[Antiphospholipid syndrome 2007. Current aspects of laboratory diagnostics and their therapeutic consequences]. / Antiphospholipid-Syndrom 2007. Aktuelle Aspekte in der Labordiagnostik und deren therapeutische Konsequenzen.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by recurrent vascular thrombosis and loss of pregnancy in association with the presence of antiphospholipid antibodies (APA) detectable as lupus anticoagulants, anticardiolipin antibodies or anti-beta2 glycoprotein I antibodies. The pathophysiological importance of APA in APS is accepted, however, the mechanisms leading to thrombosis are likely to be multifactorial and are so far unclear. Without a prior thrombosis, the risk of developing a new thrombosis in healthy patients with APA is slightly increased (<1% per year). However, the risk of a recurrent thrombosis increases considerably (>10% per year) in patients with a history of thrombosis without anticoagulation. The careful and correct identification of patients with APS is important because prophylactic anticoagulation can reduce the risk of recurrent thrombotic events, and during pregnancy can improve fetal and maternal outcome.

Effects of dexamethasone and celecoxib on calcium homeostasis and expression of cyclooxygenase-2 mRNA in MG-63 human osteosarcoma cells.

OBJECTIVE: Glucocorticoids and selective COX-2 inhibitors are potent anti-inflammatory agents. They are also suggested to influence bone physiology and remodeling. Here we searched for effects of dexamethasone and celecoxib on crucial parameters of bone physiology that could be therapeutically relevant. METHODS: The human osteosarcoma cell line MG-63 was used to measure effects of these drugs on (i) intracellular calcium concentration ([Ca2+]i) using a microfluorometric technique, (ii) alkaline phosphatase and osteocalcin levels (EIA) and (iii) the expression of cox-2 mRNA (quantitative real time PCR). Measurements were performed in Vitamine D-incubated quiescent cells and in cells stimulated with TNF-alpha and IL-1beta. RESULTS: We found the cytokine-stimulation to increase [Ca2+]i which was prevented by dexamethasone already after 30 min and still after 48 h. Dexamethasone was without any effect on [Ca2+]i in quiescent cells. Celecoxib had no measurable short-term or long-term effects neither in quiescent nor in stimulated cells. Vitamin D stimulated the expression of cox-2 mRNA which was further enhanced by TNF-alpha/IL-1beta. Dexamethasone did not have any measurable effects on COX-2 expression after 30 min, but a pronounced inhibition was seen after 48 h. In contrast, celecoxib had no effect on COX-2 expression. Neither of the drugs had any effect on the secretion of alkaline phosphatase and osteocalcin. CONCLUSION: We found dexamethasone to inhibit the [Ca2+]i increase in MG-63 cells following stimulation and to reduce considerably COX-2 expression via the genomic pathway. In contrast, celecoxib did not show any measurable short-term or long-term effects on the parameters of bone physiology measured.

Modulation of intracellular calcium signaling and mitochondrial function in cultured osteoblastic cells by dexamethasone and celecoxib during mechanical stimulation.

OBJECTIVE: Evaluation of potentially therapeutically relevant effects of dexamethasone and celecoxib on crucial parameters of bone physiology during and following mechanical stimulation in cultured osteoblasts. METHODS: An in vitro mechanical stimulation model based on the rat osteogenic cell line UMR-106 was developed to investigate glucocorticoid (dexamethasone) and selective COX-2 inhibitor (celecoxib) induced changes in the intracellular calcium concentration ([Ca2+]i) and mitochondrial membrane potential (deltapsi(m)). Microfluorometric techniques were applied to monitor [Ca2+]i (Fura-2 AM) and deltapsi(m) (rhodamine 123) online as the main parameters of the actual cellular metabolism. RESULTS: Basal [Ca2+] was found to be 92.2 +/- 3.7 nM and increased tip to 711 +/- 27 nM during mechanical stimulation under controlled conditions. Addition of 100 nM dexamethasone or 10 microM celecoxib for 24 h suppressed the increase in [Ca2+]i significantly to 530 +/- 33 nM and 546 +/- 39 nM, respectively. Dexamethasone significantly reduced, but celecoxib significantly increased the spread velocity of the mechanically induced intracellular calcium wave. Furthermore, the effects induced by dexamethasone were amplified during the inhibition of gap junction coupling and diminished following enlarged gap junction coupling. In contrast, the modulation of gap junction coupling exerted only a minor influence on the celecoxib-induced effects. Short-term application of dexamethasone (5 min) caused significantly reduced mechanically induced depolarization of the mitochondrial membrane, but long-term application (24 h) did not. In contrast, only the long-term application (24 h) of celecoxib caused such depolarization. CONCLUSION: The observed effects of dexamethasone and celecoxib on mechanically induced changes in [Ca2+] and deltapsi(m) are suggested to result from short-term changes in membrane characteristics and long-term changes in protein synthesis. This indicates an influence of these drugs on cell-to-cell communication and metabolism that may be therapeutically relevant.

