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).