Neurotropic Effect of Carbamylated Darbepoietin on the Model of Primary Hippocampal Culture

Activation of the cerebral erythropoietin system can be a promising strategy for the management of various neurodegenerative and neuropsychiatric diseases as it triggers neuroprotective mechanisms and improves cognitive functions. Lack of information about the possible neurotrophic effect of erythropoietin reduces the possibility of using the brain receptor for erythropoietin as a therapeutic target in neurodegenerative diseases associated with hypoxia and inflammation.

The investigation aims to study the influence of the erythropoietin receptor agonist — carbamylated darbepoetin (CdEpo) — on the morphofunctional features of neuron-glia networks of primary hippocampal cultures in mice under normoxia.

Materials and Methods. Primary hippocampal cell cultures dissociated from embryos (E18) of C57BL/6 mice were used to study the influence of erythropoietin receptor stimulation on the functional activity of hippocampal neuron-glia networks. The experiments were carried out on days 18–23 of culture development in vitro. CdEpo (100 ng/ml) action duration was 24 h. Functional changes were assessed based on the electrical and metabolic activity of cultured cells using patch-clamp techniques, multielectrode registration of bioelectric activity in neural networks and calcium imaging, respectively. The morphological features of primary hippocampal cell cultures were studied using transmission electron microscopy.

Results. Treatment of primary hippocampal cell cultures with CdEpo during 24 h did not affect the frequency of spontaneous single action potentials, spontaneous burst activity of cells, the patterns of neuron action potentials (the amplitude of depolarization phase, threshold potential, the amplitude of hyperpolarization phase), membrane capacity. It did not affect the bioelectric parameters of neural network activity (the number of spikes in a network burst, network burst duration, and inter-burst interval), calcium activity of neurons and glial cells determined by the duration and frequency parameters of spontaneous calcium oscillations. At the ultrastructural level, the number of mature asymmetric synaptic contacts remained unchanged under the influence of CdEpo, but there was a morphogenesis of the internal structure of dendritic spines: the number of spines with endoplasmic reticulum and/or the spine apparatus inside increased, which was a unique phenomenon for a model of primary hippocampal culture.

Conclusion. There was revealed no neurotropic effect of CdEpo on the model of primary hippocampal culture as reflected by bioelectric activity parameters of single neurons, neuronal networks, and astrocytic network activity evident in the parameters of intracellular calcium concentration changes (calcium oscillations) under normoxia conditions. However, CdEpo causes changes in the internal structure of dendritic spines in some neurons with the spine apparatus appearing in them. The absence of CdEpo effect on the functions of intact neurons and glia indicates the relative safety of using this molecule for therapeutic purposes as cytoprotection for the brain tissue.

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