[Percutaneous thermoablation of lung metastases. Indication, performance, initial results, and imaging findings]. / Perkutane Laserablation von Lungenmetastasen. Indikation, technische Durchführung, erste Ergebnisse und Bildbefunde.

Not unlike thermoablation of liver metastases, thermoablation of metastases to the lungs is gaining clinical interest. Radiofrequency ablation and laser-induced interstitial thermotherapy are both used clinically. Initially it was suspected that percutaneous treatment of lung metastases would result in a rate of pneumothoraces and tissue reactions which would not be clinically acceptable. However, this did not prove true. Fear of pneumothoraces however did lead to the desire for an applicator with a maximally reduced diameter. While clinical results are not yet available, technical success rates of laser-induced interstitial thermotherapy of lung metastases are promising. The percentage of pneumothoraces does not differ significantly from that seen in diagnostic procedures. Large metastases may be treated by simultaneous use of multiple applicators or by repositioning of an applicator (pullback technique). Surgical experience is still guiding us in deciding which primaries' metastases may be successfully treated percutaneously. The literature indicates that lung metastases from colorectal primaries are especially suited. Radiotherapy is only an alternative method in cases of lung metastases if they cause symptoms (such as pain because of thorax infiltration or difficulty in breathing because of bronchial lesion). Due to the risk of radiation-induced pneumonia general radiotherapy is not to be recommended. The possibility of stereotactic ray treatment is being considered, but because breathing shifts the metastases it is not yet feasible. Therefore, percutaneous thermoablation could be used as a minimally invasive, rather riskless therapeutic option for a relatively high percentage of inoperable lung metastases.

Coupling of neuronal activity and mitochondrial metabolism as revealed by NAD(P)H fluorescence signals in organotypic hippocampal slice cultures of the rat.

During physiological activity neurons may experience localised energy demands which require intracellular signals for stimulation of mitochondrial NADH generation and subsequent delivery of ATP. To elucidate these mechanisms, we applied microfluorimetric monitoring of cytoplasmic (Fluo-3) and mitochondrial (Rhod-2) calcium concentration ([Ca(2+)](c), [Ca(2+)](m)), as well as of mitochondrial oxidative metabolism (NAD(P)H), whilst simultaneously measuring changes in extracellular potassium concentration ([K(+)](o)), as an indicator of neuronal activity in hippocampal slice cultures. Changes in neuronal activity were induced by repetitive stimulation at different frequencies (5, 20, 100 Hz) and intensities. Stimulation parameters were chosen to elicit rises in [K(+)](o) of less than 3 mM which is comparable to physiologically occurring rises in [K(+)](o). The mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) reduced stimulus-induced changes in Rhod-2 fluorescence by 79%, indicating that Rhod-2 signals were primarily of mitochondrial origin. Repetitive stimulation at 20 Hz applied to areas CA1 or CA3 resulted in moderate rises in [K(+)](o) which were associated with stimulus-dependent elevations in [Ca(2+)](c) and [Ca(2+)](m) of up to 15%. The same stimuli also elicited biphasic changes in NAD(P)H fluorescence characterised by an initial decline and a subsequent prolonged elevation of up to 10%. Variation of stimulus parameters revealed close correlations between rises in [K(+)](o), in [Ca(2+)](m) and changes in NAD(P)H fluorescence. To elucidate the role of intracellular Ca(2+) accumulation in induction of NAD(P)H fluorescence signals, the effect of application of Ca(2+)-free solution on these signals evoked by repetitive antidromic stimulation of the alveus during recordings in area CA1 was studied. Lowering extracellular Ca(2+) led to complete blockade of postsynaptic field potential components as well as of rises in [Ca(2+)](c) and [Ca(2+)](m). Amplitudes of NAD(P)H signals were reduced by 59%, though rises in [K(+)](o) were comparable in presence and absence of extracellular Ca(2+). The results suggest i) that mitochondrial oxidative metabolism is fine-tuned to graded physiological activity in neurons and ii) that rapid mitochondrial Ca(2+) signalling represents one of the main determinants for stimulation of oxidative metabolism under physiological conditions.

