Ischemia/reperfusion injury and tissue hypoxia are of high clinical relevance, as they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms of the ischemia/reperfusion induced cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. Aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions (pO2<10mmHg) in the culture medium. Hydrogen peroxide assays, glucose measurements and westernblotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium (pO2 0 minutes: 107.57±0.99mmHg, 70 minutes: 9.00±0.58mmHg, 120 minutes: 5.00±0.00mmHg, 170 minutes: 2.00±0.00mmHg, 360 minutes: 2.00±0.00mmHg). Glucose concentrations gradually decreased ([Glc] 0 minutes: 4.50±0.02g/l, 360 minutes: 1.22±0.07g/l) while levels of hydrogen peroxide were not altered ([H2O2] 0 minutes: 9.57±0.00µM, 360 minutes: 7.96±0.67µM). Moreover, a rapid and reversible (on/off) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of LDH as a biochemical marker of cell damage (hypoxia: 0.50±0.08a.u., normoxia: 0.20±0.05a.u.; P<0.05). Hypoxic conditions also increased the amounts of cellular procaspase-3 (hypoxia: 1.45±0.19a.u., normoxia: 0.98±0.01a.u.; P<0.05) and catalase (hypoxia: 1.71±0.55a.u., normoxia: 0.61±0.09a.u.; P<0.05) as well as phosphorylation of the prosurvival kinase Akt (hypoxia: 0.65±0.14a.u., normoxia: 0.05±0.01a.u.; P<0.05), but not Erk1/2 or STAT5. In summary, we present a novel framework in investigating hypoxia mediated mechanisms on cellular level. We claim that the model, the first of its kind, enables researches to rapidly and reversibly induce hypoxic conditions in-vitro without interference of the hypoxia inducing agent with the cultured cells. The system may help to further unravel hypoxia associated mechanisms which are clinically relevant in various tissues and organs.
- Received May 29, 2013.
- Accepted September 2, 2013.
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