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hypoglycemic injury
Neuropharmacology 39 (2000) 1779–1787 www.elsevier.com/locate/neuropharm
Inhibition of different pathways influencing Na+ homeostasis protects organotypic hippocampal slice cultures from hypoxic/hypoglycemic injury Jo¨rg Breder a
a,*
, Clemens F. Sabelhaus a, Thoralf Opitz a, Klaus G. Reymann Ulrich H. Schro¨der a
a, b
,
Project Group Neuropharmacology, Leibniz Institute for Neurobiology, POB 1860, D-39008 Magdeburg, Germany b Research Institute for Applied Neurosciences, Leipziger Str. 44, D-39120 Magdeburg, Germany Accepted 7 January 2000
Abstract A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na+ and Ca2+, which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca2+ concentration [Ca2+]i, whereas the elevation of intracellular Na+ has received less attention. In the present study we investigated the effects of inhibitors of different Na+ channels and of the Na+/Ca2+ exchanger, which couples the Na+ to the Ca2+ gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na+ channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na+/Ca2+ exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca2+ accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca2+ extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca2+]i increase caused by the exchanger. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Organotypic slice cultures; Neuroprotection; Pharmacology; Ischemia; Cation channels; Na+/Ca2+ exchanger
1. Introduction Cerebral ischemia results in severe cell degeneration and consequently in loss of brain functions. It is widely accepted that ischemic neuronal cell death results from excessive intracellular accumulation of Ca2+ caused by the massive release of glutamate during the insult. Thus, a large number of studies have focused on the pathways involved in [Ca2+]i increase. In comparison, the elevation of the intracellular Na+ concentration ([Na+]i), which may also contribute to brain damage, has received little attention. Potential major routes for the rise in [Na+]i during ischemia include the influx through voltage-gated
* Corresponding author. Tel.: +49-391-6117-202; fax: +49-3916117-201. E-mail address: [email protected] (J. Breder).
and ligand-gated Na+ channels and entry through the Na+/Ca2+ exchanger. The importance of these pathways for Na+ homeostasis during ischemia and subsequent neuronal damage is still under debate. Specific inhibitors of voltage-gated Na+ channels or the ligand-gated aamino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate and N-methyl-D-aspartate (NMDA) receptor channels reduced neuronal damage in different ischemia models in vivo and in vitro (Gill et al., 1991; Gill, 1994; Newell et al., 1995; Pringle et al., 1997; Vornov et al., 1994). The Na+/Ca2+ exchanger electrogenically exchanges 3 Na+ for 1 Ca2+ and can function to cause Ca2+ accumulation (reverse mode) or Ca2+ extrusion (forward mode) depending on the concentration of each ion on either side of the membrane and on membrane potential (Pitts, 1979). During ischemia and early reperfusion the exchanger is expected to operate in reverse mode,
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because both the decrease in membrane potential and the large increase in [Na+]i would favor the entry of Ca2+. Whether or not this mechanism plays a critical role in ischemic brain damage is still a matter of debate. Inhibition of the exchanger protected optic nerves from anoxic damage (Stys et al., 1991) but exerted little protection or even exacerbated damage in models of glutamate neurotoxicity (Andreeva et al., 1991; Hoyt et al., 1998). Studies regarding the pathophysiological roles of the exchanger have been hampered by the lack of selective inhibitors. The inhibitors used to date, bepridil and amiloride analogues, not only block the forward and reverse modes of the exchanger, but also inhibit a number of ion channels, ion transporters and cell metabolism (Kleyman and Cragoe, 1988; Storozhevykh et al., 1996). Recently, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943) was reported to potently and selectively inhibit the reverse mode of the Na+/Ca2+ exchanger without affecting other Na+-dependent transport systems, Na+ channels or ionotropic glutamate receptors (Iwamoto et al., 1996; Hoyt et al., 1998). In intact cells KB-R7943 was found to act primarily on external exchanger site(s) other than the transport sites, and inhibition was non-competitive with respect to Ca2+ and Na+ (Iwamoto et al., 1996). In a recent paper we have demonstrated that the inhibition of the Na+/Ca2+ exchanger by KB-R7943 potently improved population spike recovery in freshly isolated hippocampal slices after hypoxia/hypoglycemia at a concentration preferentially inhibiting the reverse mode of the exchanger (Schro¨der et al., 1999). Here we investigated whether this short-term neuroprotective effect of KB-R7943 subsequently results in a long-term improvement of neuronal recovery after a hypoxic/hypoglycemic insult utilizing organotypic hippocampal slice cultures. Furthermore we studied the contribution to neuronal injury of other pathways influencing Na+ homeostasis.
2. Materials and methods 2.1. Tissue culture Organotypic slice cultures were prepared according to Stoppini et al. (1991). Briefly, 350 µm thick hippocampal slices were prepared from 10 day old Wistar rats (Institute breeding stock) using a McIlwain tissue chopper (The Mickle Laboratory Engineering Co., Guildford, UK) and placed by 3–4 on membrane inserts in 6-well plates (NUNC, Wiesbaden, Ger.). Each well contained 1.2 ml of tissue culture medium consisting of 50% MEM (Biochrom, Berlin, Ger.), 25% HBSS (Gibco, Eggenstein, Ger.), 25% horse serum (Gibco), 350 mg/ml NaHCO3 (Sigma, Deisenhofen, Ger.), pH 7.3–7.4. Organotypic cultures were maintained in a humidified
incubator gasified with 1% CO2 at 36°C. Culture medium was changed three times/week. 2.2. Induction of ischemic injury After 12–15 days in vitro (DIV) organotypic slice cultures were transferred to 1 ml/well of Ringer solution (composition in mM: NaCl 124, KCl 4.9, MgSO4 1.3, CaCl2 2, KH2PO4 1.2, NaHCO3 25.6, D-glucose 10) in 6-well plates. Hypoxia/hypoglycemia was induced by incubating slice cultures in a moistened gas mixture of 95% N2/5% CO2 in an incubator at 37°C for 40 min in modified Ringer containing mannitol (10 mM) instead of glucose (Ringer-mannitol). Test compounds were applied either in Ringer 1 h before hypoxia/hypoglycemia and in Ringer-mannitol during the insult, or in culture medium during the recovery period until propidium iodide staining or throughout the entire experiment (1 h before hypoxia/hypoglycemia until propidium iodide staining). After hypoxia/hypoglycemia, slice cultures were incubated in culture medium under normoxic conditions for another 24 h. 2.3. Quantification of neuronal cell death 24 h after hypoxia/hypoglycemia organotypic cultures were stained with propidium iodide (10 µg/ml) for 2 h. Propidium iodide uptake is indicative of significant membrane injury (Macklis and Madison, 1990) and has been correlated with lactate dehydrogenase release and the degree of histologic change in glutamate neurotoxicity in slice cultures (Vornov et al., 1991). Propidium iodide fluorescence was elicited at 546 nm and recorded at ⬎610 nm on an inverted fluorescence microscope (Nikon, Du¨sseldorf, Ger.). Images were captured using a CCD camera (Visitron Systems, Puchheim, Ger.), stored on CD, and subsequently analyzed on a PC with an image analysis software (LUCIA M, Nikon). The neuronal regions of control slice cultures could be easily identified from transmission images. Due to a pronounced darkening of slice cultures after hypoxia/hypoglycemia (Figs. 2a and 3a), however, the areas of the CA1 and CA3 subfields could not be unequivocally demarcated. Thus, for the quantification of neuronal damage the percentage of the CA1–CA3 area expressing propidium iodide fluorescence above background level was calculated in relation to the total area of each organotypic culture. In order to compensate for variations in propidium iodide fluorescence between different experiments, the mean values of 3–4 slice cultures treated under identical conditions were calculated and normalized against standard damage. Standard damage was obtained as the mean percentage of fluorescing CA1–CA3 area of organotypic slices subjected to hypoxia/hypoglycemia with no compound applied. This condition was contained in each experiment.
