An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

Abstract

Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing 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. The 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 in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting 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. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. 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 lactate dehydrogenase (a biochemical marker of cell damage). Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and reversibly induce hypoxic conditions in vitro without unwanted interference of the hypoxia-inducing agent on the cultured cells. The system could help to further unravel hypoxia-associated mechanisms that are clinically relevant in various tissues and organs.

Fig. 1.

Assembly and functionality of the insert-based two-enzyme hypoxia system. (A–F) Commercially available six-well inserts from which the bottom membrane was removed are used as a framework for the assembly of a semipermeable dialysis membrane. (A–D) The basic steps of insert assembly. 1 and 2 in A describe the order in which the steps are performed: 1, insert the semipermeable membrane; 2, insert the plastic ring. (E,F) The assembled insert in a six-well plate. (G) Addition of glucose oxidase and catalase into the upper compartment containing standard cell culture medium leads to the generation of hypoxia in the lower compartment. (H) Schematic depiction of the glucose-oxidase- and catalase-dependent reactions that lead to hypoxic culture conditions. CAT, catalase; GO, glucose oxidase.

Fig. 2.

Characterization of transient and long-term hypoxic conditions induced by the insert-based two-enzyme system. (A) Application of the enzyme-containing insert (red arrow) results in a rapid decrease in pO₂. After removing the insert (dark green arrow), pO₂ quickly increases to almost baseline levels. This on-off procedure can be repeated, yielding similar pO₂ profiles each time. (B) Silver staining experiments show that the hypoxia-inducing enzymes are restrained within the insert and do not pass the semipermeable membrane. (C) Applying the enzyme-containing insert for a prolonged time period results in a decrease of pO₂ to values as low as 2.00 mmHg. Hypoxic conditions are stable for at least 6 hours. During the experiment, glucose concentrations gradually decrease, whereas hydrogen peroxide levels keep at a steady state. Bars denote mean ± s.e.m. of three experiments.

Fig. 3.

Experimental setting and time frame of the study. CAT, catalase; GO, glucose oxidase.

Fig. 4.

Hypoxia generated by using the insert-based enzymatic system damages cultured neuronal cells. Cells subjected to hypoxia show morphological changes such as cell rounding and detachment from the growth surface. Scale bars: ∼30 μm. LDH measurements in the culture media confirm these results and reveal an increase of LDH in the hypoxia group. Bars denote mean ± s.e.m. of five experiments; *P<0.05.

Fig. 5.

Effects of hypoxia generated by using the insert-based enzymatic system on the expression of hypoxia-associated proteins. Several hypoxia-associated proteins are differentially expressed between the hypoxia (H) and normoxia (N) groups at 1 hour (A) and 2 hours (B) after the hypoxic insult. One representative western blotting experiment is shown above the columns. Arrowheads denote the specific protein band detected by the respective antibody. Bars denote mean ± s.e.m. of three experiments; *P<0.05; +, uncleaved PARP.

Fig. 6.

Effects of hypoxia generated by using the insert-based enzymatic system on the phosphorylation of cell signalling molecules. Several molecules associated with cellular signalling and survival events are differentially phosphorylated between the hypoxia (H) and normoxia (N) groups at 2 hours after the hypoxic insult. One representative western blotting experiment is shown above the columns. Bars denote mean ± s.e.m. of three experiments; *P<0.05.