Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Huang, Ying, Zitta, Karina, Bein, Berthold, Steinfath, Markus, Albrecht, Martin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Company of Biologists Limited 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820273/
https://www.ncbi.nlm.nih.gov/pubmed/24046359
http://dx.doi.org/10.1242/dmm.013078
_version_ 1782290115254550528
author Huang, Ying
Zitta, Karina
Bein, Berthold
Steinfath, Markus
Albrecht, Martin
author_facet Huang, Ying
Zitta, Karina
Bein, Berthold
Steinfath, Markus
Albrecht, Martin
author_sort Huang, Ying
collection PubMed
description 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 (onoff) 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.
format Online
Article
Text
id pubmed-3820273
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher The Company of Biologists Limited
record_format MEDLINE/PubMed
spelling pubmed-38202732013-11-07 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 Huang, Ying Zitta, Karina Bein, Berthold Steinfath, Markus Albrecht, Martin Dis Model Mech Resource Article 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 (onoff) 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. The Company of Biologists Limited 2013-11 2013-09-05 /pmc/articles/PMC3820273/ /pubmed/24046359 http://dx.doi.org/10.1242/dmm.013078 Text en © 2013. Published by The Company of Biologists Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
spellingShingle Resource Article
Huang, Ying
Zitta, Karina
Bein, Berthold
Steinfath, Markus
Albrecht, Martin
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
title 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Resource Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3820273/
https://www.ncbi.nlm.nih.gov/pubmed/24046359
http://dx.doi.org/10.1242/dmm.013078
work_keys_str_mv AT huangying aninsertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT zittakarina aninsertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT beinberthold aninsertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT steinfathmarkus aninsertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT albrechtmartin aninsertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT huangying insertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT zittakarina insertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT beinberthold insertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT steinfathmarkus insertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells
AT albrechtmartin insertbasedenzymaticcellculturesystemtorapidlyandreversiblyinducehypoxiainvestigationsofhypoxiainducedcelldamageproteinexpressionandphosphorylationinneuronalimr32cells