Combining 2-iminobiotin and hypothermia for reducing hypoxia-induced neuronal damage: Implications from a human cell culture model of hypoxic-ischemic brain injury

Karina Zitta1, Cacha Peeters-Scholte2, Lena Sommer1, Kerstin Parczany1, Markus Steinfath1, Martin Albrecht1
Department of Anesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Kiel, Germany 1, Neurophyxia B.V.,’s-Hertogenbosch, The Netherlands2

Background and Goal of Study: Perinatal asphyxia represents one of the major causes of neonatal morbidity and mortality. Hypothermia is currently the only established treatment for hypoxic-ischemic encephalopathy, and no specific pharmacological therapy exists so far. We have shown that the biotin analogue 2-iminobiotin (2-IB) is able to reduce neuronal cell damage both in vitro as well as in vivo. Here we evaluated whether 2-IB has the potential to attenuate hypoxia-induced injury of neuronal cells also under hypothermic conditions. 
Materials and Methods: In-vitro hypoxia was induced in cultures of IMR-32 cells. After the hypoxic injury, cells were incubated with vehicle or 2-IB (10, 30, 50, 100 and 300ng/ml) under hypothermic conditions (33.5°C). Cell damage was analyzed by LDH assays. Production of reactive oxygen species (ROS) was measured using fluorometric assays. Westernblotting for PARP and phosphorylation of akt and erk1/2 was performed. 

Results: Hypoxia led to morphological signs of cell damage. Under hypothermia, LDH measurements revealed a significant LDH increase earliest at 25h after the hypoxic insult (p<0.01 vs normoxia), while under normothermic conditions comparable cell damage was already detectable after 17h. The post-hypoxic application of 2-IB significantly reduced the hypoxia-induced LDH release under hypothermia (10ng/ml, p<0.001; 30ng/ml, p<0.01; 50ng/ml, p<0.01; 300ng/ml, p<0.05; all vs vehicle control). Application of 2-IB (30ng/ml) after hypoxia attenuated the hypoxia-induced ROS production under hypothermic conditions (hypoxia: p<0.01 vs normoxia; hypoxia+2-IB: p<0.05 vs normoxia). Higher concentrations of 2-IB were less effective in reducing hypoxia-induced ROS generation. Culture medium concentrations of hydrogen peroxide were 9-fold increased by hypoxia under  hypothermia (p<0.001 vs normoxia) but not influenced by  post-hypoxic application of 2-IB. Cleavage of PARP and akt phosphoprylation were by trend increased after the hypoxic insult, while hypoxia significantly increased the phosphorylation of the pro-survival kinase erk1/2 (p<0.05 vs normoxia). Addition of 2-IB under hypothermia did not influence cleavage or phosphorylation of any of the mentioned molecules. Conclusion: Low doses of 2-IB can even under hypothermic conditions reduce hypoxia-induced neuronal cell damage in vitro. Combination treatment of hypothermia with 2-IB is a promising strategy for reducing hypoxia-induced neuronal injury.

Combining 2-iminobiotin and hypothermia for reducing hypoxia-induced brain damage: Implications from a human cell culture model of hypoxic-ischemic brain injury