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The normalized values of neuronal cell death obtained from independent experiments are given as mean ± s.e.m. The Wilcoxon matched pairs test was used to compare neuronal damage of organotypic slices treated with compounds against standard damage (100%).
3. Results
2.4. Electrophysiological experiments
A 40 min period of combined oxygen–glucose deprivation (hypoxia/hypoglycemia) resulted in massive cell death in the neuronal cell layers, especially in the CA1 region, of slice cultures as assessed by propidium iodide uptake 24 h after the ischemic simulation (Figs. 2b and 3b). For pharmacological validation of our test system, inhibitors of different Na+ channels, which have been intensively investigated for their protective potency against ischemic damage, were tested in our in vitro ischemia model. The involvement of voltage-gated Na+ channels in mediating neuronal damage in slice cultures was investigated utilizing the Na+ channel blocker TTX, which reduced neuronal damage to 14–24% of standard damage when applied during the hypoxic/hypoglycemic insult (Figs. 1a and 2). When applied only during the recovery period, no protective effect could be detected. Inhibition of NMDA receptor channels by MK 801 also protected organotypic cultures from ischemic damage (Figs. 1b and 2). When MK 801 was applied during the ischemic insult it reduced neuronal damage dose-dependently to 50% (1 µM) or about 5% (10 or 100 µM) of standard damage, respectively. When applied only during the recovery period after the insult, the protective effect of MK 801 was still apparent, although markedly reduced. In contrast to Na+ channels and the NMDA receptor, the inhibition of AMPA/kainate receptor channels by the specific antagonist NBQX failed to protect organotypic slices from ischemic injury, even when applied during the entire experimental period, i.e. during hypoxia/hypoglycemia and recovery (Fig. 1c).
Slice cultures were placed in an interface type recording chamber and maintained at 35±1°C with a constant Ringer perfusion (1 ml/min). The surface of the slice cultures was exposed to a moist atmosphere of 95% O2/5% CO2. Population spikes were evoked by stimulation of the Schaffer collaterals and recorded in the stratum pyramidale of the CA1 region. Test stimuli (biphasic current pulses, 0.1 ms per half wave) were adjusted to elicit a population spike of about 66% of its maximum amplitude. The population spike amplitude was evaluated by calculating the voltage difference between the negative peak and the positive one preceding it. Since the amplitude of population spikes correlates over a wide range with the number of firing neurons (Andersen et al., 1971), it may serve as a measure of functional neuronal integrity. After the population spike amplitude had been stable for at least 30 min (baseline) hypoxia/hypoglycemia was induced by changing the carbogen atmosphere of the chamber to a gas mixture consisting of 95% N2/5% CO2 in the presence of Ringermannitol. After 18 min of hypoxia/hypoglycemia normal oxygen and glucose supply was re-established. The restitution of population spikes was monitored for 1 h. KBR7943 was bath applied from 10 min before hypoxia/hypoglycemia until the end of the experiment. Values of population spike recovery obtained from independent experiments are given as mean ± s.e.m. The Mann-Whitney U-test was used to compare population spike recovery of organotypic slices after hypoxia/hypoglycemia without drug treatment vs hypoxia/hypoglycemia with drug treatment.
2.5. Compounds and solutions
Stock solutions of compounds were prepared as follows: 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX, Tocris, Bristol, UK) 100 mM in dimethylsulfoxide (DMSO), (+)-MK 801 maleate (Tocris) 10 mM in H2O, propidium iodide (Sigma) 10 mM in H2O, tetrodotoxin (TTX, Alexis, Gru¨nberg, Germany) 10 mM in equimolar acetic acid, KB-R7943 100 mM in DMSO. KB-R7943 was generously provided by the Pharmaceutical R&D Center, Kanebo Ltd., Osaka, Japan.
3.1. Effects of voltage-gated Na+ channels, NMDA receptor channels and AMPA/kainate receptor channels on neuronal cell death after hypoxia/hypoglycemia
3.2. Effects of blocking Na+/Ca2+ exchangers on neuronal damage KB-R7943 is a novel specific inhibitor of Na+/Ca2+ exchangers that inhibits both the forward and the reverse mode of the exchanger in a concentration-dependent manner. At low concentrations it selectively inhibits the reverse mode, at higher concentrations it also inhibits the forward mode of the exchanger (Iwamoto et al., 1996; Watano et al., 1996). In order to find out the appropriate concentration that induces a neuroprotective effect on organotypic slice cultures, KB-R7943 was applied to slice cultures in a concentration range from 10 nM–30 µM. As shown in Figs. 3 and 4, KB-R7943 at a concentration of 100 nM, reduced neuronal cell death to 71% of standard cell death. No protective effects of KB-R7943 could be observed at concentrations of 10 nM or 1 µM. When
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Fig. 2. Inhibition of voltage-gated Na+ channels or NMDA receptor channels protects organotypic hippocampal slices from neuronal damage. Transmission images (a, c, e, g) and propidium iodide fluorescence images (b, d, f, h) of slice cultures subjected to 40 min hypoxia/hypoglycemia. (a, b) standard condition: hypoxia/ hypoglycemia without protective compound, (c, d) 10 µM TTX protecting from neuronal damage, (e, f) 1 µM MK 801 effecting only partial protection from neuronal damage, (g, h) 10 µM MK 801 effecting total protection. The white lines in (b), (d) and (f) demarcate the propidium iodide-positive areas. Space bar: 0.5 mm.
Fig. 1. Inhibition of voltage-gated Na+ channels, NMDA receptor channels and AMPA/kainate receptor channels differentially affects neuronal cell death after simulated ischemia in vitro. (a) TTX protects organotypic slice cultures from neuronal damage when present during the hypoxic/hypoglycemic insult (n=7 or 8), but not when applied only during the recovery period (n=8, each condition). (b) MK 801 reduces neuronal damage dramatically (n=6, each concentration) when applied during the hypoxic/hypoglycemic insult. This protective effect is diminished, but still apparent when MK 801 is given only during the recovery period (n=4, each condition). (c) NBQX applied to organotypic cultures for the entire experimental period (insult + recovery) fails to reduce neuronal damage (n=6 or 5). *p⬍0.05%, ** p⬍0.01% (Wilcoxon matched pairs test).
administered at higher concentrations (10 and 30 µM), however, KB-R7943 increased neurodegeneration by 38 or 40%, respectively. The deleterious effects of the high KB-R7943 concentrations appear to be specific for the hypoxic/hypoglycemic condition since the incubation of slice cultures with 30 µM KB-R7943 under control conditions, without hypoxia/hypoglycemia, did not result in propidium iodide uptake (Fig. 3h). The Na+/Ca2+ exchanger is expected to change its operation mode from forward to reverse during hypoxia/hypoglycemia and back to forward during the recovery period. Therefore, the inhibition of the exchanger might have different effects depending on the presence of the inhibitor during or after the insult. In order to test this hypothesis, KB-R7943 was administered either during the hypoxic/hypoglycemic insult or
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Fig. 3. Transmission images (a, c, e, g) and propidium iodide fluorescence images (b, d, f, h) of organotypic slice cultures subjected to hypoxia/hypoglycemia. (a, b) standard condition: 40 min hypoxia/hypoglycemia, (c, d) 0.1 µM KB-R7943 protecting from neuronal damage, (e, f) 30 µM KB-R7943 exacerbating neuronal damage, (g, h) 30 µM KB-R7943 applied to control cultures not subjected to an ischemic insult. The white lines in (b), (d) and (f) demarcate the propidium iodide-positive areas. Space bar: 0.5 mm.
