Determining Blood-Brain Barrier Disruption Using Fluorescent Dyes in an EAE Mouse Model

Abstract

Source: Tietz, S. M., et al., Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo. J. Vis. Exp. (2019).

This video demonstrates a procedure for assessing blood-brain barrier disruption in an EAE mouse model. Fluorescent dyes of different sizes are injected retro-orbitally and circulate through the brain's blood vessels. The smaller dye leaks through gaps into the brain tissue, while the larger dye enters only if the disruption is severe, correlating with the extent of barrier damage.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. In vivo permeability assay

1. Preparations of solutions:
   1. For dextran stock solutions, dissolve 10 mg of 10 kDa Dextran Alexa Fluor 488 as well as 3 kDa Dextran Texas Red in 500 µL of 0.9% sodium chloride solution (20 mg/mL).
   2. For the dextran working solution, just before injection pipet 55 µL of 10 kDa Dextran Alexa Fluor 488 stock solution (20 mg/mL) onto a piece of sealing film and add 55 µL of 3 kDa dextran Texas Red stock solution (20 mg/mL). Mix and collect 100 µL into a disposable fine syringe (final concentration 2 mg/100 µL).
   3. For the 10% formaldehyde (Paraformaldehyde, or PFA) stock solution, combine 10 g of PFA extra pure powder, 100 mL of PBS, and 200 µL of 1N Sodium hydroxide (NaOH) in a clean glass beaker and heat to precisely 56 °C under stirring using a magnetic stirrer. Keep at 56 °C for 30 min until the PFA is completely dissolved. Cool down to room temperature, adjust pH to 7.4, and filter through a paper filter. Store at—20 °C. The stock solution can be diluted further using Phosphate buffered saline or PBS.
2. Shortly anesthetize healthy C57BL/6 mice or C57BL/6 mice suffering from experimental autoimmune encephalomyelitis, or EAE with isoflurane (the mouse will be sedated for approximately 1 min). Use an automated system (mice will be exposed to 4.5% isoflurane in oxygen in an induction chamber and then transferred to a facemask with 2.1% isoflurane).
3. Place the anesthetized mouse in a lateral position on the table, then intravenously (e.g. retro-orbitally) inject 100 µL of fluorescent tracers into the mouse before the mouse wakes up.
4. Immediately remove the facemask and ensure the mouse is fully awake and motile after the short anesthesia. Let the tracer circulate for 15 minutes.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dextran Alexa Fluor 488 (10,000 MW)	e.g. Molecular probes	D22910	Store at -20 °C; protect from light
Dextran Texas Red (3000 MW)	Invitrogen	D3328	Store at -20 °C; protect from light
Maintenance food	e.g. PROVIMI KLIBA SA	3436
Paraformaldehyde	Merck	30525-89-4
NaCl 0.9 %	B. Braun	3535789
Sealing film e.g. Parafilm M	e.g Sigma-Aldrich	P7793
syringe 1 ml	e.g. PRIMO	62.1002