Cell death and metabolic activity during epileptiform discharges and status epilepticus in the hippocampus

T. Sutula and A. Pitk~inen (Eds.) Progress in Brain Research, Vol. 135 © 2002 Elsevier Science B.V. All rights reserved

CHAFFER 17

Cell death and metabolic activity during epileptiform discharges and status epilepticus in the hippocampus U. Heinemann *, K. Buchheim, S. Gabriel, O. Kann, R. Kovacs and S. Schuchmann Johannes Mailer Institute of Physiology, Charitd, Humboldt University Berlin, D-lOll7 Berlin, Germany

Abstract: Mechanisms of seizure-induced cell death were studied in organotypic hippocampal slice cultures. These develop after withdrawal of magnesium recurrent seizure-like events (SLE), which lead to intracellular and intramitochondrial calcium accumulation. The intramitochondrial Ca accumulation seems to be involved in causing increased production of NADH, measured as NAD(P)H autofluorescence. During SLEs, depolarization of mitochondria and increased production of free radicals is indicated by fluorescence measurements with appropriate dyes. During recurrent seizures, an increased failure to produce NADH is noted while at the same time free radical production seems to increase. This increase and the decline in NADH production could be involved in transition to late recurrent discharges, a phase in which status epilepticus becomes pharmacoresistant. It also coincides with increased cell death as determined with propidium iodide fluorescence. Interestingly, some of these changes can be prevented by application of a-tocopherol, a free radical scavenger, which also has neuroprotective effects under our experimental conditions. The results suggest that free radical-induced mitochondrial impairment is involved in seizure-induced cell death.

Introduction Interictal and ictal discharges indicate synchronized hyperactivity in large ensembles of neurons. These discharges are associated with significant changes of the extracellular ionic microenvironment (Lux et al., 1986). During a seizure, extracellular potassium concentration ([K+]o) can rise to 12 mM, while [Na+]o, [Ca2+]o and the size of the extracellular space decreases (Lux et al., 1986). Consequently intracellular ion concentrations change (Ballanyi et al., 1987; Gloveli et al., 1999) and transmembrane fluxes of C1- appear (Dietzel et al., 1982). Moreover, pH measurements reveal an initial transient alkalosis followed by an acidic shift (Gutschmidt et al.,

*Correspondence to: U. Heinemann, Johannes Mtiller Institute of Physiology, Charit6, Humboldt University Berlin, D-10117 Berlin, Germany. Tel.: +49-30-450528091; Fax: +49-30-4505-28962; E-mail: uwe.heinemann @charite.de

1999). After a seizure, transport processes have to be activated in order to restore ionic gradients. These processes depend on sufficient supply of ATE Biochemical evidence suggests that about 60% of cerebral ATP consumption is used for operation of the electrogenic Na,K-pump which transports three Na ions out of the cell in exchange for two K ions (Ames, 2000). The Na,K-ATPase is activated by intracellular Na accumulation, but some variants of the Na,K-ATPase, particularly those in glial cells, can also be activated by extracellular K accumulation (Grisar et al., 1979). Many other transport processes in nerve cells, such as uptake of glutamate, choline and GABA are dependent on the transmembrane Na gradient. Ca can, in addition to Na/Ca exchange, also be transported by the Ca,Mg-ATPase. The ATP content within a nerve cell is rather limited and other stores for energy production are also scarce. Neurons in the CNS can utilize GABA for ATP production through the GABA shunt and also metabolize lactate (Schousboe et al., 1997; Waagepetersen et al., 1999). Particularly consumption of GABA for ATP

