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Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
Adenoviral Vectors Expression in Human Astrocytic Glioma Cells and Primary Astrocytes Monocultures
N.V. Ponomareva*, S. A. Tutukova, E.V. Mitroshina, A.V. Lebedeva, E.A. Epifanova, T.A. Mishchenko, M.V. Vedunova, A.A. Babaev
Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia. * Presenting e-mail: ponomareva@neuro.nnov.ru
Recombinant viral vectors that are able to effectively transport a gene of interest into the cells were previously designed. Adenoviral vectors produce recombinant virus at a high titer with high-level expression of introduced genes. We intend to use adenoviral constructs in optical stimulation experiments for astrocyte cultures with laboratory animals. The aim of this study is to examine the effectiveness of adenoviral vectors application for in vivo expression in neuroglial cells.
To achieve this, on the first stage of our investigation human astrocytic glioma cell lines - a simple and effective model for testing the obtained samples- were used.
Adenoviral vectors containing promoter of the human glial fibrillary acidic protein GFAP (AVV-GFAP-ChR2-Venus, AVV-GFAP-Case12 and AVV-GFAP-GFP), which were kindly provided by Prof. Sergey Kasparov (School of Medical Sciences, University of Bristol) were used. AVV-GFAP-ChR2-Venus represents a fusion construct consisting of Channel rhodopsin 2 (ChR2) fused to Venus yellow fluorescent protein (Ex/Em 515/528) is necessary to visualize the expression. AVV-GFAP-Case12 contains a genetically encoded fluorescent biosensor for analyzing fluctuations in intracellular calcium ions concentration. The amplification of viral vectors in HEK 293FT cell culture was carried out. The viruses were purified and concentrated using Amicon ultra-15 (Merck Millipore) centrifugal filter devices and titrated (AdEasy Viral Titer Kit, Agilent Technologies).
U251 MG cell line of the human astrocytic glioma obtained from the cell collection of the Institute of Cytology (St.-Pe-tersburg) was used to perform a fast and efficient evaluation of in vitro expression. Due to its enhanced growth and ease of cultivation, this cell line is an optimal model for testing the effectiveness of viral vectors. U251 MG cells transfection by the virus was performed at glial cell density of 70-80 per cent per field of vision. Expression of fluorescent proteins within the viral constructs was evaluated by the level of their illumination intensity upon excitation with light of a certain wavelength on the 1st, 2nd and 3rd day using a Zeiss LSM510 confocal laser scanning microscope (Fig.1). It was shown the maximum GFP expression was observed on the first day after the transfection with GFAP-GFP constructs, whereas the expression of Venus fluorescent protein as part of the GFAP-ChR2-Venus construct was low and showed only on the third day after the transfection, which can be explained by the complexity and size of the optogenetic construct.
At the second stage, the efficiency of viral vectors expression in primary astrocytes monocultures we demonstrated.
Primary astrocytes monocultures were obtained from the cortex of newborn mice (P0-P2). The cultures were infected on day 10 of culture development in vitro (DIV). The viral vectors expression by using the Karl Zeiss LSM 510 confocal laser-scanning microscope was detected on DIV 5 (Fig. 2). Toxic effects of AVV-GFAP-ChR2-Venus virus at a dose of 1.2x106 TU/ml (cell swelling, the formation of cell conglomerates) were observed. Application of a lower titer of viral preparation allowed to observe an accurate expression of viral constructs in infected astrocytes in comparison with control cultures.
Control GFAP-GFP-expression Control GFAP-ChR2-Venus- expression
Fig.1 GFAP-GFP and GFAP-ChR2-Venus expression in U-251 MG human glioma cells
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v- p A /
v is.*,.
Control AVV-GFAP- Case12-expression Control AVV-GFAP-ChR2-Venus-expression
Fig.2 Astrocytes expressing AVV-GFAP-ChR2-Venus and AVV-GFAP-Case12
Therefore, the effectiveness of human glioma cultures model for viral constructs verification was demonstrated. 82 Opera Med Physiol 2016 Vol. 2 (S1)
Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain
The viruses that have been amplified and tested in human astrocytic glioma cells and in primary astrocytes monocultures are expected to be used in optical stimulation experiments in vitro and in vivo.
This work was supported by the Russian Science Foundation (project 16-14- 00201).