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

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 alpha-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.

Coupling of electrical and metabolic activity during epileptiform discharges.

Changes in electrical activity, ionic microenvironments, and intracellular Ca concentration were measured during recurrent seizures induced by low Mg in slices and slice cultures. In both preparations, initial seizure-like events (SLEs) changed after some time into drug-refractory late recurrent discharges. In slice cultures, there was considerable cell loss in all hippocampal areas after 2 h of status epilepticus. During recurrent SLEs, the NAD(P)H autofluorescence declined, as did intramitochondrial calcium signals, indicating mitochondrial damage. At the same time, ethidium signals indicated increased radical oxygen species production. These alterations could be reduced by alpha-tocopherol, which also protected slice cultures against status epilepticus-induced cell death.

Synaptic and nonsynaptic ictogenesis occurs at different temperatures in submerged and interface rat brain slices.

To investigate the temperature sensitivity of low-Ca2+-induced nonsynaptic and low-Mg2+-induced synaptic ictogenesis under submerged and interface conditions, we compared changes of extracellular field potential and extracellular potassium concentration at room temperature (23 +/- 1 degrees C; mean +/- SD) and at 35 +/- 1 degrees C in hippocampal-entorhinal cortex slices. The induction of spontaneous epileptiform activity under interface conditions occurred at 35 +/- 1 degrees C in both models. In contrast, under submerged conditions, spontaneous epileptiform activity in low-Mg2+ artificial cerebrospinal fluid (ACSF) was observed at 35 +/- 1 degrees C, whereas epileptiform discharges induced by low-Ca2+ ACSF occurred only at room temperature. To investigate the different temperature effects under submerged and interface conditions, measurements of extra- and intracellular pH and extracellular space volume were performed. Lowering the temperature from 35 +/- 1 degrees C to room temperature effected a reduction in extracellular pH under submerged and interface conditions. Under submerged conditions, temperature changes had no significant influence on the intracellular pH in presence of either normal or low-Mg2+ ACSF. In contrast, application of low-Ca2+ ACSF effected a significant increase in intracellular pH at room temperature but not at 35 +/- 1 degrees C under submerged conditions. Therefore increasing intracellular pH by lowering the temperature in low-Ca2+ ACSF may push slices to spontaneous epileptiform activity by opening gap junctions. Finally, extracellular space volume significantly decreased by switching from submerged to interface conditions. The reduced extracellular space volume under interface conditions may lead to an enlarged ephaptic transmission and therefore promotes low-Mg2+- and low-Ca2+-induced spontaneous epileptiform activity. The results of the study indicate that gas-liquid interface and total-liquid submerged slice states impart distinct physiological parameters on brain tissue.

Axo-axonal coupling. a novel mechanism for ultrafast neuronal communication.