198 synthesis may be a dangerous event, as this would lead to depletion of the GABA pool during recurring seizures. Indeed, transition of recurring seizures to drug resistant late status epilepticus (Dreier and Heinemann, 1991; Zhang et al., 1995) may depend on increased GABA consumption. It is widely held that the energy demands of a group of nerve cells are covered by local adaptation of blood flow (Mathiesen et al., 1998; Caesar et al., 1999), which indeed strongly increases (by up to a factor of seven) (Nilsson et al., 1976; Meldrum, 1983) in areas participating in seizure activity (Horton et al., 1980; Ingvar and Siesjo, 1983). Rises in [K+]o, decreases in Ca, acidosis, release of adenosine and generation of NO seem to be factors involved in this coupling process (Dirnagl et al., 1994; Dirnagl, 1997). Prolonged status epilepticus is a condition which can cause considerable cell loss (Meldrum and Chapman, 1993). This cell loss seems to include glial cells as well (Schmidt-Kastner and Ingvar, 1996). Four hypotheses were proposed to explain status epilepticus-induced cell death. The original idea that energy supply to the brain may be reduced due to systemic factors was rejected early on the basis of glucose consumption and blood flow measurements (Pinard et al., 1984). However, when status epilepticus lasts for a prolonged period, a decline in ATP content (Folbergrova et al., 1985) and a change in the redox potential (Wasterlain and Plum, 1973; Fujikawa et al., 1988) was found, suggesting that during recurring seizures, energy production, in spite of increased supply, may be hampered (Folbergrova et al., 1985). The idea that excitotoxic cell damage alone is responsible for seizure-induced cell death always faced the difficulty that glutamate-induced cell death normally spares glial cells which contribute to cell loss during status epilepticus. More recently, it was suggested that cell death could occur, when intracellular Ca is elevated, causing mitochondrial depolarization (Duchen, 1999). Depolarized mitochondria may exploit 02 incompletely, resulting in an increased production of radical oxygen species (ROS). As a result, mitochondrial function may be compromised, leading to reduced generation of NADH and subsequently reduced production of ATE On the other hand, increases in intracellular Ca concentration may lead to uptake of Ca in mitochondria and

to increased formation of NADH and FADH. Indeed, some enzymes in the tricarboxylic acid cycle are sensitive to Ca and thereby Ca may play an important role in adjusting the ATP production to a given state of neuronal activity (McCormack and Denton, 1993a,b; Hansford and Zorov, 1998). We decided to exploit imaging techniques to get an insight into possible damage cascades during glutamate exposure and during seizures. Methods

The experiments were done on three types of preparations. Studies on glutamate-induced cell damage were done in dissociated hippocampal cell cultures (Schuchmann et al., 1998; Schuchmann and Heinemann, 2000a) with some additional experiments in organotypic slice cultures. Both preparations were performed as previously described (Peacock et al., 1979; Stoppini et al., 1991). Subsequently, we turned to complex entorhinal cortex and hippocampal slices, where recurrent seizures are readily induced by lowering of extracellular Mg concentration or application of 4AP in the entorhinal cortex and neighboring structures, such as the subiculum and the temporal neocortex (Walther et al., 1986). This activity progresses after some time into late recurrent discharges (Dreier and Heinemann, 1991) which are resistant to the presently available anticonvulsant drugs (Zhang et al., 1995). All experiments with respect to ictal activity in this paper were done by removing extracellular Mg concentration. We recently exploited the advantages of organotypic slice cultures. These cultures develop a strong excitatory coupling which leads to facilitated seizure generation during application of low Mg or bicuculline in comparison to age-matched slices (Gutierrez et al., 1999). Dissociated and slice cultures offer the advantage that they can be readily bulk loaded with different dyes which permit imaging of cytosolic and mitochondrial Ca concentration changes, measurements of mitochondrial potentials, formation of ROS and of NAD(P)H. When excited with 360 nm light, NAD(P)H produces a bright autofluorescence. The recordings were done under an upright microscope equipped with a photomultiplier and a CCD camera and a monochromator suitable to generate light with

199 wavelength between 200 and 1000 nm. Most frequently, the photomultiplier was used to sample light emission from area CA3, the hilus and part of area CA1. In slices, we either injected single cells with a given dye or used the NAD(P)H autofluorescence in order to gain insight into mechanisms involved in cellular metabolism. For methodological details see Schuchmann et al. (1999, 2000, 2001) and Kovacs et al. (2001).