in the recovery period. As demonstrated in Fig. 4b, both the beneficial effect of 0.1 µM KB-R7943 as well as the deleterious effect of 30 µM were no longer detectable in either case. Thus, for conferring a neuroprotective effect KB-R7943 has to be present both during hypoxia/hypoglycemia and the recovery period. In order to compare the protection from neuronal cell death by KB-R7943 in organotypic slice cultures, which resemble juvenile tissue, to the short-term protective effect seen in hippocampal slices from adult rats (Schro¨der et al., 1999) we investigated the influence of KB-R7943 on the recovery of population spikes recorded in the CA1 region of organotypic slices. Interruption of oxygen and glucose supply of organotypic slices resulted in a complete loss of the evoked electrophysiological response 2–3 min after the onset of hypoxia/hypoglycemia. Following an 18 min hypoxia/hypoglycemia, population spikes recorded from
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Fig. 4. Na+/Ca2+ exchangers are involved in neuronal damage in organotypic cultures after hypoxia/hypoglycemia. (a) KB-R7943 reduces neuronal damage when applied at a concentration of 0.1 µM throughout the entire experiment (insult + recovery) but increases damage at 10 and 30 µM. 0.01 µM or 1 µM KB-R7943 have no effect on neuronal damage (0.01 µM: n=5, 0.1 µM: n=14, 1 µM: n=6, 10 µM: n=6, 30 µM: n=15). (b) When applied only during the hypoxic/hypoglycemic insult or only during the recovery period, the protective effect of 0.1 µM KB-R7943 and the deleterious effect of 30 µM are no longer detectable (insult: n=9, each condition, recovery: n=9 or 6). **p⬍0.01, ***p⬍0.001 (Wilcoxon matched pairs test).
untreated CA1 pyramidal cells recovered within 1 h only to about 15% of their baseline amplitude (Fig. 5). In the presence of 0.1 µM KB-R7943, population spike responses recovered to approximately 75%, significantly different from the recovery in control slices. Moreover, KB-R7943 did not affect population spike amplitude in slices not subjected to hypoxia/hypoglycemia (103±3.2% of baseline amplitude after 20 min).
4. Discussion 4.1. Involvement of glutamate receptor channels in neuronal damage in organotypic slice cultures Organotypic cultures preserve much of the synaptic connectivity and extracellular microenvironment that
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Fig. 5. The Na+/Ca2+ exchange inhibitor KB-R7943 protects organotypic slice cultures from functional neuronal damage after hypoxia/hypoglycemia. (a) Traces of electrophysiological recordings in the CA1 region after stimulation of the Schaffer collaterals of organotypic slice cultures subjected to 18 min oxygen/glucose deprivation (control) and slice cultures additionally subjected to KB-R7943 (0.1 µM). Immediately after the insult (0 min) no population spike can be evoked, after 60 min recovery the population spike amplitude after KB-R7943 treatment is larger than without treatment (control). (b) Recovery of synaptic transmission during 60 min after hypoxia/hypoglycemia of slice cultures subjected to hypoxia/hypoglycemia without protective compound (control) and with KB-R7943 treatment (n=5, each condition). *p⬍0.05 (MannWhitney U-test).
exists in vivo (Ga¨hwiler, 1981; Stoppini et al., 1991). Models of oxygen–glucose deprivation based on organotypic cultures demonstrate that many of the elements of the in vivo situation are retained, including selective vulnerability of CA1 pyramidal cells, delayed neuronal cell death, and protection by glutamate receptor antagonists (Newell et al., 1995; Pringle et al., 1997; Strasser and Fischer, 1995; Vornov et al., 1994). In our model the NMDA receptor antagonist MK 801 turned out to be protective both when applied during the
hypoxic/hypoglycemic insult and, to a lesser extent, when applied only during the recovery period. These results coincide well with findings obtained in other in vivo and in vitro models of cerebral ischemia, which have shown that the NMDA receptor contributes to neuronal damage both during the ischemic insult and the recovery period (Newell et al., 1995; Vornov et al., 1994; Hatfield et al., 1992; Pringle et al., 1997). The AMPA/kainate receptor antagonist NBQX, on the contrary, was not protective in our model. In contrast to our findings NBQX was shown to be neuroprotective in animal models of permanent and temporary middle cerebral artery occlusion and against neuronal degeneration of hippocampal CA1 neurons in animal models of severe forebrain ischemia (see Gill, 1994 for review). NBQX also exerted a neuroprotective effect on corticostriatal slice cultures against AMPA or kainate-induced excitotoxicity (Kristensen et al., 1999), but had no effect on glycine-induced neuronal damage in organotypic hippocampal slice cultures (Newell et al., 1997). In in vitro models of cerebral ischemia utilizing organotypic slice cultures the involvement of AMPA/kainate receptors in neuronal damage is still a matter of debate. The differences in the protective effects of CNQX, which was used as antagonist, may at least in part be due to the severity of the ischemic insult the slice cultures are subjected to. Pringle et al. (1997) reported on neuroprotective effects of CNQX after 60 min hypoxia/hypoglycemia, whereas Laake et al. (1996) and Tasker et al. (1992) did not find any protective effect of CNQX. Strasser and Fischer (1995) reported that CNQX exerted a protective effect only when the ischemic period was short enough to result only in partial neuronal cell death, whereas it was not protective when the insult lasted longer. From this we conclude that the insult was rather severe in our model. Furthermore it has to be taken into account that the use of CNQX as a selective antagonist of AMPA/kainate receptors is complicated because at high concentrations this compound partially blocks NMDA receptor activation through an action at the glycine modulatory site (Lester et al., 1989). 4.2. Influence of voltage gated Na+ channels on neuronal damage Blockade of voltage-gated Na+ channels by TTX increases the tolerance against anoxic/ischemic conditions both in vitro and in vivo. TTX reduced the fall in ATP concentration during anoxia and improved recovery of evoked population spikes from dentate granule cells and CA1 pyramidal neurons in rat hippocampal slices (Boening et al., 1989). TTX also delayed anoxic depolarization (Prenen et al., 1988; Rosen et al., 1994; Xie et al., 1994) and the critical ionic changes associated with it: Na+ entry, Ca2+ entry and K + efflux (Xie et al., 1994). In the isolated perfused rat brain, TTX slowed
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down extracellular acidosis indicating a reduction of the anaerobic metabolic rate (Xie et al., 1994). Furthermore TTX protected cultured hippocampal neurons against ischemia-induced injury even when applied after the insult (Tasker et al., 1992; Vornov et al., 1994). In the present study we have shown that the inhibition of voltage gated Na+ channels by TTX during an hypoxic/hypoglycemic insult was neuroprotective. When applied only after the insult, however, TTX did not improve neuronal survival. The activation of voltagegated Na+ channels thus appears to contribute to neuronal damage during the ischemic insult, but not during the recovery period. Therefore, these results support the conclusion that the ischemic insult we applied was a rather severe one, since it has been shown that the protective effect of TTX is influenced by the severity of the insult, TTX applied after the insult being protective only in the mildest forms of the ischemic simulation (Vornov et al., 1994). These findings are supported by those of Pringle et al. (1997), who evidenced that post-insult addition of TTX prevented hypoxic but not ischemic damage. 