Introduction
Fatty acids are critical for the development and function of all organisms, in particular, very long chain of polyunsaturated fatty acids, such as alpha-linolenic acid (ALA) are necessary for the health and maintenance of mammals. ALA is a potent activator of some background 2P-domain K+ channels, TREK and TRAAK, involved in the regulation of cell excitability. We have shown that (1) ALA is a powerful neuroprotector in acute cerebral ischemia and is a preconditioning inductor of delayed tolerance to cerebral ischemia; (2) TREK-1-KO mice are more sensitive to cerebral ischemia and not protected by ALA. Altogether this indicates that TREK-1 is a crucial mediator of the neuronal protection. Although there is some evidence that TREK-1 is expressed in neurons as well as in some vessels, little is know on the role of the vascular TREK-1 in the resistance to ischemia. We have therefore analyzed the brain vessel distribution of TREK-1 mRNA using RT-PCR, in situ hybridization and electron microscopy, as well as TREK-1 protein localization by immunohistochemistry. Moreover, we are investigating the role of this channel in association with cerebral blood flow and ALA-induced acute cerebral protection.
Methods
RT-PCR was performed on different mouse and rat vessel mRNAs and visualized on 2% agarose gel. For in situ hybridization, the oligonucleotide probe (CAC AAT GGT CCT CTG GGA AAT CTC CTG AGG) was DIG-dUTP labelled. TREK-1 mRNA was detected with a Fluorescein-conjugated anti-DIG antibody. For electron microscopy, probes were immunogold-labeled with an anti-DIG antibody and silver enhanced. Ultrathin sections were examined using a JEOL 6700F field emission scanning electron microscope. For immunostaining, small fixed arteries were detected with an anti-TREK-1 primary antibody (Alomone) and observed using an epifluorescence microscope. Monoclonal mouse anti-CD31 antibody and Hoechst, a blue-fluorescent nuclei acid stain, were used to determine which cell type expresses TREK-1. To assess the effect of a neuroprotective dose of ALA on the CBF, we monitored it using a laser-Doppler in WT and TREK-1-KO animals within the protection window described for a single dose of 500 nM ALA.
Results
This study shows that TREK-1channel is present throughout all the mouse vessels and more abundant in brain arteries. In situ techniques using epifluorescence and electron microscope combined with immunohistochemistry provide compelling evidence for the expression of these channels throughout the vessels in both myocytes and endothelial cell layers. CBF-monitoring shows that an injection of ALA increased the CBF in rats as well as in WT mice. Further experiments on TREK-1 KO should establish if TREK-1 account for the vasodilatation and CBF increase induced by ALA.
Conclusion
These results provide evidence for the presence of two-pore domain K+ channels, TREK-1 in rat and mice brain arteries, in both myocytes and endothelial cells and for a modulation of the LIN-induced CBF at a dose known to be neuroprotective. Collectively, they strongly suggest that activation of vascular TREK-1channels by ALA is most likely to participate to the brain protection observed versus ischemia, in association with the neuronal activation of this channel.