Cell death determinations In order to determine cell death in organotypic and dissociated cultures we used propidium iodide staining (Kov~ics et al., 1999). This dye is normally excluded from healthy cells which can be marked by acridine orange, for example. When the plasma membrane is damaged the propidium iodide enters nerve cells and forms a bright fluorescence after binding to RNA and DNA. The intensity of this staining was used to determine the degree of cell loss after 2 h of status epilepticus or exposure to glutamate. Results

Glutamate-induced fluorescence signals in hippocampal dissociated cell cultures and organotypic slice cultures Glutamate dose-dependently induced an increase in cytosolic Ca concentration from a baseline concentration of about 80 nM as determined by ratiometric Fura-2 measurements. Application of 100 ttM of glutamate induced an increase in cytosolic Ca concentration in the order of 400 nM in cultures older then 2 weeks (Schuchmann et al., 1998). This compared to a 300-1000 nM increase in intracellular free Ca concentration in slices during seizure-like events (Gloveli et al., 1999). Application of 100 IxM glutamate for 1 h led to a reduction of viable cells by roughly 60% within 6 h of glutamate exposure and by roughly 80% after 24 h (Schuchmann and Heinemann, 2000a). Application of cyclosporin A but not of tocopherol could protect against this cell death. This suggested an involvement of mitochondria and perhaps development of transition pores in the glutamate-induced cell death.

We therefore decided to obtain more information on the effects of glutamate on mitochondrial potential. For this we employed the fluorescent dye rhodamine-123 which is positively charged and accumulated, therefore, within mitochondria where the fluorescence is quenched. When mitochondria become depolarized, part of the rhodamine-123 leaves the mitochondria resulting in a rhodamine-123 fluorescence increase (see e.g. Schuchmann et al., 1998, 2000). Application of 100 IxM glutamate-induced a pronounced mitochondrial depolarization which was absent when glutamate was applied in the presence of lowered extracellular Ca concentration. The rhodamine-123 fluorescence increase amounted to about 10% in cultures older than 2 weeks. The fluorescence increase was, moreover, dose- and agedependent as well as being dependent on application time. The depolarization of mitochondria might interfere with their capability to generate NADH. We therefore determined the NAD(P)H autofluorescence in cultured hippocampal cells and found that following an initial decrease in NAD(P)H autofluorescence there was a subsequent increase in NAD(P)H fluorescence, suggesting that in spite of mitochondrial depolarizations, the cells were able to generate NAD(P)H. This increase lasted for some 200 s before it returned to baseline (Schuchmann et al., 1998). It was about 3%. in cultures older than 2 weeks. In the presence of depolarized mitochondria, utilization of 02 is less complete and the formation of free radicals is facilitated. We therefore determined whether glutamate-induced Ca load leads to an increased formation of free radical oxygen species. Unfortunately dyes which are used to measure ROS production are not very specific. We therefore compared the oxidation of three dyes which become fluorescent upon oxidation. These were dihydroethidine (HEt), 2'-7'-dichloro dihydroftuorescein (DCF) and dihydrorhodamine (DHR). Upon exposure to 100 IzM glutamate, all three dyes became rapidly oxidized and thereby fluorescent (Fig. 1). Control measurements with biochemical methods indicated there was, indeed, an increased production of ROS species (Schuchmann and Heinemann, 2000b). The increased production of ROS will eventually lead to increased consumption of glutathione. We therefore also studied the effect of glutamate

200

A =~

100 pM glutamate

I

B o~"

100 pM glutamate

n, -1- 0 J 121

C o~"

100 pM glutamate

._~ 0

OJ

o

I I 100 s

Fig. 1. Measurements of ROS production induced by glutamate in cultured hippocampal neurons using different ROS indicators. Application of 100 IxM glutamate for 100 s induced an increase of the fluorescence signal of ethidium, the oxidized form of hydroethidium (HEt, A), rhodamine-123, the oxidized form of dihydrorhodamine (DHR, B) and dichlorofluorescein (DCF, C). All signals were expressed as changes in baseline signal in %.

exposure on the glutathione content in cultured hippocampal neurons. For this, we employed the dye monochlorobimane (MBCL) which predominantly reacts with glutathione - - but only in its reduced form - - to emit a bright fluorescence (Stabel-Burow et al., 1997; Schuchmann and Heinemann, 2000a; Reichelt et al., 1997; Huster et al., 2000). Glutamate applied with 100 txM for 1 h leads to a fall in GSH content by about 5%, which slowly recovers to baseline within 6 h. In the presence of glutamate receptor antagonists (NBQX and 2APV), glutamate instead caused an increase in MBCL fluorescence due to a glutamate-dependent increased synthesis of glutathione which could be further augmented by

cystine or cysteine (Schuchmann and Heinemann, 2000a). The findings suggested that exposure of neurons to elevated glutamate levels induces a Ca-dependent depolarization of mitochondria leading to an increased formation of ROS. The glutamate-induced cell death could be prevented in part by upregulation of glutathione or by application of cyclosporin A, an inhibitor of transition pores in the mitochondria. If such pores are formed, release of cytochromes is expected which might be involved in induction of apoptosis (Bernardi, 1996; Zamzami et al., 1996).