4.3. Contribution of the Na+/Ca2+ exchanger to neuronal damage The presence of Na+/Ca2+ exchanger in the brain is well established. In the hippocampus, the Na+/Ca2+ exchanger is mainly distributed in the synaptic fields with only sparse labeling found in the cell body layers (Juhaszova et al., 1996). Besides neurons, astrocytes also express a Na+/Ca2+ exchanger (Goldman et al., 1994), but it is much more prevalent in neurons than in astrocytes (Juhaszova et al., 1996). In neurons the exchanger apparently plays an important role in extruding Ca2+ following cell activation and in controlling [Ca2+]i. In the present study we have demonstrated modulatory effects of the novel Na+/Ca2+ exchange inhibitor KB-R7943 on neuronal damage after an ischemic simulation in organotypic slice cultures. At a rather low concentration (0.1 µM) KB-R7943 was neuroprotective, whereas at higher concentrations (10–30 µM) the compound exacerbated neuronal damage. The protective effect of KBR7943 is in accordance with the findings of Stys et al. (1991), showing that direct pharmacological blockade of the Na+/Ca2+ exchanger protected the optic nerve from anoxic damage, and with our own results obtained on hippocampal slices of adult rats (Schro¨der et al., 1999). The Na+/Ca2+ exchanger can function to cause Ca2+ influx (reverse mode) or Ca2+ extrusion (forward mode) depending on the concentration of each ion on either side of the membrane and on membrane potential (Pitts, 1979). KB-R7943 inhibits the reverse mode of the exchanger at low concentrations. The IC50 ranges from 0.32 µM in cardiac ventricular cells (Watano et al., 1996) to 1.6–2.4 µM in transfected fibroblasts, smooth
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muscle cells or cardiomyocytes (Iwamoto et al., 1996). Hoyt et al. (1998) reported an IC50 of 0.7 µM for the reverse mode in isolated rat forebrain neurons. At higher concentrations, KB-R7943 also inhibits the forward mode of the exchanger, the IC50 ranging from 17–⬎30 µM (Iwamoto et al., 1996; Watano et al., 1996). We found a rather low concentration of 0.1 µM KB-R7943 to be protective in organotypic slice cultures both measured by propidium iodide uptake and by electrophysiological recordings of population spikes in the CA1 region. The same concentration, which is thought to inhibit the reverse mode of the exchanger in our model, was shown to be protective in hippocampal slices of adult rats (Schro¨der et al., 1999). At higher concentrations (10–30 µM), that probably also inhibit the forward mode of the exchanger in organotypic slice cultures, KB-R7943 exaggerated neuronal damage. The Na+/Ca2+ exchanger thus appears to contribute to neuronal damage by increasing [Ca2+]i during and after the ischemic insult. KB-R7943 has been reported to be highly specific for the Na+/Ca2+ exchanger. Iwamoto et al. (1996) demonstrated that KB-R7943 does not inhibit Na+/H+ exchange, passive 22Na+ uptake, sarcolemmal and sarcoplasmic reticulum Ca2+-ATPases or Na+,K +-ATPase. At high concentrations (ⱖ30 µM) KB-R7943 also inhibits Ltype Ca2+ channels in cultured smooth muscle cells and cardiac voltage gated Na+ channels, but inhibition by low concentrations (0.3–10 µM) of KB-R7943 is selective for the Na+/Ca2+ exchanger. The data of Meder et al. (1997) on rat cerebral cortical synaptosomes also strongly imply that KB-R7943 (3 µM) does not inhibit voltage dependent Ca2+ channels. These findings are supported by Hoyt et al. (1998) who reported that KBR7943 inhibits voltage-sensitive Ca2+ currents in neurons, but the magnitude of inhibition is rather low (7.7 or 24.3% at 10 or 30 µM, respectively) and can not explain the neuroprotection seen in this study. Recently, Sobolevsky and Khodorov (1999) reported that KBR7943 effectively blocks NMDA channels in rat hippocampal slices. They found evidences for the existence of two populations of NMDA channels differing by a high (IC50 =0.8 µM) and low (IC50 =11 µM) affinity for KBR7943. These findings are in contrast to results obtained by Hoyt et al. (1998), who demonstrated that 10 or 30 µM KB-R7943 did not alter NMDA-induced whole cell currents in rat forebrain neurons. We found out that KBR7943 exerts its protective effects at a concentration of 0.1 µM, which fails to block NMDA channels according to Sobolevsky and Khodorov (1999). Furthermore the effects of high concentrations of KB-R7943 are deleterious and not protective as would be expected if KBR7943 would block NMDA-gated channels. The protection by blockade of NMDA-gated channels could be clearly demonstrated by application of MK 801 in our study. Contrary to the finding that MK 801 is neuroprotective when applied only during the insult, KB-R7943
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was neuroprotective only when applied throughout the entire experiment. For these reasons we favor the hypothesis that the effects of KB-R7943 are due to a selective blockade of the Na+/Ca2+ exchanger instead of blocking NMDA channels. The relatively small protective effect of KB-R7943 measured as a reduction in propidium iodide fluorescence 24 h after the ischemic simulation may be explained by the fact that the ischemic insult the organotypic slice cultures were subjected to was rather severe. In the short term range the protective effect of KB-R7943 appears to be more pronounced, since it enhanced recovery of synaptic transmission in the CA1 region from about 15% to about 75%. This is in the same range of protection we found in hippocampal slices of adult rats (Schro¨der et al., 1999). The degree of protection measured by propidium iodide uptake, however, is expected to be more reliable since it represents protection from neuronal cell death and not a functional aspect of neuronal activity measured in the short-term range. In order to test the hypothesis that the inhibition of the exchanger might have different effects depending on the time of inhibition in relation to the hypoxic/hypoglycemic insult, KB-R7943 was administered only during the hypoxic/hypoglycemic insult or during the recovery period. In either case the beneficial effect of 0.1 µM as well as the deleterious effect of 30 µM were no longer detectable. This finding may be explained by the consideration that the exchanger works in the forward mode at the beginning of the ischemic simulation and changes its direction of transport to the reverse mode only some time after the beginning of hypoxia/hypoglycemia, in response to membrane depolarization and increase of [Na+]i. At the end of the ischemic insult the exchanger is expected to be still in reverse mode until the membrane potential and the ionic conditions favor the switch back to the forward mode during the recovery period. Our results strongly suggest that it might be best to inhibit the exchanger during the entire period it operates in the reverse mode. This is only possible if the inhibitor is present both during the insult and the early recovery period. In the same way, high concentrations of KB-R7943 might inhibit the forward mode of the exchanger sufficiently only when present during the entire time period the exchanger works in the forward mode, i.e. both at the beginning of the hypoxic/hypoglycemic insult and some time after the beginning of the recovery period. In conclusion our data suggest that, besides Na+ channels and the NMDA receptor, the Na+/Ca2+ exchanger operating in reverse mode contributes significantly to hypoxia/hypoglycemia-induced neuronal damage in the hippocampus. Specific inhibitors of the reverse mode of the exchanger might therefore be valuable means to reduce neuronal damage in cerebral ischemia.
Acknowledgements The authors wish to thank the Pharmaceutical R&D Center, Kanebo Ltd., Osaka, Japan, for generously providing us with KB-R7943 and Mrs. Heidi Herold and Anke Bo¨cker for expert technical assistance. This research was supported by the German Ministry for Science and Technology (BMBF) grant BEO 210319998B.
