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.