The Influence of Glial Cell Line-Derived Neurotrophic Factor and Modulated Activity of Endocannabinoid System on g3h Mice Sustainability to Ischemic Brain Injury In Vivo
E.V. Mitroshina1,2 *, B.ZH. Abogessimengane1, M.D. Urazov1,I. Khamray1, T.A. Mishchenko2,1,
T.A. Astrakhanova1, N.A. Shchelchkova2,1, R.D. Lapshin2, I.I. Belousova2,1.V. Mukhina2,1, M.V. Vedunova1,2
1 The Institute of Biology and Biomedicine, Lobachevsky State University of Nizhny Novgorod, Nizhny Novgorod, Russia
2 Molecular and cell technologies group, Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia * Presenting e-mail: helenmitroshina@gmail.com
Nowadays, ischemia considered as one of the main causes of death and disability in the population all over the world. Among the consequences of brain ischemia an acute deterioration of memory and neurological status, learning and cognitive functions' impairment should be especially noted. Therefore, development of methods aimed at enhancing the adaptive capacity of the organism to ischemia is extremely urgent scientific issue. The modulation of endogenous systems, contributing to the survival of nervous cells under the influence of stress factors and maintaining their functional activity seems to be promising.
The aim of the investigation was to study the effects of Glial cell line-derived neurotrophic factor (GDNF) and the endogenous cannabinoid system activation by jzl 195 (an inhibitor of endocannabinoid biodegradation enzymes MAGL and FAAH) on the animal resistance to ischemic damage.
Materials and Methods
The study was conducted on 120 adult male C3H mice weighing 25-40 g. Animals were divided into the following groups: 1) Intact (n = 16), 2) Sham - falsely operated mice subjected to tissues incision with subsequent finding the artery without occlusion (n = 16), 3) Control - the animals with bilateral carotid arteries occlusion (n = 40), 4) The animals with bilateral carotid arteries occlusion and a single jzl 195 (10 mg/kg) intraperitoneal injection 45 minutes before surgery (n= 9), 5) The animals with bilateral carotid arteries occlusion and intranasal GDNF (0.08 mg/ml) administration 45 minutes before and during 3 days after the surgery (n = 22), 6) The animals with bilateral carotid arteries occlusion and intranasal GDNF (0.8 ml/ml) injection 45 min before and within 3 days after surgery (n = 17).
In order to develop ischemic lesions in different brain structures, an experimental animal model of bilateral carotid arteries occlusion was performed. The animals were anesthetized with pentobarbital (70 mg/kg) and were placed on an operating table 15 minutes after injection. The left and right carotid artery were allocated and then the simultaneous vessels ligation using non-absorbable ligature strands was carried out. Hereafter, the wound was sutured and sprinkled by streptocid powder to prevent inflammation.
To assess the animal's physiological state after ischemic brain damage, the neurological status (standard neurological scale and Garcia scale) was evaluated. To identify the mechanisms of ischemic brain damage, the mitochondrias' functional state by measuring of oxygen consumption rate was conducted. Mitochondrial dysfunction is a key component of the brain cell damages induced by ischemia. Mitochondrias was isolated by a standard differential centrifugation. Brain mitochondrial respiration rate was recorded using a high-resolution respirometer Oroboros Oxygraph-2k (Oroboros Instruments Corp, Austria).
The carried out experiments revealed that the bilateral carotid arteries occlusion causes the animal mortality up to 7080%. There was no difference in neurological status between intact and falsely operated animals during the observation period. For all experimental groups (except for "Sham") a significant neurological status deterioration in comparison with intact animals 24 hours after surgery was shown. The neurological status of experimental animals treated by jzl 195 (10 mg/kg) and GDNF (0.8 mg/kg) did not differ from intact mice on day 3 after ischemia modeling (Figure 1). However, there was no difference between the experimental groups on day 7 after ischemia modeling. Thus, it can be assumed that GDNF treatment during 3 days after surgery has a positive effect on the animal sustainability to ischemic brain injury. Further experiments will be focused on the selection of optimal scheme of jzl and GDNF administration to achieve a maximum effect. The mitochondrial oxygen consumption rate was conducted on day 4 after ischemia modeling. Our studies revealed that mitochondrias oxygen consumption rate in the intact group was 254.8 pmol/(s*ml) whereas in control group this parameter was decreased up to 208.8 pmol/(s*ml) after ischemia modeling. The rate of mitochondrias oxygen consumption in the group treated by GDNF 0.8 mg/kg did not differ from the intact animal (243.5 pmol/(s*ml)), and in animals treated by jzl195 was significantly increased (465.7 pmol/(s*ml)).
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Opera Med Physiol 2016 Vol. 2 (S1) 83