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Inhibition of different pathways influencing Na+ homeostasis protects organotypic hippocampal slice cultures from hypoxic/hypoglycemic injury Jo¨rg Breder a
a,*
, Clemens F. Sabelhaus a, Thoralf Opitz a, Klaus G. Reymann Ulrich H. Schro¨der a
a, b
,
Project Group Neuropharmacology, Leibniz Institute for Neurobiology, POB 1860, D-39008 Magdeburg, Germany b Research Institute for Applied Neurosciences, Leipziger Str. 44, D-39120 Magdeburg, Germany Accepted 7 January 2000
Abstract A prominent feature of cerebral ischemia is the excessive intracellular accumulation of both Na+ and Ca2+, which results in subsequent cell death. A large number of studies have focused on pathways involved in the increase of the intracellular Ca2+ concentration [Ca2+]i, whereas the elevation of intracellular Na+ has received less attention. In the present study we investigated the effects of inhibitors of different Na+ channels and of the Na+/Ca2+ exchanger, which couples the Na+ to the Ca2+ gradient, on ischemic damage in organotypic hippocampal slice cultures. The synaptically evoked population spike in the CA1 region was taken as a functional measure of neuronal integrity. Neuronal cell death was assessed by propidium iodide staining. The Na+ channel blocker tetrodotoxin, and the NMDA receptor blocker MK 801, but not the AMPA/kainate receptor blocker NBQX prevented ischemic cell death. The novel Na+/Ca2+ exchange inhibitor 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), which preferentially acts on the reverse mode of the exchanger, leading to Ca2+ accumulation, also reduced neuronal damage. At higher concentrations, KB-R7943 also inhibits Ca2+ extrusion by the forward mode of the exchanger and exaggerates neuronal cell death. Neuroprotection by KB-R7943 may be due to reducing the [Ca2+]i increase caused by the exchanger. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Organotypic slice cultures; Neuroprotection; Pharmacology; Ischemia; Cation channels; Na+/Ca2+ exchanger
1. Introduction Cerebral ischemia results in severe cell degeneration and consequently in loss of brain functions. It is widely accepted that ischemic neuronal cell death results from excessive intracellular accumulation of Ca2+ caused by the massive release of glutamate during the insult. Thus, a large number of studies have focused on the pathways involved in [Ca2+]i increase. In comparison, the elevation of the intracellular Na+ concentration ([Na+]i), which may also contribute to brain damage, has received little attention. Potential major routes for the rise in [Na+]i during ischemia include the influx through voltage-gated
* Corresponding author. Tel.: +49-391-6117-202; fax: +49-3916117-201. E-mail address: [email protected] (J. Breder).
and ligand-gated Na+ channels and entry through the Na+/Ca2+ exchanger. The importance of these pathways for Na+ homeostasis during ischemia and subsequent neuronal damage is still under debate. Specific inhibitors of voltage-gated Na+ channels or the ligand-gated aamino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate and N-methyl-D-aspartate (NMDA) receptor channels reduced neuronal damage in different ischemia models in vivo and in vitro (Gill et al., 1991; Gill, 1994; Newell et al., 1995; Pringle et al., 1997; Vornov et al., 1994). The Na+/Ca2+ exchanger electrogenically exchanges 3 Na+ for 1 Ca2+ and can function to cause Ca2+ accumulation (reverse mode) or Ca2+ extrusion (forward mode) depending on the concentration of each ion on either side of the membrane and on membrane potential (Pitts, 1979). During ischemia and early reperfusion the exchanger is expected to operate in reverse mode,
0028-3908/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 8 - 3 9 0 8 ( 0 0 ) 0 0 0 2 7 - 7
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because both the decrease in membrane potential and the large increase in [Na+]i would favor the entry of Ca2+. Whether or not this mechanism plays a critical role in ischemic brain damage is still a matter of debate. Inhibition of the exchanger protected optic nerves from anoxic damage (Stys et al., 1991) but exerted little protection or even exacerbated damage in models of glutamate neurotoxicity (Andreeva et al., 1991; Hoyt et al., 1998). Studies regarding the pathophysiological roles of the exchanger have been hampered by the lack of selective inhibitors. The inhibitors used to date, bepridil and amiloride analogues, not only block the forward and reverse modes of the exchanger, but also inhibit a number of ion channels, ion transporters and cell metabolism (Kleyman and Cragoe, 1988; Storozhevykh et al., 1996). Recently, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943) was reported to potently and selectively inhibit the reverse mode of the Na+/Ca2+ exchanger without affecting other Na+-dependent transport systems, Na+ channels or ionotropic glutamate receptors (Iwamoto et al., 1996; Hoyt et al., 1998). In intact cells KB-R7943 was found to act primarily on external exchanger site(s) other than the transport sites, and inhibition was non-competitive with respect to Ca2+ and Na+ (Iwamoto et al., 1996). In a recent paper we have demonstrated that the inhibition of the Na+/Ca2+ exchanger by KB-R7943 potently improved population spike recovery in freshly isolated hippocampal slices after hypoxia/hypoglycemia at a concentration preferentially inhibiting the reverse mode of the exchanger (Schro¨der et al., 1999). Here we investigated whether this short-term neuroprotective effect of KB-R7943 subsequently results in a long-term improvement of neuronal recovery after a hypoxic/hypoglycemic insult utilizing organotypic hippocampal slice cultures. Furthermore we studied the contribution to neuronal injury of other pathways influencing Na+ homeostasis.
2. Materials and methods 2.1. Tissue culture Organotypic slice cultures were prepared according to Stoppini et al. (1991). Briefly, 350 µm thick hippocampal slices were prepared from 10 day old Wistar rats (Institute breeding stock) using a McIlwain tissue chopper (The Mickle Laboratory Engineering Co., Guildford, UK) and placed by 3–4 on membrane inserts in 6-well plates (NUNC, Wiesbaden, Ger.). Each well contained 1.2 ml of tissue culture medium consisting of 50% MEM (Biochrom, Berlin, Ger.), 25% HBSS (Gibco, Eggenstein, Ger.), 25% horse serum (Gibco), 350 mg/ml NaHCO3 (Sigma, Deisenhofen, Ger.), pH 7.3–7.4. Organotypic cultures were maintained in a humidified
incubator gasified with 1% CO2 at 36°C. Culture medium was changed three times/week. 2.2. Induction of ischemic injury After 12–15 days in vitro (DIV) organotypic slice cultures were transferred to 1 ml/well of Ringer solution (composition in mM: NaCl 124, KCl 4.9, MgSO4 1.3, CaCl2 2, KH2PO4 1.2, NaHCO3 25.6, D-glucose 10) in 6-well plates. Hypoxia/hypoglycemia was induced by incubating slice cultures in a moistened gas mixture of 95% N2/5% CO2 in an incubator at 37°C for 40 min in modified Ringer containing mannitol (10 mM) instead of glucose (Ringer-mannitol). Test compounds were applied either in Ringer 1 h before hypoxia/hypoglycemia and in Ringer-mannitol during the insult, or in culture medium during the recovery period until propidium iodide staining or throughout the entire experiment (1 h before hypoxia/hypoglycemia until propidium iodide staining). After hypoxia/hypoglycemia, slice cultures were incubated in culture medium under normoxic conditions for another 24 h. 2.3. Quantification of neuronal cell death 24 h after hypoxia/hypoglycemia organotypic cultures were stained with propidium iodide (10 µg/ml) for 2 h. Propidium iodide uptake is indicative of significant membrane injury (Macklis and Madison, 1990) and has been correlated with lactate dehydrogenase release and the degree of histologic change in glutamate neurotoxicity in slice cultures (Vornov et al., 1991). Propidium iodide fluorescence was elicited at 546 nm and recorded at ⬎610 nm on an inverted fluorescence microscope (Nikon, Du¨sseldorf, Ger.). Images were captured using a CCD camera (Visitron Systems, Puchheim, Ger.), stored on CD, and subsequently analyzed on a PC with an image analysis software (LUCIA M, Nikon). The neuronal regions of control slice cultures could be easily identified from transmission images. Due to a pronounced darkening of slice cultures after hypoxia/hypoglycemia (Figs. 2a and 3a), however, the areas of the CA1 and CA3 subfields could not be unequivocally demarcated. Thus, for the quantification of neuronal damage the percentage of the CA1–CA3 area expressing propidium iodide fluorescence above background level was calculated in relation to the total area of each organotypic culture. In order to compensate for variations in propidium iodide fluorescence between different experiments, the mean values of 3–4 slice cultures treated under identical conditions were calculated and normalized against standard damage. Standard damage was obtained as the mean percentage of fluorescing CA1–CA3 area of organotypic slices subjected to hypoxia/hypoglycemia with no compound applied. This condition was contained in each experiment.
