Endocannabinoid System Participates in Neuron-Glial Networks Adaptation to Modeled Ischemia Factors in vitro

The aim of the study was to evaluate the role of a cannabinoid receptor agonist and blockers of key enzymes of endocannabinoid biodegradation in the adaptation of neuron-glial networks to ischemia factors influence.

Materials and Methods. Dissociated hippocampal cells obtained from C57BL/6 murine embryos on the 18^th day of gestation were cultivated as primary cultures. Two major factors of ischemia — hypoxia and glucose deprivation — were modeled on day 14 of culture development in vitro. Synthetic endocannabinoid N-arachidonoyl dopamine (NADA) at concentration of 10 μM, irreversible inhibitor of monoacylglycerol lipase JZL184 (1 μM) and inhibitor of two key enzymes of endocannabinoid biodegradation JZL195 (1 μM) were added into the culture medium at the beginning of ischemia factors modeling or immediately after it. The cell viability was assessed on days 1, 3, and 7 after the influence of ischemia factors. On day 7, the spontaneous calcium activity in primary hippocampal cells was analyzed using the multicellular functional calcium imaging. To reveal the molecular neuroprotective mechanisms of the tested compounds, expression of mRNA CB_1R receptor and mRNA hypoxia-inducible factor HIF-1α were measured using fluorescent mRNA detection probes (Smart Flare Merck, Germany).

Results. Application of NADA (the agonist of the cannabinoid type 1 and 2 receptors) or JZL195 (the MAGL/FAAH enzymes blocker) into the culture medium has been found to have a neuroprotective effect manifesting itself in a consistent cell death decrease compared with the control cultures. In addition, activation of the endocannabinoid system partially neutralizes the changes induced by hypoxia and glucose deprivation in vitro: the spontaneous calcium activity was detected in a greater number of cells (vs the control group), and the duration and frequency of calcium oscillations were partially normalized.

It was also found that NADA application during hypoxia significantly increased the type 1 cannabinoid receptors expression. When NADA and JZL195 were added the expression level of mRNA HIF-1α factor did not differ from that of the intact cultures.

Conclusion. The synthetic endocannabinoid NADA and the enzyme blocker JZL195 have a pronounced neuroprotective effect on the modeled ischemia factors consisting in the increase of neuronal cell viability and a good functional calcium activity of primary hippocampal cultures in a long-term period after injury.

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Mitroshina Е.V., Mishchenko T.A., Voronova N.V., Yarkov R.S., Asyutin D.S., Mishchenko M.A., Vedunova M.V. Endocannabinoid System Participates in Neuron-Glial Networks Adaptation to Modeled Ischemia Factors in vitro. Sovremennye tehnologii v medicine 2017; 9(4): 66, https://doi.org/10.17691/stm2017.9.4.08