201

Properties of status epilepticus in combined entorhinal cortex hippocampal slices We noted earlier (Walther et al., 1986) that the lowering of Mg can induce different patterns of epileptiform activity in combined slices of the hippocampus and neighboring structures, such as the ento- and perirhinal cortex. Lowering of extracellular Mg concentration in these preparations induces recurrent seizure-like events characterized by slow negative shifts superimposed by tonic- and clonic-like discharges. The SLEs are accompanied by similar ionic changes, as in vivo, and are blocked by anticonvulsant drugs (Zhang et al., 1995; Dreier et al., 1998). These events recur regularly, but after some 20-40 repetitions they change their appearance (Dreier and Heinemann, 1991). The late recurrent discharges are shorter in duration and recur with a relatively high frequency. Studies on the pharmacological sensitivity to clinically employed anticonvulsants has revealed that these late recurrent discharges no longer respond to clinically employed anticonvulsants and thus seem to model the late pharmacoresistant status epilepticus which presents with considerable problems in clinical care. In slices, it was shown that this drugresistant status epilepticus can readily be reversed to treatable status epilepticus, when GABA is supplemented (Pfeiffer et al., 1996). This is in contrast to high levels of midazolam or phenobarbital, which are without effect. The finding that GABA and muscimol can stop the late recurrent discharges in this model of drug-resistant discharges then points to a loss of GABA during recurrent seizures, presumably due to consumption by neurons and glia in the GABA shunt of the tricarboxylic acid cycle. To test this hypothesis further, we studied the effects of anticonvulsants on 4AP-induced seizurelike events. These are similar in appearance to low Mg-induced SLEs, but recur, in our hands, in a somewhat lower frequency (Brtickner and Heinemann, 2000; Buchheim et al., 2000; Schuchmann et al., 1999). They differ from those induced by low Mg in that one type of interictal discharge can persist (albeit reduced in amplitude) when the seizurelike events are blocked by CNQX combined with 2APV, antagonists of ionotropic glutamate receptors (Briickner et al., 2000). These interictal discharges are further reduced in amplitude when bicuculline

is applied (Perreault and Avoli, 1991). This suggests an involvement of GABA in the generation of these events. However, the used concentrations also lead to blockade of glycinergic currents (Shirasaki et al., 1991) and to blockade of Ca-dependent small conductance K channels (Khawaled et al., 1999; Strobaek et al., 2000). The interictal discharges of this type frequently occur just at the onset of a SLE (Lucke et al., 1995). However, thorough counting reveals that this varies from slice to slice and may also change during the course of recurring SLEs. Also in the 4AP model transition to late recurrent discharges is frequently observed. This process can be accelerated when bicuculline is employed together with 4AP (Brtickner et al., 1999). Under that condition, SLEs change almost immediately to late recurrent discharges. The same is observed with the combined application of low Mg and bicuculline (Pfeiffer et al., 1996). Tests on the pharmacological sensitivity of these late discharges revealed that they are also insensitive to clinically employed anticonvulsants, even in the toxic concentration range (Zhang et al., 1995).

Seizure-induced changes in NAD(P )H autofluorescence in entorhinal cortex slices The seizure-like events in parahippocampal structures, such as the subiculum the entorhinal cortex, the perirhinal cortex and neighboring temporal neocortex were characterized by 30-90-s-long negative potential shifts superimposed by initial tonic-like and then clonic-like field potential transients. These events were followed by interictal discharges. Fura-2 measurements revealed rises in [Ca2+]i by 200-900 txM, depending on cell type (Gloveli et al., 1999). Interestingly, the rises in [Ca2+]i were particularly large in layer III neurons, a cell group which is particularly vulnerable during status epilepticus (Du et al., 1993; Du et al., 1995). The SLEs were usually initiated in medial entorhinal cortex from where they spread to neighboring areas (Buchheim et al., 2000). In adult tissue from normal rats, invasion of SLEs into the hippocampus were usually not observed. This was different in slices from juvenile animals (Weissinger et al., 2000) and from adult animals which had previously experienced a pilocarpine status epilepticus or which were kindled by recur-

