The overall goal of this procedure is to assess the permeability of brain vasculature in mice using fluorescently-labeled tracers to assess blood-brain barrier dysfunction in animal models. This method can help answer key questions in the blood-brain barrier field pertaining to neuro-vascular permeability in diseases and its recovery upon therapy. The main advantage of this method is that it is simple and quantitative.
It gives the possibility for concurrent assessment of permeability by fluorometry, which is quantitative, and also fluorescence microscopy for regional defenses. The implications of this technique extend towards diagnosis and therapy of several neurological disorders, including brain tumors, stroke, and Alzheimer's disease because these diseases are associated with life-threatening brain edema and improvement of BBD function as a key therapeutic target. Demonstrating part of the procedure will be Miss Jadranka Macas, a scientific technology officer at the institute.
Intraperitoneally inject mice with 100 microliters of two-millimolar tracer solution. Choose lysine-fixable tracers, such as dextrans labeled with FITC and TMR with distinct excitation emission spectra for minimal interference between the dyes. If combining tracers, inject 200 microliters of combined tracer solution intraperitoneally.
Include vehicle-only injections to serve as sham controls for autofluorescence background subtraction. After each injection, allow five minutes before deeply anesthetizing the mice according to the approved institutional protocols. Prepare the animals for cardiac perfusion 10 minutes after anesthetic administration.
Check the absence of paw twitch response to ensure that the mouse has reached a surgical plane of anesthesia. Lay the mouse to be perfused on its back and apply 80%ethanol on the skin of the abdominal area. After using small scissors to create a two-centimeter incision in the abdominal wall, separate the liver from the diaphragm, and then slowly cut through the diaphragm, exposing the plural cavity.
Cut the ribcage bilaterally and fix the cut sternum to expose the left ventricle. Insert a 21-gauge butterfly needle connected to a peristaltic perfusion system in the posterior of the left ventricle. Puncture the right atrium and then use a one-milliliter pipette tip with the end cut off to quickly collect the 200 to 300 microliters of released blood and transfer to a serum collection tube.
Store the collected blood on ice. As soon as the blood is collected, switch on the perfusion system and perfuse the animal for three minutes with room temperature calcium and magnesium ion-free PBS. Assess perfusion quality by noting the color of the liver and kidneys, which appear pale after perfusion.
After harvesting the brain, verify that there is no blood visible in vessels of the meninges. The sample should be excluded from the permeability analysis if the perfusion quality is poor. Use a scalpel to separate the brain into two hemi-brains.
Then dissect the cerebellum and olfactory lobe from one hemi-brain, and transfer the hemi-cerebrum to a two-milliliter tube. Place one harvested kidney to another two-milliliter tube. Immediately place the samples on dry ice.
Natively embed the remaining kidney and hemi-brain with cerebellum and olfactory lobes intact in TissueTech optimal cutting temperature compound, and put it on dry ice. Lastly, after centrifuging the blood samples at 10, 000 G for 10 minutes at four degrees Celsius, transfer the serum supernatants to 1.5-milliliter tubes and place them in the dry ice container. Transfer the samples to the minus 80 degrees Celsius freezer as homogenization efficiency increases after freeze-thawing.
Place the hemi-cerebrum and kidney samples on ice to thaw, and then weigh the tubes containing the organs. From these weights, subtract the mean value of approximately 20 empty tubes to get the tissue weight. Add 300 microliters of cold PBS to the tube containing kidney and 200 microliters to the tubes containing hemi-cerebrum.
Homogenize each sample in the original microfuge tube with a PTFE pestle attached to an electric overhead stirrer, using about 15 strokes. Store the homogenized samples on ice, protected from light. Rinse the pestle with PBS in between samples and wipe dry before proceeding to the next sample.
When all samples have been homogenized, centrifuge for 20 minutes at 15, 000 G and four degrees Celsius. Following centrifugation, transfer the supernatants to new 1.5-milliliter tubes on ice before proceeding to fluorescence measurement and quantification. Transfer the supernatant to a 384-well plate and insert the plate into fluorescence reader.
Make sure the right excitation and emission wavelengths are included for the tracer and set the gain to Optimal. Start the measurement, and export the data as a spreadsheet to perform the calculations as described in the protocol. On the day of staining, thaw 10-micron cryostat sections mounted on slides at 37 degrees Celsius for 10 minutes.
Then, fix the sections with 4%paraformaldehyde for 10 minutes at room temperature, followed by quick-washing in PBS. Permeabilize and block non-specific binding in the sections by incubating in sterile PBS containing 1%PSA and 0.5%Triton X-100 for one hour at room temperature. After blocking, incubate the slides with a one-to-100 dilution of CD31 primary antibody for 1.5 hours at room temperature.
After washing with PBS three times for five minutes each, perform secondary antibody incubation for one hour at room temperature with species-specific fluorescently labeled antibodies. Mount the stained sections with Aqua-Poly/Mount and leave overnight for polymerization at room temperature in the dark. Finally, acquire images using a spectral imaging confocal laser scanning microscope system.
In order to establish the tracer circulation time for the permeability assay, a long circulation time of two hours is compared to a short circulation time of 15 minutes post tracer injection. The longer circulation time of two hours leads to low amounts of tracers accumulation in kidney compared to a short 15-minute circulation time potentially due to a high clearance FITC dextran shown in green from the vascular compartment. This effect is even more dramatic in the brain due to the tight blood-brain barrier.
CD31 staining seen in the red channel confirmed the presence of vessels in the kidney at both time points examined and in the brain. Once mastered, this technique can be performed within five to six hours for 10 mice with two scientists working in tandem. While attempting this technique, it is important to remember to use tracers of right molecular weight, chart and fluorescence characteristics in order to answer the questions regarding the blood-brain barrier permeability.
Following this procedure, other methods like immuno-histochemistry for BBD and disease fill on factors can be performed to answer additional questions, like severity and localization of disease site.
