Brain-derived neurotrophic factor (BDNF) is one of the key signaling molecules that supports the viability of neural cells in various brain pathologies, and can be considered a potential therapeutic agent. However, several methodological difficulties, such as overcoming the blood⁻brain barrier and the short half-life period, challenge the potential use of BDNF in clinical practice. Gene therapy could overcome these limitations. Investigating the influence of viral vectors on the neural network level is of particular interest because viral overexpression affects different aspects of cell metabolism and interactions between neurons. The present work aimed to investigate the influence of the adeno-associated virus (AAV)-Syn-BDNF-EGFP virus construct on neural network activity parameters in an acute hypobaric hypoxia model in vitro.
Materials and methods: An adeno-associated virus vector carrying the BDNF gene was constructed using the following plasmids: AAV-Syn-EGFP, pDP5, DJvector, and pHelper. The developed virus vector was then tested on primary hippocampal cultures obtained from C57BL/6 mouse embryos (E18). Acute hypobaric hypoxia was induced on day 21 in vitro. Spontaneous bioelectrical and calcium activity of neural networks in primary cultures and viability tests were analysed during normoxia and during the posthypoxic period.
Results: BDNF overexpression by AAV-Syn-BDNF-EGFP does not affect cell viability or the main parameters of spontaneous bioelectrical activity in normoxia. Application of the developed virus construct partially eliminates the negative hypoxic consequences by preserving cell viability and maintaining spontaneous bioelectrical activity in the cultures. Moreover, the internal functional structure, including the activation pattern of network bursts, the number of hubs, and the number of connections within network elements, is also partially preserved. BDNF overexpression prevents a decrease in the number of cells exhibiting calcium activity and maintains the frequency of calcium oscillations.
Conclusion: This study revealed the pronounced antihypoxic and neuroprotective effects of AAV-Syn-BDNF-EGFP virus transduction in an acute normobaric hypoxia model.
Keywords: BDNF; adeno-associated virus vector; brain-derived neurotrophic factor; calcium imaging; cerebral hypoxia; multielectrode arrays; neuroprotection; primary hippocampal cell cultures.