%A Zitta,Karina %A Peeters-Scholte,Cacha %A Sommer,Lena %A Gruenewald,Matthias %A Hummitzsch,Lars %A Parczany,Kerstin %A Steinfath,Markus %A Albrecht,Martin %D 2018 %J Frontiers in Pharmacology %C %F %G English %K Hypothermia,Neuroprotection,Asphyxia,2-iminobiotin,Cell damage,Hypoxia-Ischemia, Brain,Apoptosis,In-vitro models %Q %R 10.3389/fphar.2017.00971 %W %L %M %P %7 %8 2018-January-11 %9 Original Research %+ Martin Albrecht,Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein,Germany,martin.albrecht@uksh.de %# %! Attenuation of hypoxia-induced cell damage by 2-iminobiotin and hypothermia %* %< %T 2-Iminobiotin Superimposed on Hypothermia Protects Human Neuronal Cells from Hypoxia-Induced Cell Damage: An in Vitro Study %U https://www.frontiersin.org/articles/10.3389/fphar.2017.00971 %V 8 %0 JOURNAL ARTICLE %@ 1663-9812 %X Perinatal asphyxia represents one of the major causes of neonatal morbidity and mortality. Hypothermia is currently the only established treatment for hypoxic-ischemic encephalopathy (HIE), but additional pharmacological strategies are being explored to further reduce the damage after perinatal asphyxia. The aim of this study was to evaluate whether 2-iminobiotin (2-IB) superimposed on hypothermia has the potential to attenuate hypoxia-induced injury of neuronal cells. In vitro hypoxia was induced for 7 h in neuronal IMR-32 cell cultures. Afterwards, all cultures were subjected to 25 h of hypothermia (33.5°C), and incubated with vehicle or 2-IB (10, 30, 50, 100, and 300 ng/ml). Cell morphology was evaluated by brightfield microscopy. Cell damage was analyzed by LDH assays. Production of reactive oxygen species (ROS) was measured using fluorometric assays. Western blotting for PARP, Caspase-3, and the phosphorylated forms of akt and erk1/2 was conducted. To evaluate early apoptotic events and signaling, cell protein was isolated 4 h post-hypoxia and human apoptosis proteome profiler arrays were performed. Twenty-five hour after the hypoxic insult, clear morphological signs of cell damage were visible and significant LDH release as well as ROS production were observed even under hypothermic conditions. Post-hypoxic application of 2-IB (10 and 30 ng/ml) reduced the hypoxia-induced LDH release but not ROS production. Phosphorylation of erk1/2 was significantly increased after hypoxia, while phosphorylation of akt, protein expression of Caspase-3 and cleavage of PARP were only slightly increased. Addition of 2-IB did not affect any of the investigated proteins. Apoptosis proteome profiler arrays performed with cellular protein obtained 4 h after hypoxia revealed that post-hypoxic application of 2-IB resulted in a ≥ 25% down regulation of 10/35 apoptosis-related proteins: Bad, Bax, Bcl-2, cleaved Caspase-3, TRAILR1, TRAILR2, PON2, p21, p27, and phospho Rad17. In summary, addition of 2-IB during hypothermia is able to attenuate hypoxia-induced neuronal cell damage in vitro. Combination treatment of hypothermia with 2-IB could be a promising strategy to reduce hypoxia-induced neuronal cell damage and should be considered in further animal and clinical studies.