The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis
Abstract Background The blood–brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases’ progression. This functional damage is one key event which...
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| Format: | Article |
| Language: | English |
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BMC
2020-03-01
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| Series: | Fluids and Barriers of the CNS |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12987-020-00179-3 |
| _version_ | 1798043058921537536 |
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| author | Anna Gerhartl Nadja Pracser Alexandra Vladetic Sabrina Hendrikx Heinz-Peter Friedl Winfried Neuhaus |
| author_facet | Anna Gerhartl Nadja Pracser Alexandra Vladetic Sabrina Hendrikx Heinz-Peter Friedl Winfried Neuhaus |
| author_sort | Anna Gerhartl |
| collection | DOAJ |
| description | Abstract Background The blood–brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases’ progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis. Methods Transwell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments. Results In contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations. Conclusions The co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly. |
| first_indexed | 2024-04-11T22:44:19Z |
| format | Article |
| id | doaj.art-687954031d7a449f93e6273a5c6f9a8d |
| institution | Directory Open Access Journal |
| issn | 2045-8118 |
| language | English |
| last_indexed | 2024-04-11T22:44:19Z |
| publishDate | 2020-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Fluids and Barriers of the CNS |
| spelling | doaj.art-687954031d7a449f93e6273a5c6f9a8d2022-12-22T03:58:51ZengBMCFluids and Barriers of the CNS2045-81182020-03-0117111710.1186/s12987-020-00179-3The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysisAnna Gerhartl0Nadja Pracser1Alexandra Vladetic2Sabrina Hendrikx3Heinz-Peter Friedl4Winfried Neuhaus5Competence Unit Molecular Diagnostics, Center Health and Bioresources, AIT–Austrian Institute of Technology GmbHCompetence Unit Molecular Diagnostics, Center Health and Bioresources, AIT–Austrian Institute of Technology GmbHCompetence Unit Molecular Diagnostics, Center Health and Bioresources, AIT–Austrian Institute of Technology GmbHCompetence Unit Molecular Diagnostics, Center Health and Bioresources, AIT–Austrian Institute of Technology GmbHCompetence Unit Molecular Diagnostics, Center Health and Bioresources, AIT–Austrian Institute of Technology GmbHCompetence Unit Molecular Diagnostics, Center Health and Bioresources, AIT–Austrian Institute of Technology GmbHAbstract Background The blood–brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases’ progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis. Methods Transwell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments. Results In contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations. Conclusions The co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly.http://link.springer.com/article/10.1186/s12987-020-00179-3Brain endothelial cellsClaudinCerebral ischemiaStrokeTraumatic brain injury |
| spellingShingle | Anna Gerhartl Nadja Pracser Alexandra Vladetic Sabrina Hendrikx Heinz-Peter Friedl Winfried Neuhaus The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis Fluids and Barriers of the CNS Brain endothelial cells Claudin Cerebral ischemia Stroke Traumatic brain injury |
| title | The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis |
| title_full | The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis |
| title_fullStr | The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis |
| title_full_unstemmed | The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis |
| title_short | The pivotal role of micro-environmental cells in a human blood–brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis |
| title_sort | pivotal role of micro environmental cells in a human blood brain barrier in vitro model of cerebral ischemia functional and transcriptomic analysis |
| topic | Brain endothelial cells Claudin Cerebral ischemia Stroke Traumatic brain injury |
| url | http://link.springer.com/article/10.1186/s12987-020-00179-3 |
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