We provide physiological, pharmacological, and structural evidence that axons of hippocampal principal cells are electrically coupled, with prepotentials or spikelets forming the physiological substrate of electrical coupling as observed in cell somata. Antidromic activation of neighboring axons induced somatic spikelet potentials in neurons of CA3, CA1, and dentate gyrus areas of rat hippocampal slices. Somatic invasion by these spikelets was dependent on the activation of fast Na(+) channels in the postjunctional neuron. Antidromically elicited spikelets were suppressed by gap junction blockers and low intracellular pH. Paired axo-somatic and somato-dendritic recordings revealed that the coupling potentials appeared in the axon before invading the soma and the dendrite. Using confocal laser scanning microscopy we found that putative axons of principal cells were dye coupled. Our data thus suggest that hippocampal neurons are coupled by axo-axonal junctions, providing a novel mechanism for very fast electrical communication.

Monitoring NAD(P)H autofluorescence to assess mitochondrial metabolic functions in rat hippocampal-entorhinal cortex slices.

Changes in neuronal energy metabolism, mitochondrial functions and homeostasis of reactive oxygen species are often supposed to induce alterations in neuronal activity in hippocampal slice models. In order to investigate the NAD(P)H autofluorescence signal in brain slice models, methods to monitor NAD(P)H signal in isolated mitochondria as described by Chance et al. [J. Biol. Chem. 254 (1979) 4764] and dissociated neurons as described by Duchen [Biochem. J. 283 (1992) 41] were adapted to recording conditions required for brain slices. Considering different experimental questions, we established an approach to monitor NAD(P)H autofluorescence signals from hippocampal slices of 400 microm thickness under either submerged or interface conditions. Therefore the procedure described here allows the measurement of NAD(P)H autofluorescence under conditions typically required in electrophysiological experiments. Depolarization of plasma membrane caused by electrical stimulation or application of glutamate (100 microM) resulted in a characteristic initial decrease followed by a long-lasting increase in the NAD(P)H autofluorescence signal. H(2)O(2) (100 microM) evoked a strong NAD(P)H signal decrease indicating direct oxidation to the nonfluorescencend NAD(P)(+). In contrast, the increase in NAD(P)H signal that followed a brief inhibition of mitochondrial respiratory chain complex I using rotenone (1 microM) indicated an accumulation of NAD(P)H. However, in presence of rotenone (1 microM) electrically evoked long-lasting NAD(P)H signal overshoot decreased progressively, due to a negative feedback of accumulated NAD(P)H to the citrate cycle. A comparable reduction in NAD(P)H signal increase were observed during low-Mg(2+) induced epileptiform activity, indicating a relative energy failure. In conclusion, the method presented here allows to monitor NAD(P)H autofluorescence signals to gain insight into the coupling of neuronal activity, energy metabolism and mitochondrial function in brain slice models.

Enhanced temporal stability of cholinergic hippocampal gamma oscillations following respiratory alkalosis in vitro.

The decrease in brain CO(2) partial pressure (pCO(2)) that takes place both during voluntary and during pathological hyperventilation is known to induce gross alterations in cortical functions that lead to subjective sensations and altered states of consciousness. The mechanisms that mediate the effects of the decrease in pCO(2) at the neuronal network level are largely unexplored. In the present work, the modulation of gamma oscillations by hypocapnia was studied in rat hippocampal slices. Field potential oscillations were induced by the cholinergic agonist carbachol under an N-methyl-D-aspartate (NMDA)-receptor blockade and were recorded in the dendritic layer of the CA3 region with parallel measurements of changes in interstitial and intraneuronal pH (pH(o) and pH(i), respectively). Hypocapnia from 5 to 1% CO(2) led to a stable monophasic increase of 0.5 and 0.2 units in pH(o) and pH(i), respectively. The mean oscillation frequency increased slightly but significantly from 32 to 34 Hz and the mean gamma-band amplitude (20 to 80 Hz) decreased by 20%. Hypocapnia induced a dramatic enhancement of the temporal stability of the oscillations, as was indicated by a two-fold increase in the exponential decay time constant fitted to the autocorrelogram. A rise in pH(i) evoked by the weak base trimethylamine (TriMA) was associated with a slight increase in oscillation frequency (37 to 39 Hz) and a decrease in amplitude (30%). Temporal stability, on the other hand, was decreased by TriMA, which suggests that its enhancement in 1% CO(2) was related to the rise in pH(o). In 1% CO(2), the decay-time constant of the evoked monosynaptic pyramidal inhibitory postsynaptic current (IPSC) was unaltered but its amplitude was enhanced. This increase in IPSC amplitude seems to significantly contribute to the enhancement of temporal stability because the enhancement was almost fully reversed by a low concentration of bicuculline. These results suggest that changes in brain pCO(2) can have a strong influence on the temporal modulation of gamma rhythms.