202 ring stimulation of the amygdala (Behr et al., 1998; Wozny et al., 2000). With time, the appearance of the SLEs changed and after roughly 20-40 SLEs, the activity changed abruptly into late recurrent discharges. In order to test for the hypothesis that metabolism is altered during recurring seizure-like events and may be compromised during transition to late drug-resistant discharges, we measured the changes in NAD(P)H autofluorescence during recurring seizure-like events. We found that each seizure-like event was accompanied by an initial decrease in NAD(P)H autofluorescence followed by a long-lasting increase. With recurring numbers of SLEs, the amplitude of the rises in NAD(P)H autofluorescence declined, while the initial decreases in these signals remained constant. At the time when seizure-like events were replaced by late recurrent discharges, the NAD(P)H overshoots had disappeared (Schuchmann et al., 1999). These findings suggested that recurrent seizures can damage mitochondrial functions and that this process may be involved in causing cell death. Unfortunately, detailed studies with respect to this damage cascade cannot be readily performed in slices. This is due to the fact that slices underwent a period of hypoxia during preparation, that they have damaged axons and dendrites and that due to these alterations also microglial cells become activated. Moreover, the oxygen tension in the slice is variable depending on distance to the cut surface of the slice. Staining of slices with fluorescent probes is also not readily performed as exposure to dyes in stagnant chambers may alter viability of slices further. We therefore took advantage of organotypic slice cultures which also develop seizure-like events when exposed to low Mg concentration (Gutierrez et al., 1999).

Recurring seizure-like events in organotypic hippocampal cultures Unlike hippocampal slices from rats aged 2-3 weeks, where lowering of extracellular Mg concentration induces SLEs only in area CA1 and the subiculum, slice cultures of similar developmental age generate SLEs, which rapidly synchronize throughout the preparation. These events involve the DG, the hilus, area CA3 and CA1 (Gutierrez et al.,

1999; Kov~ics et al., 1999). This is due to the development of aberrant connectivity in the slice culture presumably due to deafferentation and deefferentation in the isolation procedure. In such cultures, recurrent axon collaterals can be demonstrated for mossy fibers and mutual connections between CA1 and CA3 and CA1 and the DG also exist (Gutierrez and Heinemann, 1999). This is actually comparable to the synaptic organization in slices from rats with pilocarpine- or kainate-status epilepticusinduced hippocampal sclerosis where similar aberrant connectivities were also demonstrated (Esclapez et al., 1999; Lehmann et al., 2000, 2001; Smith and Dudek, 2001). The SLEs induced by exposure to Mg free ACSF are rather similar to the SLEs induced in the entorhinal cortex. They recur with an average frequency of one SLE every 15 min. By applying short-stimulus trains to the mossy fibers, such events can also be electrically triggered (Fig. 2). They consist of an initial bursting discharge followed by a tonic- and clonic-like discharge period and a postictal depression (Kovacs et al., 2001) Before and after a SLE, interictal discharges appeared. The ionic changes accompanying such SLEs are quite comparable to those which we observed in intact animals. The [Ca2+]o drops by, on average, 0.6 mM and [K+]o rises to about 9 mM. After the 15th to 20th SLE, recurrent late discharges develop. We preloaded, in the incubator, slice cultures with different dyes and used photomultiplier and imaging techniques to follow the intracellular events during recurring SLEs. Staining with Ca green, an indicator which signals cytosolic Ca concentration changes, revealed that each seizure-like event was characterized by typical intracellular Ca fluctuations. The Ca concentration rose rapidly during the IBP, declined just before the tonic discharge period during which the Ca climbed to a plateau level. During the CLADE the Ca declined slowly although each clonic discharge was accompanied by a transient increase in Ca concentration. These kinetics were very similar from SLE to SLE. However, the amplitudes of Ca green signals declined rapidly by about 30% from the first to 3rd seizure-like event and then remained constant (Kovacs et al., 2001) (Fig. 3A). When we stained the cultures with Rhod-2, a Casensing fluorescent probe, which, due to its positive charge, accumulates within mitochondria, we were

