presence of cofactors reduces TTC, colorless when oxidized, into water-insoluble, red formazan, staining viable cells deep red. However, necrotic cells lack dehydrogenase activity; thus, TTC remains in oxidized form, staining the cells white.
Post-incubation, transfer the brain slices into an imaging tray. Separate the hemispheres in the sagittal plane. Image the brain slices.
Healthy tissue appears red, while damaged tissue appears white, differentiating necrotic tissue from viable tissue and enabling estimation of the extent of tissue damage.
After removing the brain, including the brain stem, from the skull, place the brain, with its ventral side up, in the brain matrix. Depending on the weight of the animals, choose the correct size of the brain stainless-steel matrix. Using blades, restrict the frontal and caudal parts of the brain. Then, put the blades partially into the channels between the first and the last blades.
When all the blades are inserted and arranged in parallel, press all the blades down with the palm at the same time to cut the brain into 2-millimeter coronal slices. Then, grasp the blades firmly along the sides with two fingers, and remove them together with the sliced brain from the matrix. Arrange the brain slices one by one in a tray, ensuring that the anterior surface of each slice is always facing up.
Next, pour warm 1% triphenyl tetrazolium chloride solution and PBS onto the brain slices, and incubate for 8 minutes at 37 degrees Celsius in the dark. After incubation, transfer the brain slices into a blue plastic tray to capture images. Arrange the brain slices in sequential order from the frontal to the caudal part, and use a scalpel to separate the hemispheres in the sagittal plane.
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