Hi, I am searching for a good protocol to measure the Blood brain barrier permeability in mice model without using the Evans blue dye.
Sodium fluorescein is often used as a smaller molecular weight tracer. It allows you to do a simple fluorescence assay with your brain homogenate to assess extent (or use a microscope if you want to assess visually) rather than evans blue which you have to score. You could also look up sucrose permeability studies and the wet weight/dry weight method of brain edema
is you can get the tisuues, I think the more good tool is the ussing chamber
http://www.harvardapparatus.com/ussing_chamber_systems.pdf
and if you can made the culture of the barrier maybe you can used TEER sytem
cheers
Sweilem,
Sodium fluorescein works as suggested by Kate above. Per a suggested protocol - after injecting the fluorescein (1 h prior to sacrifice), you should perfuse the brain with ice-cold saline until all blood is removed (runs clear), but do not perfuse with a fixing agent. Rather, snap freeze the brains (to minimize degradation), cryosection the tissue, and visualize on a fluorescence microscope.
Alternatively, if you suspect significant BBB permeation, you may also use immunofluorescence / immunohistochem microscopy to probe for serum albumin. Again, you will need to perfuse with saline post-mortem to remove all blood, after which you may follow a standard immunofluorescence procedure with a anti-SA primary antibody.
Best of luck,
Andrew
You can evaluate the physical and functional integrity of the BBB in mice using the protocol described in this paper:
http://www.ncbi.nlm.nih.gov/pubmed/24631967
Thank you very Much!
Your recommendations helped a lot.
Best wishes
Sweilem
I would suggest these 2 papers:
•Alata, W., Paris-Robidas, S., Emond, V., Bourasset, F. and Calon, F., Brain uptake of a fluorescent vector targeting the transferrin receptor: a novel application of in situ brain perfusion., Mol Pharm, 11 (2014) 243-253.
•Bourasset, F., Ouellet, M., Tremblay, C., Julien, C., Do, T.M., Oddo, S., LaFerla, F. and Calon, F., Reduction of the cerebrovascular volume in a transgenic mouse model of Alzheimer’s disease., Neuropharmacology, 56 (2009) 808-813.
Thank you ALL very much for your help
I really appreciate that.
You suggestions helped me a lot.
best wishes
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