Blood-brain barrier permeability assay.

The protocol used to assess blood-brain barrier permeability was adapted from the protocol previously described by Devraj et al. (81). Briefly, 100 μl 2 mM sodium fluorescein (F6377-100G; Sigma) was administered intraperitoneally to newborn mice at the indicated time points using a 1-ml U-100 syringe (28 gauge by 1/2 inch). After 1 h, blood was collected using heparinized capillaries, and brain tissue was collected following perfusion with cold PBS. Blood samples were centrifuged, and serum was collected for fluorescein intensity measurement. Brain tissue was flash frozen and then thawed and homogenized in sterile PBS. Homogenized brain samples were centrifuged, and the supernatant was collected for fluorescein intensity measurement. Fluorescein intensity was measured in a VICTOR Nivo multimode microplate reader (Perkin Elmer) at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Serum and brain samples from sham-treated newborn mice were used as blanks for all samples. The blood-brain barrier permeability index was calculated, using raw fluorescence units (RFUs), as permeability index (ml/g) = (tissue RFUs/g tissue weight)/(serum RFUs/ml serum).