(555.27) The Cold Receptor TRPM8 Mediates the Antioxidant and Cytoprotective Effects of Mild Hypothermia in Cerebral Endothelial Cells During Inflammation

Poster Board Number: E87

Rong Zhang (University of Tennessee Health Science Center), Jianxiong Liu (University of Tennessee Health Science Center), Helena Parfenova (University of Tennessee Health Science Center)

Cerebral vascular endothelium, the key component of the neurovascular unit, is most vulnerable to oxidative stress injury. Our previous reports in newborn pigs indicated that brain hypothermia prevents cerebral endothelial injury by oxidative stress caused by neonatal seizures and asphyxia. We used primary cerebral vascular endothelial cells (CVEC) from newborn pigs as an in vitro model of oxidative stress condition in the brain caused by inflammation. During normothermia (37˚C), CVEC respond to a pro-inflammatory cytokine TNF-alpha (30 ng/ml) by excessive formation of reactive oxygen species (ROS) leading to cell death by apoptosis (caspase-3 activation, DNA fragmentation, loss of cell-cell contacts).  Remarkably, mild hypothermia (24.5°C) reduced ROS and prevented apoptosis in CVEC exposed to TNF-alpha. We tested the hypothesis that the cold sensing menthol receptor TRPM8 mediates the cytoprotective effects of mild hypothermia in cerebral endothelium. TRPM8 (mRNA and protein) is expressed in CVEC. The TRPM8 agonist icilin (5 µM) reduced ROS and prevented apoptosis in CVEC exposed to TNF-alpha at 37˚C, thus mimicking the cytoprotective effects of mild cooling. The TRPM8 antagonist AMTB (20 µM) reversed the icilin-mediated cytoprotective effects during inflammation. The cell surface biotinylation technique suggested that mild hypothermia and icilin (at 37˚C) promote a time-dependent TRPM8 translocation from an intracellular compartment to the plasma membrane concomitantly with the receptor activation. We used the in vitro model of the blood-brain barrier (BBB) to test the functional consequences of TRPM8-mediated cytoprotection. Confluent monolayers of CVEC cultured on transwell inserts with semipermeable membranes were exposed to TNF-alpha during normothermia and mild hypothermia conditions. The BBB properties were detected by transendothelial electrical resistance (TEER) and the permeability to 3 kDa fluorescent Dextran.  During a 2-6 h exposure to TNF-alpha at 37°C, TEER was time-dependently reduced, and the Dextran permeability was elevated indicating the BBB opening. Mild hypothermia (24.5°C) and icilin (at 37˚C) largely preserved the BBB properties during inflammatory conditions. Overall, our findings demonstrate that mild hypothermia, via a TRPM8-mediated mechanism, reduces oxidative stress, promotes cell survival, and prevents BBB damage during inflammation. The TRPM8 targeting approaches may provide a promising therapeutic strategy for cerebrovascular disease caused by brain inflammation.

Support or Funding Information

The National Institutes of Health Grants NS101717 and NS105655 (to H. Parfenova)