Visualizing the Gatekeeper: Evan's Blue Dye-Based Assessment of Blood-Brain Barrier Permeability in Adult Zebrafish

This research focuses on developing a reliable, simple, and cost-effective method to assess blood-brain barrier integrity in adult zebrafish. Most zebrafish studies don't have an easy, reliable way to measure blood-brain barrier disruption, and existing methods are often complex or costly. So this protocol offers a practical and affordable solution.

This protocol is simple, cost-effective, and combines both visual and measurable analysis, all without the need for expensive equipment or complicated steps. By providing a simple, reproducible protocol, it will make it easier for labs to explore neurovascular changes in adult zebrafish disease models. To begin, anesthetize the zebrafish by gradually lowering the water temperature from 12 degrees Celsius to seven degrees Celsius until the fish no longer responds to tactile stimuli.

Using a 31-gauge insulin syringe, inject 20 microliters of 1%Evans blue, or EB dye, into the intraperitoneal cavity of the fish, and apply gentle pressure at the injection site for 10 seconds to prevent backflow. Now transfer the fish to a recovery tank and allow recovery for 30 minutes. Monitor the fish for injury, mortality, or behavioral changes during this time.

For establishing a positive control, prepare a sterile 31-gauge needle attached to an insulin syringe to perform a stab wound injury procedure. After a 30-minute recovery period, anesthetize the fish again, as demonstrated earlier. Position the fish upright on a flat, cold surface, such as an ice gel pack.

Insert the needle into one side of the cephalon at a 90-degree angle to a depth not exceeding 1.5 millimeters. Then transfer the fish to a recovery tank and let it recover for 30 minutes. After euthanizing the fish by hypothermia, use a sterile blade or surgical scissors to decapitate the euthanized fish.

Rinse the head twice in 1X PBS using a 1:20 ratio of fish to PBS to remove excess blood and Evans blue dye. Then fix the head in 4%paraformaldehyde using a 1:20 ratio of fish to fixative, and incubate at four degrees Celsius overnight. Place the fixed head in a clean Petri dish and dissect the brain using pointed-end tweezers.

Continuously add PBS dropwise during dissection to keep the tissue hydrated and prevent drying. Rinse the dissected brain twice in two separate PBS baths and place the tissue on a 1%agarose plate to retain moisture and prevent shrinkage. Observe the dissected brain tissues under a stereo microscope.

To begin quantitative analysis, download and install Fiji software and open the brain image file in Fiji. Perform color deconvolution by selecting Image, followed by Color, Color Deconvolution, and choose RGB. This will result in three separate images representing red, green, and blue hues.

Now, activate the blue channel image, usually labeled Color 3, by clicking on the image. Convert it to 8-bit grayscale by selecting Image, followed by Type, and 8-bit. Next, to measure the gray intensity of the 8-bit image, set the measurement parameters by selecting Analyze and Set Measurements.

Then choose the area and mean gray value. Using the rectangle selection tool, draw a uniform-sized region of interest and measure the gray intensity by selecting Analyze, followed by Measure. Normal zebrafish displayed a typical brownish skin tone, and the EB-injected zebrafish exhibited a noticeable bluish hue.

To investigate blood-brain barrier integrity in the adult zebrafish Parkinsonian model, EB dye extravasation into brain tissues was compared between saline-injected control and Parkinsonian zebrafish. In the negative control group, blood vessels in the brain appeared reddish, indicating the absence of EB dye extravasation. In contrast, the saline-injected control, positive control, and Parkinsonian groups all showed bluish coloration in brain vasculature, confirming successful systemic circulation of EB dye.

Dissected brains from the negative control group appeared whitish with clearly visible blood vessels, confirming the absence of EB staining. In the positive control injury group, intense blue coloration surrounding the brain tissue indicated dye leakage due to deliberate blood-brain barrier disruption. In the saline-injected group, EB staining remained confined to the blood vessels, indicating an intact blood-brain barrier.

The Parkinsonian group showed a darker, more diffuse blue staining pattern, indicating significant EB dye leakage and blood-brain barrier compromise. Quantitative grayscale analysis revealed significantly lower gray intensity values in the Parkinsonian group compared to the saline-injected group.