CpG-C does not affect BBB leakage or cellular permeability.

(a-c) Biocytin-TMR was intravenously injected into animals expressing GFP under the claudin-5 promotor 24 hours following CpG-C or control treatment, and 90 minutes later brains were perfused and removed. Biocytin-TMR intensity (normalized to intensity levels in the liver, not shown) and IgG staining intensity were similar in CpG-C–treated and control animals (two-tailed unpaired Student t test, t(22) = 0.3758, p = 0.7106; a-b). Moreover, no difference in number of gaps in claudin-5 strands was found between control and CpG-C–treated animals (two-tailed unpaired Student t test, t(22) = 0.4283, p = 0.6726; c). (d) Brain sections of WT animals treated with CpG-C or PBS were stained for CD4 or CD68 24 hours following treatment. No infiltration of immune cells was detected (spinal cords of EAE mice served as positive controls; right panels). (e-f) Using two-photon imaging, we followed leakage of a low (NaF; 376 Da) and a high (Texas-Red; 70kDa) molecular weight dextrans. Intensities of representative images (e) were auto-adjusted in Fiji for display purposes only. No differences were found between control and CpG-C–treated animals at baseline (p = 0.8567 and p = 0.8421 for NaF and Texas-Red, respectively), following a single CpG-C treatment (p = 0.9243 and p = 0.2419 for NaF and Texas-Red, respectively), and following two CpG-C treatments (p = 0.4656 and p = 0.3918 for NaF and Texas-Red, respectively. See Methods for an explanation of the quantification; f). For (a-c), each sample consisted of an average of at least five images that were analyzed. Samples were taken from four different anatomical brain regions (cortex, midbrain, cerebellum, and hippocampus) in three mice/group (See S5 Fig for regional presentations). Scale bar is 50 μm. Box plot whiskers represent minimum–maximum range (a-c), and data in (f) are presented as mean (±SEM). The underlying data for this figure can be found in S1 Data. A.U., arbitrary units; BBB, blood-brain barrier; CD, cluster of differentiation; EAE, experimental autoimmune encephalomyelitis; GFP, green fluorescent protein; IgG, immunoglobulin G; TMR, tetramethylrhodamine; WT, wild type.