The aim of the investigation was to assess antihypoxic and neuroprotective properties of the glial cell line-derived neurotrophic factor (GDNF) in hypoxia models in vitro and in vivo. In vitro experiments were carried out on primary hippocampal cultures. Hypoxia modeling was performed on day 14 of culture development in vitro (DIV) by replacing the normoxic cultural medium with a medium containing low oxygen for 10 minutes. Registration of extracellular action potentials was conducted by MEA systems (Multichannel Systems, Germany) application. Study the effect of GDNF on synaptic plasticity was performed using SmartFlare RNA Detection Probes (Merck Millipore, France) and fluorescent microscopy. In vivo experiments were carried out on C57BL/6j male mice. For acute hypobaric hypoxia a vacuum flow-through chamber was used at the ambient temperature of 20–22°C. We have investigated the resistance of animals to hypoxia and their spatial memory retention in Morris water maze test 24 hours after hypoxia. In vitro and in vivo data demonstrated that GDNF has strong antihypoxic and neuroprotective properties. Preventive GDNF application before hypoxia contributed to the animal survival and spatial memory retention as well as the maintenance of cells viability in primary hippocampal cultures.
Nowadays investigations concerning the searching endogenous factors for the nervous cells protection from hypoxic damage is one of the topical issues in modern neuroscience and medicine. Glial cell line-derived neurotrophic factor (GDNF) considered as a possible endogenous substance able to control cellular metabolic rates under low oxygen conditions and promotes neuronal survival. However, a question about the mechanisms of neuroprotective and antihypoxic actions of GDNF during hypoxia is still open. The aim of the investigation was to study antihypoxic and neuroprotective GDNF actions in hypoxia models in vitro and in vivo.
MATERIAL AND METHODS
In vitro studies were performed using hippocampal cells dissociated from 18-day embryonic CBA mice. The cells were cultured on multielectrode arrays (Multichannel Systems, Germany). Hypoxia modeling was performed on day 14 of culture development in vitro by replacing the normoxic cultural medium with a medium containing low oxygen for 10 min. The oxygen was displaced from the medium in sealed chamber in which the air was replaced with an inert gas. The neurotrophic factor was added to the medium 20 min before hypoxia. In the control group hypoxia was induced without additional treatment. The viability of dissociated hippocampal cells was evaluated according to the percentage ratio between the number of dead cells stained by propidium iodide (Sigma, Germany) and the total number of cells stained by bisBenzimide (Invitrogen, USA) for 7 days after hypoxia. In vivo experiments were performed on 86 C57BL/6j sexually mature male mice weighing 18–20 g. For modeling of acute hypobaric hypoxia a vacuum flow-through chamber was used at the ambient temperature of 20–22oC. Mice were placed under conditions corresponding altitude10 000–10 500 m (170–185 mm Hg) with a lifting speed 183 m/s . The long-term memory retention test (Morris water maze re-test) was conducted 24 hours after hypoxia.
In vitro experiments showed that 10-minutes acute hypoxia caused the decreasing of cellular viability in primary hippocampal cultures approximately 4.5 times (р<0.01). A preventive GDNF application reduced the number of dead cells in 2 times in comparison with control cultures (р<0.01). Electrophysiological data demonstrated that hypoxia led to spontaneous bioelectrical activity violations and to the destruction of pattern of spontaneous network activity. Preventive application of neurotrophic factor GDNF (1 ng/ml) partially neutralizes the negative hypoxic effects on the spontaneous bioelectrical activity. By the day 7 of the post-hypoxic period in group, which received preventive doses of neurotrophic factor, there was a restoration in the number of small network bursts and in the average number of spikes per burst up to the baseline. At the same time the parameters of spontaneous bioelectrical activity in control cultures, without preventive GDNF treatment, were significantly (p<0.05) lower than in experimental groups. To identify the possible GDNF influence on synaptic plasticity, the level of the expression of mRNA GluR2-subunits of AMPA-receptors in normoxic conditions and after acute oxygen deficiency was evaluated. The received data showed that hypoxia significantly decreased the number mRNA GluR2-positive cells. Preventive GDNF application contributed to the preservation the level of cells, expressing mRNA GluR2-subunits of AMPA-receptors, whether in normal conditions GDNF injection resulted in increased expression of mRNA GluR2. The following step was to assess the effect of GDNF on animal survival to acute hypobaric hypoxia. It was shown that preventive intranasal application of the neurotrophic factor (4 μg/kg) increased animal resistance to acute hypobaric hypoxia which is manifested as significantly elevated the lifetime on the “height”.
Our data revealed that glial cell line-derived neurotrophic factor has strong antihypoxic and neuroprotective properties. Preventive GDNF application neutralizes the negative effects of oxygen deficiency by increasing the cell viability and maintaining of functional network activity in primary hippocampal cultures at a certain functional level.
The research was supported by the Federal Target Program "Research and development in priority areas of the development of the scientific and technological complex of Russia for 2014–2020” of the Ministry of Education and Science of Russia (Project ID RFMEFI60715X0117).
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