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The normalized values of neuronal cell death obtained from independent experiments are given as mean ± s.e.m. The Wilcoxon matched pairs test was used to compare neuronal damage of organotypic slices treated with compounds against standard damage (100%).
3. Results
2.4. Electrophysiological experiments
A 40 min period of combined oxygen–glucose deprivation (hypoxia/hypoglycemia) resulted in massive cell death in the neuronal cell layers, especially in the CA1 region, of slice cultures as assessed by propidium iodide uptake 24 h after the ischemic simulation (Figs. 2b and 3b). For pharmacological validation of our test system, inhibitors of different Na+ channels, which have been intensively investigated for their protective potency against ischemic damage, were tested in our in vitro ischemia model. The involvement of voltage-gated Na+ channels in mediating neuronal damage in slice cultures was investigated utilizing the Na+ channel blocker TTX, which reduced neuronal damage to 14–24% of standard damage when applied during the hypoxic/hypoglycemic insult (Figs. 1a and 2). When applied only during the recovery period, no protective effect could be detected. Inhibition of NMDA receptor channels by MK 801 also protected organotypic cultures from ischemic damage (Figs. 1b and 2). When MK 801 was applied during the ischemic insult it reduced neuronal damage dose-dependently to 50% (1 µM) or about 5% (10 or 100 µM) of standard damage, respectively. When applied only during the recovery period after the insult, the protective effect of MK 801 was still apparent, although markedly reduced. In contrast to Na+ channels and the NMDA receptor, the inhibition of AMPA/kainate receptor channels by the specific antagonist NBQX failed to protect organotypic slices from ischemic injury, even when applied during the entire experimental period, i.e. during hypoxia/hypoglycemia and recovery (Fig. 1c).
Slice cultures were placed in an interface type recording chamber and maintained at 35±1°C with a constant Ringer perfusion (1 ml/min). The surface of the slice cultures was exposed to a moist atmosphere of 95% O2/5% CO2. Population spikes were evoked by stimulation of the Schaffer collaterals and recorded in the stratum pyramidale of the CA1 region. Test stimuli (biphasic current pulses, 0.1 ms per half wave) were adjusted to elicit a population spike of about 66% of its maximum amplitude. The population spike amplitude was evaluated by calculating the voltage difference between the negative peak and the positive one preceding it. Since the amplitude of population spikes correlates over a wide range with the number of firing neurons (Andersen et al., 1971), it may serve as a measure of functional neuronal integrity. After the population spike amplitude had been stable for at least 30 min (baseline) hypoxia/hypoglycemia was induced by changing the carbogen atmosphere of the chamber to a gas mixture consisting of 95% N2/5% CO2 in the presence of Ringermannitol. After 18 min of hypoxia/hypoglycemia normal oxygen and glucose supply was re-established. The restitution of population spikes was monitored for 1 h. KBR7943 was bath applied from 10 min before hypoxia/hypoglycemia until the end of the experiment. Values of population spike recovery obtained from independent experiments are given as mean ± s.e.m. The Mann-Whitney U-test was used to compare population spike recovery of organotypic slices after hypoxia/hypoglycemia without drug treatment vs hypoxia/hypoglycemia with drug treatment.
2.5. Compounds and solutions
Stock solutions of compounds were prepared as follows: 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX, Tocris, Bristol, UK) 100 mM in dimethylsulfoxide (DMSO), (+)-MK 801 maleate (Tocris) 10 mM in H2O, propidium iodide (Sigma) 10 mM in H2O, tetrodotoxin (TTX, Alexis, Gru¨nberg, Germany) 10 mM in equimolar acetic acid, KB-R7943 100 mM in DMSO. KB-R7943 was generously provided by the Pharmaceutical R&D Center, Kanebo Ltd., Osaka, Japan.
3.1. Effects of voltage-gated Na+ channels, NMDA receptor channels and AMPA/kainate receptor channels on neuronal cell death after hypoxia/hypoglycemia
3.2. Effects of blocking Na+/Ca2+ exchangers on neuronal damage KB-R7943 is a novel specific inhibitor of Na+/Ca2+ exchangers that inhibits both the forward and the reverse mode of the exchanger in a concentration-dependent manner. At low concentrations it selectively inhibits the reverse mode, at higher concentrations it also inhibits the forward mode of the exchanger (Iwamoto et al., 1996; Watano et al., 1996). In order to find out the appropriate concentration that induces a neuroprotective effect on organotypic slice cultures, KB-R7943 was applied to slice cultures in a concentration range from 10 nM–30 µM. As shown in Figs. 3 and 4, KB-R7943 at a concentration of 100 nM, reduced neuronal cell death to 71% of standard cell death. No protective effects of KB-R7943 could be observed at concentrations of 10 nM or 1 µM. When
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Fig. 2. Inhibition of voltage-gated Na+ channels or NMDA receptor channels protects organotypic hippocampal slices from neuronal damage. Transmission images (a, c, e, g) and propidium iodide fluorescence images (b, d, f, h) of slice cultures subjected to 40 min hypoxia/hypoglycemia. (a, b) standard condition: hypoxia/ hypoglycemia without protective compound, (c, d) 10 µM TTX protecting from neuronal damage, (e, f) 1 µM MK 801 effecting only partial protection from neuronal damage, (g, h) 10 µM MK 801 effecting total protection. The white lines in (b), (d) and (f) demarcate the propidium iodide-positive areas. Space bar: 0.5 mm.
Fig. 1. Inhibition of voltage-gated Na+ channels, NMDA receptor channels and AMPA/kainate receptor channels differentially affects neuronal cell death after simulated ischemia in vitro. (a) TTX protects organotypic slice cultures from neuronal damage when present during the hypoxic/hypoglycemic insult (n=7 or 8), but not when applied only during the recovery period (n=8, each condition). (b) MK 801 reduces neuronal damage dramatically (n=6, each concentration) when applied during the hypoxic/hypoglycemic insult. This protective effect is diminished, but still apparent when MK 801 is given only during the recovery period (n=4, each condition). (c) NBQX applied to organotypic cultures for the entire experimental period (insult + recovery) fails to reduce neuronal damage (n=6 or 5). *p⬍0.05%, ** p⬍0.01% (Wilcoxon matched pairs test).
administered at higher concentrations (10 and 30 µM), however, KB-R7943 increased neurodegeneration by 38 or 40%, respectively. The deleterious effects of the high KB-R7943 concentrations appear to be specific for the hypoxic/hypoglycemic condition since the incubation of slice cultures with 30 µM KB-R7943 under control conditions, without hypoxia/hypoglycemia, did not result in propidium iodide uptake (Fig. 3h). The Na+/Ca2+ exchanger is expected to change its operation mode from forward to reverse during hypoxia/hypoglycemia and back to forward during the recovery period. Therefore, the inhibition of the exchanger might have different effects depending on the presence of the inhibitor during or after the insult. In order to test this hypothesis, KB-R7943 was administered either during the hypoxic/hypoglycemic insult or
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Fig. 3. Transmission images (a, c, e, g) and propidium iodide fluorescence images (b, d, f, h) of organotypic slice cultures subjected to hypoxia/hypoglycemia. (a, b) standard condition: 40 min hypoxia/hypoglycemia, (c, d) 0.1 µM KB-R7943 protecting from neuronal damage, (e, f) 30 µM KB-R7943 exacerbating neuronal damage, (g, h) 30 µM KB-R7943 applied to control cultures not subjected to an ischemic insult. The white lines in (b), (d) and (f) demarcate the propidium iodide-positive areas. Space bar: 0.5 mm.
