Neuroprotective Effect of HIF Prolyl Hydroxylase Inhibition in an In Vitro Hypoxia Model

Abstract

A novel potent analog of the branched tail oxyquinoline group of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, neuradapt, has been studied in two treatment regimes in an in vitro hypoxia model on murine primary hippocampal cultures. Neuradapt activates the expression of HIF1 and HIF2 target genes and shows no toxicity up to 20 μM, which is more than an order of magnitude higher than its biologically active concentration. Cell viability, functional activity, and network connectivity between the elements of neuronal networks have been studied using a pairwise correlation analysis of the intracellular calcium fluctuations in the individual cells. An immediate treatment with 1 μM and 15 μM neuradapt right at the onset of hypoxia not only protects from the death, but also maintains the spontaneous calcium activity in nervous cells at the level of the intact cultures. A similar neuroprotective effect in the post-treatment scenario is observed for 15 μM, but not for 1 μM neuradapt. Network connectivity is better preserved with immediate treatment using 1 μM neuradapt than with 15 μM, which is still beneficial. Post-treatment with neuradapt did not restore the network connectivity despite the observation that neuradapt significantly increased cell viability at 1 μM and functional activity at 15 μM. The preservation of cell viability and functional activity makes neuradapt promising for further studies in a post-treatment scenario, since it can be combined with other drugs and treatments restoring the network connectivity of functionally competent cells.

Neuroprotective Effect of Kinase Inhibition in Ischemic Factor Modeling In Vitro

“…The spontaneous calcium activity of primary hippocampal cultures was recorded on day 7 post-ischemic damage. The following main parameters were analyzed: duration of calcium events (time period from the beginning to the end of an oscillation, s), frequency of calcium oscillations (average number of oscillations per min), and percentage of active cells (the cells number with at least one recorded oscillation divided by the total cell number, %) [ 18 , 21 ].…”

“…A 100% value characterizes the maximally possible level of significant correlations in the network. An increase in the last two parameters considered above indicates the presence of a long-term significantly correlated response of the cell network, whereas their low levels characterize its absence [ 18 , 19 ].…”

“…Calcium ions play a crucial role in signaling between nerve cells and the regulation of synaptic activity, and their imaging gives a means to estimate the functional state of neuronal networks with cellular resolution. The recently developed algorithm for analyzing calcium imaging data employs correlation analysis to reconstruct and follow the dynamical architecture of neuron-glial networks [ 18 , 19 ]. It provides with a powerful tool to characterize the collective and coordinated activity of these networks on the functional level and infer its response to model conditions, and evaluate the effect of damaging and protective factors.…”

Signatures of the Consolidated Response of Astrocytes to Ischemic Factors In Vitro

“…Hypoxia is one of the most common experimental models of brain cell damage [ 18 ]. Several studies have shown a significant decrease in the viability and functional activity of brain cells in models of 10-min acute hypoxia in vitro [ 18 , 19 , 20 ]. However, astrocytes are considered more resistant to hypoxic damage than neurons.…”

“…Several studies addressed the changes in the functional activity of neuronal networks in hypoxic and ischemic lesions. Earlier, we demonstrated that hypoxia leads to the dramatic suppression of network interactions between neural cells, up to complete disruption, that is manifested in the alterations of both the calcium and bioelectric activity, as recorded by multi-electrode matrices [ 19 , 20 ]. At the same time, little is known regarding the network activity of mono-astrocytic cultures.…”