203

TLP

CLADP

IBP

2 mV

0.45 mM

Fig. 2. Typical seizure-likeevents in an organotypichippocampal slice culture, prepared at around P7 and studied about 1 week later. IBP, initial bursting discharge; TLP, tonic-likedischarge phase; CLADP,clonic-likeafter discharge period; fp, field potential recording. The Ca signal was linearizedby us using the Nernst equation. able to monitor changes in intramitochondrial Ca concentration (Fig. 3B). During SLEs, the Rhod-2 fluorescence signals indicated a rapid rise of intramitochondrial Ca during the initial burst discharge followed by a secondary rise during the tonic discharge phase. By contrast to Ca green and Fluo signals single afterdischarges were not reflected in the Rhod-2 signals. Moreover, the decay of the Rhod-2 signals during and following the clonic afterdischarge period was much slower than that of the Ca green signals. These findings suggest that the Rhod-2 fluorescence came from a different compartment than that of the Ca green signals and reliably reflected intramitochondrial rises in [Ca 2+] (see Fig. 3B). During the course of recurring SLEs, the intramitochondrial Ca concentration signals also declined in amplitude. The amplitudes decreased by roughly 60% from the first to the 15th SLE. This is much more than indicated by the cytosolic Ca signals and may point to a loss of mitochondrial function. We also used rhodamine-123 to follow changes in mitochondrial membrane potential. It turned out that each SLE was associated with a mitochondrial depolarization, which during late recurrent discharges reflected in a steadily increased mitochondrial potential. To determine whether production of ROS signals is increased during single seizure-like events, we measured the changes in HEt fluorescence (Fig. 4). We found that each SLE was accompanied by an in-

crease in ethidium fluorescence. However, while Ca signals declined in amplitude these signals increased in amplitude from seizure to seizure. In a further step, we analyzed the NAD(P)H fluorescence signals (Fig. 5). As was the case in slices, these signals also declined in amplitude during recurring seizures and rises in NAD(P)H autofluorescence were abolished shortly before or at the time of transition into late recurrent discharges. These findings indicated that during status epilepticus, Ca enters not only the cytoplasm, but also the mitochondria where they probably stimulated Casensitive enzymes in the tricarboxylic acid cycle, resulting in increased production of NADH. However, as the amplitudes of these signals declined with time, we hypothesized that production of free radicals might have affected the mitochondrial function. In order to test this hypothesis, we pretreated our slice cultures with ct-tocopherol, which is a widely used free radical scavenger acting mostly at lipid membranes. In the presence of ct-tocopherol, the rises in HEt fluorescence were initially reduced while the decline in NAD(P)H autofluorescence signals no longer occurred (Figs. 4 and 5) and transition to late recurrent discharges was protracted. This corroborated the idea that the HEt fluorescence increase was indeed due to increases in ROS production and that ROS-induced impairment of mitochondrial function might be involved in a reduced capability of nerve cells and glia to adapt their cellu-

204

A

CaGreen

avf0

2%

fp ~

~

I 1 mV

ICa"l.

O.4Sm 50 s

SLE, No 1

B

SLE, No 15

Rhod-2

~ 1

Af/fo

2%

[Ca Is

I 0.45mM S

SLE, No 1

SLE, No 15

Fig. 3. Fluorescence signals of Ca green and Rhod-2 during the first (SLE, No. 1) and 15th SLE (SLE, No. 15). Ca green indicates changes in cytosolic Ca concentration and Rhod-2 predominantly changes in intramitochondrial Ca concentration. Simultaneously recorded changes in field potentials (fp) and extracellularCa concentrationare also displayed.

lar metabolism to the energy demands imposed onto cells by increased activity. It was therefore of interest to test whether tocopherol also protected against seizure-induced cell loss.

