Figure 8. Endothelial cell TRPA1 channel activity protects against ischemic strokes.

(A) Representative photographs of brain slices (left) and summary data (right) showing significantly greater ischemic damage 24 hr after MCAO in Trpa1 ecKO mice compared with Trpa1^fl/fl. Brain slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC), which stains metabolically active tissue red, whereas infarcted tissue remains unstained (white). Infarcted areas were quantified and expressed as a percentage of total hemisphere area (*p<0.05, Student’s t-test; n = 5–5 mice). (B) Representative photographs of brain slices (left) and summary data (right) showing reduced cerebral ischemic damage in wildtype C57/bl6 mice treated with the TRPA1 channel activator cinnamaldehyde (CinA, 50 mg/kg i.p.), injected 15 min after MCAO (*p<0.05 for CinA vs. vehicle, Student’s t-test; n = 5–6 mice). (C) Representative photographs of brain slices (left) and summary data (right) showing that the protective effects of CinA were blunted in Trpa1 ecKO mice (*p<0.05 for CinA-treated Trpa1 ecKO mice vs. CinA-treated Trpa1^fl/fl mice, Student’s t-test; n = 6–5 mice). Legends for Supplemental Figures.

Figure 8—figure supplement 1. Post-MCAO perfusion is not significantly different between all experimental groups.

(A) Representative laser Doppler flowmetry trace showing a sharp reduction in perfusion of the middle cerebral artery vascular territory after MCAO. Perfusion data are shown as a % of pre-occlusion perfusion. (B–D) Laser Doppler flowmetry assessment of cerebral perfusion to the middle cerebral artery territory showed that the extent of MCA occlusion was not significantly different between mice from all treatments and genetic backgrounds.

Figure 8—figure supplement 2. Cinnamaldehyde causes cerebral artery and arteriolar dilation by activating TRPA1 channels in endothelial cells.

(A) Representative traces of the lumen diameter of pressurized cerebral arteries incubated with increasing concentrations of CinA (1 nM to 100 µM) in Trpa1^fl/fl (top trace, black) and Trpa1 ecKO mice (bottom trace, red). (B) Summary graph showing that CinA induces a concentration dependent dilation of cerebral arteries via TRPA1 channels located in endothelial cells. *p<0.05, n = 6–6 arteries from three different Trpa1^fl/fl and three different Trpa1 ecKO mice. (C–D) CinA (30 µM) induces dilation of pressurized penetrating arterioles via TRPA1 channels, as evidenced by the representative traces (C) and the summary data (D). *p<0.05. n = 5 arterioles from three different mice. Arteries and arterioles used for pressure myography experiments were not incubated with EGTA-AM or CPA.

Figure 8—figure supplement 3. Mechanism of endothelial TRPA1 channel-mediated neuroprotection.

(A) Acute hypoxia induces a mitochondria-dependent increase in superoxide production, which leads to accumulation of the lipid peroxide 4-hydroxynonenal (4-HNE) in endothelial cells. 4-HNE is an endogenous activator of the TRPA1 channel, increasing the frequency of Ca²⁺ influx events – TRPA1 sparklets, which is amplified by Ca²⁺-induced Ca²⁺-release from the endoplasmic reticulum. The subcellular increase in intracellular Ca²⁺ activates K_Ca2.3 and K_Ca3.1, resulting in K⁺ efflux and vasodilation. (B) In vivo, endothelial cell TRPA1 channel activity may act as an adaptive mechanism to prevent infarct expansion (red) by promoting vasodilation in the ischemic penumbra (orange). Endothelial-cell specific deletion of Trpa1 removes this protection, ultimately leading to an increase in infarct volume following an ischemic stroke.