New Technique for Measuring BBB Breakdown, Brain Infarct Volume and Brain Edema

For the rats in Group 3, 2% Evans blue in saline (4 ml/kg) was injected through the cannulated tail vein as a blood-brain permeability tracer and was allowed to circulate for 60 min. The rats’ chests were opened and the animals were perfused with cooled saline through the left ventricle. Their brains were quickly isolated and sectioned into 6 coronal slices, each 2 mm of thickness. The set of slices from each brain was then scanned (this scan was needed to later assess the effects of Evans blue staining on the accuracy of measuring infarct zone and is not required in the final protocol) and incubated for 30 min at 37°C in 0.05% TTC. The slices were again scanned with an optical scanner. Infarct zone and brain edema was measured with the National Institutes of Health ImageJ software 1.37v (Boyko et al., 2011c, 2019a). For these measurements, the computer program converts the scan into a black and white image and then uses a threshold function to mask and calculate the pixels that are either black or white (see Figure 1). In order to remove the effects of the Evans dye on this process, we added a blue filter using the Channel Mixer function (Image > Adjustments > Channel Mixer) from the Adobe Photoshop CS2 software program prior to calculating brain infarct zone and brain edema. After scanned, the following was performed in order to measure BBB disruption. The brain slice samples were divided into the left and right hemisphere and measurements of vascular permeability were made by comparing their weight with pre-weighed loci in the six slices. Each brain area was weighted and homogenized in 1 ml of 50% trichloroacetic acid (weight/volume) and was centrifuged at 10,000 × g for 20 min and the supernatant was diluted 1:3 with 96% ethanol. A fluorescence detector was used at an excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 10 nm).

Histological brain scans from sham-operated rats and rats post-MCAO. Column one is a sham-operated brain slice without staining. Column two is a sham-operated brain slice with TTC staining. Column three is a post-MCAO brain slice with Evans blue staining. Column four is a post-MCAO brain slice with Evans blue and TTC staining.