Научная статья на тему 'The influence of Glial cell line-derived neurotrophic factor and modulated activity of endocannabinoid system on с3h mice sustainability to ischemic brain injuryin vivo'

The influence of Glial cell line-derived neurotrophic factor and modulated activity of endocannabinoid system on с3h mice sustainability to ischemic brain injuryin vivo Текст научной статьи по специальности «Биотехнологии в медицине»

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Похожие темы научных работ по биотехнологиям в медицине , автор научной работы — E.V. Mitroshina, B.Zh. Abogessimengane, M.D. Urazov, I. Khamray, T.A. Mishchenko

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Текст научной работы на тему «The influence of Glial cell line-derived neurotrophic factor and modulated activity of endocannabinoid system on с3h mice sustainability to ischemic brain injuryin vivo»

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)).



Volga Neuroscience School 2016 Astroglial control of rhythm genesis in the brain

Fig. 1. Neurological status of experimental animals on day 3 after total cerebral ischemia modeling (standard scale of neurological status); * - statistical significance with intact animals, p<0.05, ANOVA

Thus, we have shown that GDNF (0.8 mg/kg) application normalizes the parameters of mitochondrial respiration, and activation of the cannabinoid system stimulates the mitochondrias functioning under ischemic conditions. This effect refers to the post-ischemic adaptation and could potentially serve as one of the approaches for ischemic brain injury correction.


The research was supported by grants of Russian Foundation for Basic Research № 16-34-00301, №16-04-00245 and prepared as a part of the state project "Provision Scientific Research".

Ephrin Class a Reverse Signaling Guides Callosal Axon Growth via Efna 4-Ntrk 2 Receptor Complex Downstream of Neurod 2/6

K. Yan*, O. Grishina, G. Camarero, E. Bessa, U. Günther, R. Wunderlich, I. Bormuth, V. Tarabykin

Institute of Cell- and Neurobiology, Charité - Medical University Berlin, Germany. * Presenting e-mail: kuo.yan@charite.de

Abstract. Callosal axon guidance during cerebral cortex development is complicated and far from being fully understood. Previous work in our lab has shown that basic helix loop helix transcription factors Neurod2 and Neurod6 are essential regulators for the fasciculation and guidance of callosal axons. Here we use acallosal Neurod2/6 deficient mice as a model system to selectively study callosal axon pathfinding in vivo. We identify Efna4 as transcriptional target of Neurod2/6 in developing neocortex. In utero electroporation of Efna4 into neocortical pyramidal neurons of Neurod2/6 deficient embryos is sufficient to cell-autonomously rescue callosal axon fasciculation and migration along the normal callosal path towards the midsagittal plane. Mechanistically, Efna4 forms a co-receptor complex with Ntrk2 (TrkB) in reverse signaling, and hence regulate AKT cascades in vitro and in vivo via Ntrk2's SHC-binding tyrosine. Co-electro-poration of dominant negative Ntrk2 K571N or Ntrk2 Y515F completely abolishes the ability of Efna4 to rescue callosal axon guidance in Neurod2/6 deficient mice. We also show that the Eph receptors are abundantly expressed in the cortical plate and ventricular zone, but minimally expressed in the intermediated zone (IZ) of the cortex, while ephrinA ligands are largely present on the callosal axons in the IZ. In addition, reverse signaling from extracellular domain of EphA receptors to Efna4 leads to active axonal retraction in vivo. The complementary expression and repulsive interaction of EphA receptors and ephrinA ligands suggest a permissive channel for callosal axon navigation before midline crossing. Thus, ephrinA ligands coordinate fasciculate growth and guidance of callosal axons via interaction with Ntrk2 in cis and with EphA receptors in trans.

Keywords: NeurodD2; NeurodD6; ephrin A ligands; Ntrk2; corpus callosal agenesis.

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