3.5 FFA attenuated BBB disruption after CA/CPR
To further characterize the mechanism of neuroprotection by FFA treatment, the severity of BBB disruption, a crucial cause and marker of brain edema, was examined using IgG extravasation assay in brain sections. As illustrated, visible IgG penetration into cortex, striatum, and hippocampus following CA/CPR was observed, which was remarkably reduced after treatment with FFA (Fig.6A, C).
Since endothelial tight junctions are major components in maintaining the normal barrier function of BBB, we next measured the expression levels of Occludin, Claudin-5 and ZO-1 by immunofluorescence staining. Compared with sham operation, the fluorescence intensity of Occludin, Claudin-5 and ZO-1 covering the brain vessels in the vehicle-treated post-CA mice was significantly reduced, and FFA treatment partially reversed the decrease in fluorescence intensity (Fig. 6B, D). Overall, our results clearly demonstrated that FFA could restore BBB function, partly ascribed to protection of the endothelial tight junctions following CA/CPR.
3.6 FFA downregulated pro-inflammatory cytokines expressions and upregulatedinflammation resolution-associated molecules expressions in the brain
In addition to BBB breakdown and formation of brain edema, drastic neuroinflammation occurs following CA/CPR, leading to neurological deterioration [34]. Pro-inflammatory microglia/macrophages have been reported to engage in the neuroinflammatory reaction and subsequential destruction of BBB [11]. We thus examined the polarization of microglia/macrophages, by co-labeling a pro-inflammatory marker CD16/32 and an anti-inflammatory marker Arg1 with Iba1, and measured the expression levels of molecules relevant to pro-inflammatory response and inflammation resolution. As shown, after CA/CPR, the percentage of CD16/32 and Iba1 double-positive microglia/macrophages was dramatically elevated in the vehicle group, which was decreased in the FFA group (Fig. 7A, B). In contrast, the percentage of Arg1 and Iba1 double-positive microglia/macrophages was substantially increased in the FFA group (Fig. 7A, B). Similarly, FFA treatment significantly inhibited the mRNA levels of pro-inflammatory markers IL-1β ,TNF- α ,iNOS while enhancing the levels of anti-inflammatory markersTGF-β , Arg1 , IL-10 (Fig. 7C). Consistently, further Western blot analysis showed increased expression of Arg1 and decreased expression of TNF-α and iNOS after CA/CPR in brains with FFA treatment compared with vehicle group (Fig. 7D, E).
Anti-inflammatory microglia/macrophages are associated with pro-phagocytic function towards damaged cells, which is essential for resolving inflammation. Clearance of damaged neurons was assessed by detecting the neuronal marker NeuN within Iba1+microglia/macrophages. We found that FFA strengthened the efferocytic capacity of microglia/macrophages, manifesting as the increased number of Iba1+NeuN+ cells in the FFA group compared to the vehicle group (Fig. 7F, G). Additionally, FFA treatment was associated with significantly higher levels of ICAM-1 in the brain compared with the vehicle group (Fig. 7H, I). Hence, the neuroprotective effect of FFA was correlated with reduced pro-inflammatory response and enhanced anti-inflammatory property of microglia/macrophages.