Diversity of neurodegenerative processes in the model of brain cortex tissue ischemia

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Abstract

Stroke is known to induce massive cell death in the ischemic brain. Either necrotic or apoptotic types of cell death program were observed in neurons in zone of ischemia. We suggest that spatial heterogeneity of glucose and oxygen distribution plays a crucial role in this phenomenon. In order to elucidate the role of glucose and oxygen in ischemic neurons choice of cell death pathway, conditions corresponding to different areas of insult were reproduced in vitro in the model of surviving brain cortex tissue slices. Three zones were modeled in vitro by varying glucose and oxygen concentration in surviving slices incubation media. Modeled ischemic area I (MIA I) was corresponded to the center of suggested ischemic zone where the levels of glucose and oxygen were considered to be extremely low. MIA II was assigned as intermediate area where oxygen concentration was still very low but glucose was present (this area was also divided into two sub-areas MIA IIa and MIA IIb with physiologically low (5 mM) and normal (10 mM) level of glucose respectively). MIA III was considered as a periphery area where glucose concentration was close to physiological level and high level of ROS production had been induced by reoxygenation after anoxia. Analysis of molecular mechanisms of cell death in MIA I, IIa, IIb and III was carried out. Cell death in MIA I was found to proceed by necrotic manner. Apoptosis characterized by cyt c release, caspase 3 activation and internucleosomal DNA fragmentation was observed in MIA III. Cell death in MIA II was accompanied by several (not all) hallmarks of apoptosis. Mechanisms of cell death in MIA IIa and MIA IIb were found to be different. Internucleosomal DNA fragmentation in MIA IIa but not in MIA IIb was sensitive to glycine (5 mM), inhibitor of NMDA receptor MK-801 (10 μM) and PTP inhibitor cyclosporine A (10 μM). Activation of caspase 3 was detected in MIA IIb but not in MIA IIa. However cytochrome c release was observed neither in MIA IIa nor in MIA IIb. In MIAs II–III apoptosis was accompanied by uncoupling of oxidative phosphorylation, which was induced by rise of intracellular Ca2+ and intensive ROS production. Results obtained in present study allow us to propose existence of at least four molecular pathways of cell death development in brain ischemic zone. The choice of cell death pathway is determined by oxygen and glucose concentration in the particular area of the ischemic zone.

Introduction

Local disturbance of blood circulation in the brain results in stroke development (Adams et al., 1993, Caplan, 2000). Deficiency in blood supply leads to reduction of life substrates in the ischemic zone (Back et al., 1995, Siesjo, 1984). The reduction of energetic substrates such as glucose and oxygen is the most critical. Depletion of oxygen leads to anoxia in the ischemic zone, which occurs within minutes as the brain has extremely high respiration activity.

The ischemic zone is heterogeneous (Astrup et al., 1981). Using autoradiographic local cerebral blood flow method (LCBF) it could be defined the penumbra as a region of intermediate CBF (20–40% of control) surrounding the ischemic core (less then 20% of control) (Belayev et al., 1997, Ginsberg, 2003, Hossmann, 1994). Thereby glucose distribution in the ischemic zone is not uniform. According to Belayev et al. (1997) the ratio of local cerebral glucose metabolic rate divided by local cerebral blood flow sufficiently increases in the center of stroke. Variation of LCBF in the ischemic zone results in morphological changes in core and penumbra. It also leads to appearance of differences in the course of neurodegenerative processes developing in these areas. Study of the biochemical markers of cell death in these areas indicates that caspase-dependent pathway of apoptosis develops in the penumbra. Programmed cell death in this area is characterized by increased expression of Bcl-2, Bax and AIF, Bax translocation from the cytosol to the mitochondria, cytochrome c release and caspase 3 activation. In contrast, necrosis is realized in the ischemic core. It is characterized by reduced Bax expression but not Bax translocation and cytochrome c release (Ferrer et al., 2003).

Taken together, the autoradiographic data on LCBF and data on molecular pathways of cell death in stroke argue that differences in cell death features in the core and the penumbra can be caused by various glucose concentrations in the mentioned areas and reoxygenation occurring in periphery of the ischemic zone. This hypothesis is indirectly supported by results of the study of glucose level influence on neurons tolerance to short time ischemia (Takata et al., 2004). It was shown that the increase of glucose level from 5 to 10 mM qualitatively alters neurons ability to maintain electrophysiological activity after incubation in oxygen depleted media for 10 min.

Our aims were to investigate features of neurons cell death program in different ischemic areas and to determine if local oxygen and glucose concentration can define molecular cell death pathway choice in the ischemic brain. For this purpose, we modeled several areas of ischemic zone using brain cortex tissue slices surviving in vitro in media with different oxygen and glucose concentrations.

Section snippets

In vitro ischemia model

In order to model brain ischemia in vitro we divided ischemic zone into 3 areas with different oxygen and glucose concentrations (Fig. 1). We have modeled several conditions:

  • (1)

    Modeled ischemic area I (MIA I)—the center of the ischemic zone where levels of both glucose and oxygen were close to zero. In order to reproduce this area in vitro brain slices were incubated under anoxic conditions (see Section 2.2) with suppressed glycolysis. Incubation medium contained (in mM): NaCl 125, KCl 3.5, MgSO4

DNA fragmentation as a hallmark of apoptosis in brain cortex slices under anoxia or anoxia/reoxygenation

Rate and type of DNA destruction in the model ischemic areas (MIAs I–III) were analyzed. The type of DNA destruction was considered as a criterion of apoptosis or necrosis developing in ischemic areas in the case of oxygen and glucose deficiency.

Electrophoregrams of DNA extracted from brain cortex tissue slices after incubation under anoxia or anoxia/reoxygenation conditions for 24 h are shown in Fig. 2. Internucleosomal fragmentation of DNA, which is a common hallmark of apoptosis, was observed

Discussion

In the current study it has been shown that availability of energetic substrates (oxygen and glucose) could be a crucial factor in brain cortex cell death pathway choice in vitro. It is well-known that cytochrome c release from mitochondria to cytosol is a key evidence of classical apoptosis program development (Brown and Borutaite, 2008, Gogvadze et al., 2006, Li et al., 1997, Yang et al., 1997). Two different signal transduction pathways could be involved in apoptosis initiation under

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