Increased BBB Hyperpermeability with Mast Cell Activation and Cuprizone

BBB impairment seen with Cuprizone administration.

(A) Fluorescent images of vessels stained with Evans blue showing increased extravasation of the dye during  cuprizone treatment. 200x mag, Scale bar = 200 μm. (B) Intensity measures showed highest levels in vessel permeability at 3 days with significant differences measured at all time points (mean ± s.e.m.). n = 6, Asterisk denotes significance (p = < 0.05) (C) Western Blot analysis of corpus callosum. Occludin at 3 days of cuprizone administration. Representative blots of Occludin and GAPDH in mice after 3 days of cuprizone administration. (D) Densitometry of western blot shows significant decrease in Occludin levels compared to controls. Data are expressed as a percentage of control with the highest control set at 100. n = 3. Asterisk denotes significance (p = < 0.05).

Myelin signal measures through time period of cuprizone treatment.

(A) T2-RARE MR imaging of cuprizone induced demyelination in the corpus callosum. No visible changes in myelin signal in the corpus callosum was detected after 2 weeks of treatment and these did not differ between rostral and caudal regions as has been previously reported.[41,42] (B) Quantification of corpus callosum signal intensity shows no increases in T2 signal between groups or across time (mean± s.e.m.). Acquisition parameters: TR = 85 ms, TE = 2800.218 ms. White arrows indicate corpus callosum position. (n = 6, p < 0.05) (C) Fluoromyelin fluorescent staining of cortical and corpus callosum tissue in 3 days to 6 weeks of cuprizone treated tissue. (D-E) Quantification of cortex and corpus callosum showed no significant decrease in fluorescent signal through 2 weeks of treatment with cuprizone (mean± s.e.m.). Scale bar = 500 μm, n = 6. Asterisk denotes significance (p = < 0.05). 

A) 4x images show changes across cortical and corpus callosum. Cell counts were taken from 20x Cortex and 20x Corpus Callosum regions of each slice. (B-C) Quantification of GFAP+ astrocytes within both regions (mean ± s.e.m.). Reactive gliosis was significantly increased by 2 weeks in cortical tissue but not in the corpus callosum over the control tissue. 4x Scale bar = 500 μm, 20x Scale bar = 200 μm, n = 6. Asterisk denotes significance (p = < 0.05).    

(A) Fluorescent images of IBA-1 stained microglia in both cortical and corpus callosum regions. (B-C) Total counts of microglia increased significantly in both cortex and corpus callosum after 1 week of cuprizone administration. Within the total counts, there was significant increases at 1 week for both inactive and active morphologies of microglia in both regions (mean ± s.e.m.). 20x Scale bar = 200 μm, n = 6. Asterisk denotes significance (p = < 0.05).