in the recovery period. As demonstrated in Fig. 4b, both the beneficial effect of 0.1 µM KB-R7943 as well as the deleterious effect of 30 µM were no longer detectable in either case. Thus, for conferring a neuroprotective effect KB-R7943 has to be present both during hypoxia/hypoglycemia and the recovery period. In order to compare the protection from neuronal cell death by KB-R7943 in organotypic slice cultures, which resemble juvenile tissue, to the short-term protective effect seen in hippocampal slices from adult rats (Schro¨der et al., 1999) we investigated the influence of KB-R7943 on the recovery of population spikes recorded in the CA1 region of organotypic slices. Interruption of oxygen and glucose supply of organotypic slices resulted in a complete loss of the evoked electrophysiological response 2–3 min after the onset of hypoxia/hypoglycemia. Following an 18 min hypoxia/hypoglycemia, population spikes recorded from
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Fig. 4. Na+/Ca2+ exchangers are involved in neuronal damage in organotypic cultures after hypoxia/hypoglycemia. (a) KB-R7943 reduces neuronal damage when applied at a concentration of 0.1 µM throughout the entire experiment (insult + recovery) but increases damage at 10 and 30 µM. 0.01 µM or 1 µM KB-R7943 have no effect on neuronal damage (0.01 µM: n=5, 0.1 µM: n=14, 1 µM: n=6, 10 µM: n=6, 30 µM: n=15). (b) When applied only during the hypoxic/hypoglycemic insult or only during the recovery period, the protective effect of 0.1 µM KB-R7943 and the deleterious effect of 30 µM are no longer detectable (insult: n=9, each condition, recovery: n=9 or 6). **p⬍0.01, ***p⬍0.001 (Wilcoxon matched pairs test).
untreated CA1 pyramidal cells recovered within 1 h only to about 15% of their baseline amplitude (Fig. 5). In the presence of 0.1 µM KB-R7943, population spike responses recovered to approximately 75%, significantly different from the recovery in control slices. Moreover, KB-R7943 did not affect population spike amplitude in slices not subjected to hypoxia/hypoglycemia (103±3.2% of baseline amplitude after 20 min).
4. Discussion 4.1. Involvement of glutamate receptor channels in neuronal damage in organotypic slice cultures Organotypic cultures preserve much of the synaptic connectivity and extracellular microenvironment that
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Fig. 5. The Na+/Ca2+ exchange inhibitor KB-R7943 protects organotypic slice cultures from functional neuronal damage after hypoxia/hypoglycemia. (a) Traces of electrophysiological recordings in the CA1 region after stimulation of the Schaffer collaterals of organotypic slice cultures subjected to 18 min oxygen/glucose deprivation (control) and slice cultures additionally subjected to KB-R7943 (0.1 µM). Immediately after the insult (0 min) no population spike can be evoked, after 60 min recovery the population spike amplitude after KB-R7943 treatment is larger than without treatment (control). (b) Recovery of synaptic transmission during 60 min after hypoxia/hypoglycemia of slice cultures subjected to hypoxia/hypoglycemia without protective compound (control) and with KB-R7943 treatment (n=5, each condition). *p⬍0.05 (MannWhitney U-test).
exists in vivo (Ga¨hwiler, 1981; Stoppini et al., 1991). Models of oxygen–glucose deprivation based on organotypic cultures demonstrate that many of the elements of the in vivo situation are retained, including selective vulnerability of CA1 pyramidal cells, delayed neuronal cell death, and protection by glutamate receptor antagonists (Newell et al., 1995; Pringle et al., 1997; Strasser and Fischer, 1995; Vornov et al., 1994). In our model the NMDA receptor antagonist MK 801 turned out to be protective both when applied during the
hypoxic/hypoglycemic insult and, to a lesser extent, when applied only during the recovery period. These results coincide well with findings obtained in other in vivo and in vitro models of cerebral ischemia, which have shown that the NMDA receptor contributes to neuronal damage both during the ischemic insult and the recovery period (Newell et al., 1995; Vornov et al., 1994; Hatfield et al., 1992; Pringle et al., 1997). The AMPA/kainate receptor antagonist NBQX, on the contrary, was not protective in our model. In contrast to our findings NBQX was shown to be neuroprotective in animal models of permanent and temporary middle cerebral artery occlusion and against neuronal degeneration of hippocampal CA1 neurons in animal models of severe forebrain ischemia (see Gill, 1994 for review). NBQX also exerted a neuroprotective effect on corticostriatal slice cultures against AMPA or kainate-induced excitotoxicity (Kristensen et al., 1999), but had no effect on glycine-induced neuronal damage in organotypic hippocampal slice cultures (Newell et al., 1997). In in vitro models of cerebral ischemia utilizing organotypic slice cultures the involvement of AMPA/kainate receptors in neuronal damage is still a matter of debate. The differences in the protective effects of CNQX, which was used as antagonist, may at least in part be due to the severity of the ischemic insult the slice cultures are subjected to. Pringle et al. (1997) reported on neuroprotective effects of CNQX after 60 min hypoxia/hypoglycemia, whereas Laake et al. (1996) and Tasker et al. (1992) did not find any protective effect of CNQX. Strasser and Fischer (1995) reported that CNQX exerted a protective effect only when the ischemic period was short enough to result only in partial neuronal cell death, whereas it was not protective when the insult lasted longer. From this we conclude that the insult was rather severe in our model. Furthermore it has to be taken into account that the use of CNQX as a selective antagonist of AMPA/kainate receptors is complicated because at high concentrations this compound partially blocks NMDA receptor activation through an action at the glycine modulatory site (Lester et al., 1989). 4.2. Influence of voltage gated Na+ channels on neuronal damage Blockade of voltage-gated Na+ channels by TTX increases the tolerance against anoxic/ischemic conditions both in vitro and in vivo. TTX reduced the fall in ATP concentration during anoxia and improved recovery of evoked population spikes from dentate granule cells and CA1 pyramidal neurons in rat hippocampal slices (Boening et al., 1989). TTX also delayed anoxic depolarization (Prenen et al., 1988; Rosen et al., 1994; Xie et al., 1994) and the critical ionic changes associated with it: Na+ entry, Ca2+ entry and K + efflux (Xie et al., 1994). In the isolated perfused rat brain, TTX slowed
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down extracellular acidosis indicating a reduction of the anaerobic metabolic rate (Xie et al., 1994). Furthermore TTX protected cultured hippocampal neurons against ischemia-induced injury even when applied after the insult (Tasker et al., 1992; Vornov et al., 1994). In the present study we have shown that the inhibition of voltage gated Na+ channels by TTX during an hypoxic/hypoglycemic insult was neuroprotective. When applied only after the insult, however, TTX did not improve neuronal survival. The activation of voltagegated Na+ channels thus appears to contribute to neuronal damage during the ischemic insult, but not during the recovery period. Therefore, these results support the conclusion that the ischemic insult we applied was a rather severe one, since it has been shown that the protective effect of TTX is influenced by the severity of the insult, TTX applied after the insult being protective only in the mildest forms of the ischemic simulation (Vornov et al., 1994). These findings are supported by those of Pringle et al. (1997), who evidenced that post-insult addition of TTX prevented hypoxic but not ischemic damage. 