3.7 FFA inhibited the upregulation of TRPM4 induced by CA/CPR in
the neurovascular unit
Our previous studies have shown that glibenclamide, a selective
inhibitor of SUR1, improved neurologic function after CA/CPR
[18-20]. FFA has an established function in inhibiting the TRPM4
channel in the micromolar range [35]. We hypothesized that FFA may
prime the brain to better stand ischemic injury by blocking the
overactivated TRPM4 channel. As expected, both the mRNA and protein
levels of TRPM4 were upregulated at 24 h after ROSC (Fig. 8A, B). Double
immunofluorescence staining indicated that TRPM4 was expressed and
localized in neurons (NeuN-positive), microglia (Iba1-positive),
astrocytes (GFAP-positive) and endothelia cells (CD31-positive) (Fig.
8C). FFA treatment significantly inhibited the expression of TRPM4 after
CA/CPR (Fig. 8A, B).
To further verify the role of TRPM4 in the development of post-CA brain
injury, we used Trpm4−/− mice to receive CA/CPR
operation and observed the neurologic outcome and neuropathological
manifestations. The genotype of mice was confirmed by extracting DNA
from mice tail and then separating DNA in agarose gel (Fig. 9A).
Compared with WT mice,Trpm4−/−mice presented statistically higher neurological function
score
after CA/CPR (Fig. 9B). Furthermore, in comparison to WT mice,Trpm4−/− mice exhibited lighter histological
damages and less IgG leakage (Fig. 9C-D, 10A-B). Besides, gene deletion
of Trpm4distinctly
decreased the percentage of
CD16/32+Iba1+ microglia/macrophages,
while increasing the percentage of
Arg1+Iba1+ microglia/macrophages,
when compared with WT (Fig. 10C, D). However, no additional
neuroprotection was observed in Trpm4−/− mice
treated with FFA (Fig. 9B-D, 10). These results, when taken together,
proved that the TRPM4 channel was critically involved in brain injury
after CA/CPR. Meanwhile, FFA exerted neuroprotection, at least in part,
via targeting the TRPM4 channel (Fig. 11).