Propidium iodide staining following 2 h of status epilepticus We have previously shown that slice cultures, when stained with propidium iodide after 2 h of status

205

A Ethidium

untreated

~'~

AV,o ~

fp [Ca2*]e

~

1

k"~"~'J

i 2 o/° 1mY

0.2 mM

SLE, No 15

SLE, No 1

B Ethidium

(~-tocopherol

Af/fo

12%

f0

I 1mY

[Ca2*]e

0.2 mM

SLE, No 1

SLE, No 15

Fig. 4. Effects of recurrent seizures on increases in ethidium fluorescence after staining with hydroethidium(HEt). (A) Note increase in fluorescence signal from the first to 15th seizure-likeevent, while fp and changes in calcium concentrationremain largely unaltered. (B) In the presence of a-tocopherol, the increases in HEt fluorescence are reduced while fp and Ca concentrationchanges are not largely altered.

epilepticus, developed an intense increase in PI fluorescence. This affected all principal cell layers in the organotypic slice culture, namely the granule cell layer, the CA3 region and the CA1 region. In the presence of c~-tocopherol, this fluorescence increase

was much reduced suggesting that seizure associated production of free radicals were indeed involved in status epilepticus-induced cell loss (Fig. 6).

206

A

NAD(P)H untreated

Af/fo __~~

fp

[Ca2+]e

~

SLE, Nol

12mV 0.2 mM

SLE, No15

B

NAD(P)H ~-tocopherol

Af/fo J

~

fp 4~ll~

-~~12mv

[Ca2+]e SLE, No 1

12%

--~Js

0.2 mM

SLE, No 15

Fig. 5. Changes in NAD(P)H autofluorescence during the first and 15th SLE in untreated slice cultures (A) and in slice cultures pretreated with a-tocopherol (B). Note reduced decline in NAD(P)H autofluorescence from the first to the 15th SLE in B.

Discussion and conclusions Our findings suggest that during status epilepticus, Ca has a role in adapting N A D H synthesis in mitochondria and thereby ATP synthesis to the needs for ion homeostasis which require activation of the

Na,K-ATPase. This depends on oxygen and glucose. As during seizures mitochondria depolarize, there is the risk of increased production of free radicals. Due to the increase in blood flow during seizures, oxygen supply may be augmented and consequently the risk of ROS generation could be further enhanced. This

207

nontreated

o~-tocopherol

CA1

CA3 Fig. 6. Exampleof propidium iodide staining in an untreated and c~-tocopherol-treatedslice culture after 2 h of status epilepticus. Note neuroprotectiveeffect of the free radical scavenger.

seems to result in damage of mitochondrial function as indicated by the reduced capability to take up Ca, the decrease in NAD(P)H autofluorescence and the increase in HEt-fluorescence. The fact that some of these alterations as well as cell death can be prevented by application of a free radical scavenger raises the interesting possibility that cell loss during status epilepticus involves generation of free radicals. We have previously shown that the intracellular levels of glutathione influence sensitivity to free

radical-induced cell death (Schuchmann and Heinemann, 2000a). The synthesis of GSH depends on uptake of glycine, glutamate and cystine in glial cells while neurons require glycine and glutamylcysteine for GSH synthesis. The cystine uptake into glia and the cysteine uptake into neurons depend on exchange transport against glutamate. As during seizures extracellular glutamate is elevated, the efficacy of glutamate-cystine antiport and the supply of neurons with cysteine may be hampered while, at the same time, due to increased ROS production, GSH may become oxidized. It was shown in oligodendrocyte cultures as well as in hippocampal dissociated cell cultures (Schuchmann and Heinemann, 2000a) that supply of cysteine can enhance intracellular glutathione levels. In oligodendrocyte cultures, the clinically well known N-acetylcysteine was also effective in cell protection. This might imply that N-acetylcysteine and c~-tocopherol could exert some neuroprotection during status epilepticus. We are on the way to test these interesting hypotheses. The increases in intracellular Ca concentration correspond to those observed during application of about 100 IxM glutamate in dissociated cultures. In addition, this treatment induces cell death which likely involves activation of apoptosis. One signal commonly considered to stimulate apoptosis is mitochondrial release of cytochrome c. We do not yet know whether this also occurs during status epilepticus, but it will be interesting to see whether SE-induced cell death can also be prevented by inhibitors of mitochondrial transition pore formation.

Acknowledgements This research was supported by the BMBF, the SFB 507 C3 and the Graduate College 238: Damage processes in the central nervous system: studies with imaging techniques. We are grateful to H. Siegmund and H.-J. Gabriel for technical assistance.

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