Imaging pathological activities of human brain tissue in organotypic culture

Publication date: 15 March 2018
Source:Journal of Neuroscience Methods, Volume 298
Author(s): Caroline Le Duigou, Etienne Savary, Mélanie Morin-Brureau, Daniel Gomez-Dominguez, André Sobczyk, Farah Chali, Giampaolo Milior, Larissa Kraus, Jochen C. Meier, Dimitri M. Kullmann, Bertrand Mathon, Liset Menendez de la Prida, Georg Dorfmuller, Johan Pallud, Emmanuel Eugène, Stéphane Clemenceau, Richard Miles
BackgroundInsights into human brain diseases may emerge from tissue obtained after operations on patients. However techniques requiring transduction of transgenes carried by viral vectors cannot be applied to acute human tissue.New methodWe show that organotypic culture techniques can be used to maintain tissue from patients with three different neurological syndromes for several weeks in vitro. Optimized viral vector techniques and promoters for transgene expression are described.ResultsRegion-specific differences in neuronal form, firing pattern and organization as well as pathological activities were maintained over 40–50 days in culture. Both adeno-associated virus and lentivirus based vectors were persistently expressed from ∼10 days after application, providing 30–40 days to exploit genetically expressed constructs. Different promoters, including hSyn, e/hSyn, CMV and CaMKII, provided cell-type specific transgene expression. The Ca probe GCaMP let us explore epileptogenic synchrony and a FRET-based probe was used to follow activity of the kinase mTORC1.Comparison with existing methodsThe use of a defined culture medium, with low concentrations of amino acids and no growth factors, permitted organotypic culture of tissue from humans aged 3–62 years. Epileptic activity was maintained and excitability changed relatively little until ∼6 weeks in culture.ConclusionsCharacteristic morphology and region-specific neuronal activities are maintained in organotypic culture of tissue from patients diagnosed with mesial temporal lobe epilepsy, cortical dysplasia and cortical glioblastoma. Viral vector techniques permit expression of probes for long-term measurements of multi-cellular activity and intra-cellular signaling.

Graphical abstract

from #ORL-AlexandrosSfakianakis via ola Kala on Inoreader http://ift.tt/2CDE7hk