4.3. Contribution of the Na+/Ca2+ exchanger to neuronal damage The presence of Na+/Ca2+ exchanger in the brain is well established. In the hippocampus, the Na+/Ca2+ exchanger is mainly distributed in the synaptic fields with only sparse labeling found in the cell body layers (Juhaszova et al., 1996). Besides neurons, astrocytes also express a Na+/Ca2+ exchanger (Goldman et al., 1994), but it is much more prevalent in neurons than in astrocytes (Juhaszova et al., 1996). In neurons the exchanger apparently plays an important role in extruding Ca2+ following cell activation and in controlling [Ca2+]i. In the present study we have demonstrated modulatory effects of the novel Na+/Ca2+ exchange inhibitor KB-R7943 on neuronal damage after an ischemic simulation in organotypic slice cultures. At a rather low concentration (0.1 µM) KB-R7943 was neuroprotective, whereas at higher concentrations (10–30 µM) the compound exacerbated neuronal damage. The protective effect of KBR7943 is in accordance with the findings of Stys et al. (1991), showing that direct pharmacological blockade of the Na+/Ca2+ exchanger protected the optic nerve from anoxic damage, and with our own results obtained on hippocampal slices of adult rats (Schro¨der et al., 1999). The Na+/Ca2+ exchanger can function to cause Ca2+ influx (reverse mode) or Ca2+ extrusion (forward mode) depending on the concentration of each ion on either side of the membrane and on membrane potential (Pitts, 1979). KB-R7943 inhibits the reverse mode of the exchanger at low concentrations. The IC50 ranges from 0.32 µM in cardiac ventricular cells (Watano et al., 1996) to 1.6–2.4 µM in transfected fibroblasts, smooth
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muscle cells or cardiomyocytes (Iwamoto et al., 1996). Hoyt et al. (1998) reported an IC50 of 0.7 µM for the reverse mode in isolated rat forebrain neurons. At higher concentrations, KB-R7943 also inhibits the forward mode of the exchanger, the IC50 ranging from 17–⬎30 µM (Iwamoto et al., 1996; Watano et al., 1996). We found a rather low concentration of 0.1 µM KB-R7943 to be protective in organotypic slice cultures both measured by propidium iodide uptake and by electrophysiological recordings of population spikes in the CA1 region. The same concentration, which is thought to inhibit the reverse mode of the exchanger in our model, was shown to be protective in hippocampal slices of adult rats (Schro¨der et al., 1999). At higher concentrations (10–30 µM), that probably also inhibit the forward mode of the exchanger in organotypic slice cultures, KB-R7943 exaggerated neuronal damage. The Na+/Ca2+ exchanger thus appears to contribute to neuronal damage by increasing [Ca2+]i during and after the ischemic insult. KB-R7943 has been reported to be highly specific for the Na+/Ca2+ exchanger. Iwamoto et al. (1996) demonstrated that KB-R7943 does not inhibit Na+/H+ exchange, passive 22Na+ uptake, sarcolemmal and sarcoplasmic reticulum Ca2+-ATPases or Na+,K +-ATPase. At high concentrations (ⱖ30 µM) KB-R7943 also inhibits Ltype Ca2+ channels in cultured smooth muscle cells and cardiac voltage gated Na+ channels, but inhibition by low concentrations (0.3–10 µM) of KB-R7943 is selective for the Na+/Ca2+ exchanger. The data of Meder et al. (1997) on rat cerebral cortical synaptosomes also strongly imply that KB-R7943 (3 µM) does not inhibit voltage dependent Ca2+ channels. These findings are supported by Hoyt et al. (1998) who reported that KBR7943 inhibits voltage-sensitive Ca2+ currents in neurons, but the magnitude of inhibition is rather low (7.7 or 24.3% at 10 or 30 µM, respectively) and can not explain the neuroprotection seen in this study. Recently, Sobolevsky and Khodorov (1999) reported that KBR7943 effectively blocks NMDA channels in rat hippocampal slices. They found evidences for the existence of two populations of NMDA channels differing by a high (IC50 =0.8 µM) and low (IC50 =11 µM) affinity for KBR7943. These findings are in contrast to results obtained by Hoyt et al. (1998), who demonstrated that 10 or 30 µM KB-R7943 did not alter NMDA-induced whole cell currents in rat forebrain neurons. We found out that KBR7943 exerts its protective effects at a concentration of 0.1 µM, which fails to block NMDA channels according to Sobolevsky and Khodorov (1999). Furthermore the effects of high concentrations of KB-R7943 are deleterious and not protective as would be expected if KBR7943 would block NMDA-gated channels. The protection by blockade of NMDA-gated channels could be clearly demonstrated by application of MK 801 in our study. Contrary to the finding that MK 801 is neuroprotective when applied only during the insult, KB-R7943
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was neuroprotective only when applied throughout the entire experiment. For these reasons we favor the hypothesis that the effects of KB-R7943 are due to a selective blockade of the Na+/Ca2+ exchanger instead of blocking NMDA channels. The relatively small protective effect of KB-R7943 measured as a reduction in propidium iodide fluorescence 24 h after the ischemic simulation may be explained by the fact that the ischemic insult the organotypic slice cultures were subjected to was rather severe. In the short term range the protective effect of KB-R7943 appears to be more pronounced, since it enhanced recovery of synaptic transmission in the CA1 region from about 15% to about 75%. This is in the same range of protection we found in hippocampal slices of adult rats (Schro¨der et al., 1999). The degree of protection measured by propidium iodide uptake, however, is expected to be more reliable since it represents protection from neuronal cell death and not a functional aspect of neuronal activity measured in the short-term range. In order to test the hypothesis that the inhibition of the exchanger might have different effects depending on the time of inhibition in relation to the hypoxic/hypoglycemic insult, KB-R7943 was administered only during the hypoxic/hypoglycemic insult or during the recovery period. In either case the beneficial effect of 0.1 µM as well as the deleterious effect of 30 µM were no longer detectable. This finding may be explained by the consideration that the exchanger works in the forward mode at the beginning of the ischemic simulation and changes its direction of transport to the reverse mode only some time after the beginning of hypoxia/hypoglycemia, in response to membrane depolarization and increase of [Na+]i. At the end of the ischemic insult the exchanger is expected to be still in reverse mode until the membrane potential and the ionic conditions favor the switch back to the forward mode during the recovery period. Our results strongly suggest that it might be best to inhibit the exchanger during the entire period it operates in the reverse mode. This is only possible if the inhibitor is present both during the insult and the early recovery period. In the same way, high concentrations of KB-R7943 might inhibit the forward mode of the exchanger sufficiently only when present during the entire time period the exchanger works in the forward mode, i.e. both at the beginning of the hypoxic/hypoglycemic insult and some time after the beginning of the recovery period. In conclusion our data suggest that, besides Na+ channels and the NMDA receptor, the Na+/Ca2+ exchanger operating in reverse mode contributes significantly to hypoxia/hypoglycemia-induced neuronal damage in the hippocampus. Specific inhibitors of the reverse mode of the exchanger might therefore be valuable means to reduce neuronal damage in cerebral ischemia.
Acknowledgements The authors wish to thank the Pharmaceutical R&D Center, Kanebo Ltd., Osaka, Japan, for generously providing us with KB-R7943 and Mrs. Heidi Herold and Anke Bo¨cker for expert technical assistance. This research was supported by the German Ministry for Science and Technology (BMBF) grant BEO 210319998B.
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