Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo

The methods presented here allow us to study the molecular mechanisms of immune cell migration across the brain barriers in experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. By investigating blood-brain barrier permeability and matrix-metalloprotease activity at the same time in spatial correlation to immune cell infiltration in brain tissue sections, we can precisely associate these neuroinflammatory events to the clinical signs of EAE. Professional handling of diseased mice needs in-depth training, as well as the correct scoring of the clinical signs of EAE.

Thus, the visual demonstration is beneficial. Begin this procedure with anesthetization of C57-Black/6 mice that have been housed in specific-pathogen-free conditions as described in the text protocol. Fix the anesthetized mouse in one hand, and inject 30 microliters of MOG peptide and complete Freund's adjuvant emulsion subcutaneously into each of the hind leg flanks, in close proximity to the inguinal lymph nodes.

Now, place the mouse on its belly, and inject 20 microliters of the MOG-peptide emulsion into the soft fatty tissue on both the left and right at the tail root. Also, inject a little droplet of the emulsion into the neck of the mouse. Inject 100 microliters of pertussis toxin solution intraperitoneally into the mouse.

Hold the head of the mouse below the body center to avoid injection into the intestine. Replace the maintenance diet to the breeding diet in order to provide the mice with food of higher energy content before and during the expected clinical disease. Repeat the pertussis toxin injection 48 hours after the first treatment.

Check the health status of EAE mice every morning by taking a look inside the cages. Score EAE mice every afternoon. Take every individual mouse included in the EAE experiment out of the cage, and check whether the tail has tonus.

Move the tail upward with a finger. A healthy mouse will keep its tail up. If clinical EAE has started, the tail tonus will be lower, visible by a gradual drop of the tail.

Eventually, the mouse will not be able to lift its tail at all. Place every individual mouse included in the EAE experiment on the clean bench to observe and document the walking behavior. See the text for scoring criteria for the assessment of disease severity, which is the EAE score.

Assess and document the weight of every mouse included in the experiment. For preparing dextran stock solutions, dissolve 10 milligrams of three-kilodalton Dextran Texas Red in 500 microliters of 0.9%sodium chloride solution and five milligrams of 10-kilodalton Dextran Alexa Fluor 488 in 250 microliters of 0.9%sodium chloride solution. For the dextran working solution, just before injection, pipette 55 microliters of 10-kilodalton Dextran Alexa Fluor 488 stock solution onto a piece of sealing film.

Then, add 55 microliters of three-kilodalton Dextran Texas Red stock solution. Mix and collect 100 microliters into a disposable fine syringe. Next, place an anesthetized mouse in the lateral position on the table.

Intravenously inject 100 microliters of fluorescent tracers into the mouse before the mouse wakes up. Let the tracer circulate for 15 minutes before performing perfusion of mice as detailed in the text protocol. Fill a flat dewar container with crushed dry ice, and add 2-methylbutane until the liquid reaches about one centimeter above the dry ice pack.

Cover with a loose lid, such as an ice bucket cover. After clipping off the head of the perfused mouse with sharp scissors, remove the skin and ears using a smaller set of scissors. Carefully dissect the skullcap by cutting from the foramen magnum towards the front at the left and the right side, to allow upfolding of the skullcap over the brain.

Then, carefully lift out the intact brain from the base of the skull while severing the optical nerves using a flat metal spatula. Place the brain on aluminum foil. Cut the brain in three pieces by placing two coronal cuts.

The three pieces represent the frontal brain, middle brain, and cerebellum with brain stem. Fill a Cryomold to the first quarter with optimum-temperature cutting matrix. Then, place the brain slices into the Cryomold, with the anterior side of each brain piece facing downwards.

Cover the tissue completely with matrix. Place the Cryomold with the tissue in a horizontal orientation into the 2-methylbutane bath. Ensure that the tissue freezes from the bottom to the top within one minute by avoiding dipping the entire tissue block into the bath.

Transfer the frozen tissue to minus 80 degrees Celsius for storage. See the text protocol for preparation of the frozen tissue sections. Retrieve six-micron, non-fixed brain tissue sections that had been prepared from EAE mice.

In a fume hood, thaw the sections inside the plastic freezing box containing silica gel in the lid to avoid retention of water in the tissue. Separate two tissue sections on one slide by drawing lines with a water-repelling pen. Centrifuge reaction solutions for five minutes at 13, 300 times g at room temperature, and pre-warm to 37 degrees Celsius using a water bath.

Then, rehydrate the tissue sections for five minutes at 37 degrees Celsius using 1X reaction buffer. After five minutes, pour off the 1X reaction buffer. Add reaction solution on the tissue sections, and incubate for four hours at 37 degrees Celsius.

Then, wash the slides five times in double-distilled water. Now, fix the sections for five minutes with ice-cold methanol at minus 20 degrees Celsius before washing them once with PBS at room temperature. Add 1%BSA in PBS to the section, and incubate for 20 minutes at room temperature.

Then, discard the buffer from the section by flipping the slide on a tissue. Add primary antibody cocktail to the section, and incubate for one hour at room temperature. After washing the sections twice with PBS, add secondary antibody cocktail, and incubate for one hour at room temperature.

Then, wash the sections twice with PBS, and mount the slides with embedding solution. After letting the mounted sections dry overnight at room temperature, analyze stained tissue sections using a fluorescent microscope. The EAE disease severity increases until the peak of the disease, which can be expected between days 16 and 20 after EAE induction.

At the peak of clinical EAE, mice also showed the lowest body weight, which increases in correlation to the amelioration of EAE in the remission phase of the disease. Shown here is a representative picture of the choroid plexus and the adjacent brain parenchyma of a C57-Black/6 mouse suffering from EAE. The mouse was intravenously injected 15 minutes before sacrificing.

Tracers readily diffuse across the fenestrated microvessels into the stoma of the choroid plexus, while the blood-brain barrier does not allow the diffusion of circulating tracers into the brain parenchyma, which is thus devoid of any fluorescent signal. These images show representative leaky blood vessels in the cerebellum of a C57-Black/6 mouse suffering from EAE. Arrowheads indicate tracers diffused across the brain microvessels into the CNS parenchyma, suggesting that the blood-brain barrier function is impaired in these vessels.

In the CNS parenchyma, green and red fluorescent signal is visible outside of the blood vessel walls. Correct subcutaneous placement of the MOG-CFA emulsion is essential to mount the appropriate immune response leading to EAE. Intraperitoneal and intramuscular injection should be avoided.

Prepare CFA under a fume hood to avoid inhalation of the attenuated Mycobacterium. Also, avoid one's self-injection with the MOG-CFA emulsion, as this may cause condyloma or even lead to EAE in individuals that are genetically predisposed. Brain-infiltrating immune cells can be isolated from the CNS of mice suffering from EAE to quantify infiltrating immune cell subsets during neuroinflammation by flow cytometry.

Assessing the integrity of the endothelial and the glial brain barriers can be translated to the analysis of genetically modified mouse models or to models of other neurological disorders.