Ca2+ signalling and changes of mitochondrial function during low-Mg2+-induced epileptiform activity in organotypic hippocampal slice cultures.

Several lines of evidence indicate that augmented neuronal activity is associated with increased mitochondrial function, however, the mechanisms of coupling are still unclear. In this study we used a low extracellular Mg2+ concentration and short stimulus trains to evoke neuronal hyperactivity in the form of seizure-like events (SLE) in hippocampal slice cultures. Simultaneous microfluorimetric and electrophysiological techniques were applied to gain insight into changes of Ca2+ concentration in different compartments and into mitochondrial function. SLEs were associated with a large decrease of the extracellular Ca2+ concentration ([Ca2+]e), a spiking increase of the cytoplasmic and a smoothed elevation of the mitochondrial Ca2+ concentration (cytoplasmic concentration [Ca2+]i; intramitrochondrial concentration [Ca2+]m). Following an initial apparent decline in the mitochondrial membrane potential (DeltaPsi) and NAD(P)H autofluorescence, mitochondria depolarized and NADH production was augmented. Furthermore, SLEs were associated with increased oxidation of dihydroethidine (HEt). Our data suggest that intramitochondrial Ca2+ accumulation stimulates NADH production and production of radical oxygen species (ROS). Interestingly, mitochondrial depolarization followed [Ca2+]i and [Ca2+]m changes with a delay implying that electrogenic extrusion of Ca2+ from the mitochondrial matrix might be responsible for the depolarization of the mitochondrial membrane.

Comparison of intrinsic optical signals associated with low Mg2+-and 4-aminopyridine-induced seizure-like events reveals characteristic features in adult rat limbic system.

PURPOSE: To analyze the intrinsic optical signal change associated with seizure-like events in two frequently used in vitro models-the low-Mg2+ and the 4-aminopyridine (4-AP) models-and to monitor regions of onset and spread patterns of these discharges by using imaging of intrinsic optical signals (IOS). METHODS: Combined hippocampal-entorhinal-cortex slices of adult rats were exposed to two different treatments: lowering extracellular Mg2+ concentrations or application of 100 microM 4-AP. The electrographic features of the discharges were monitored using extracellular microelectrodes. Optical imaging was achieved by infrared transillumination of the slice and analysis of changes in light transmission using a subtraction approach. The electrographic features were compared with the optical changes. Regions of onset and spread patterns were analyzed in relevant anatomic regions of the slice. RESULTS: Both lowering extracellular Mg2+ concentrations and application of 4-AP induced seizure-like events. The relative duration of the intrinsic optical signal change associated with seizure-like events in the low-Mg2+ model was significantly longer compared with that seen with those occurring in the 4-AP model, although duration of field potentials did not differ significantly in the two models. Seizure-like events of the low-Mg2+ model originated predominantly in the entorhinal cortex, with subsequent propagation toward the subiculum and neocortical structures. In contrast, no consistent region of onset or spread patterns were seen in the 4-AP model, indicating that the seizure initiation is not confined to a particular region in this model. CONCLUSIONS: We conclude that different forms of spontaneous epileptiform activity are associated with characteristic optical signal changes and that optical imaging represents an excellent method to assess regions of seizure onset and spread patterns.

Ca(2+)- and metabolism-related changes of mitochondrial potential in voltage-clamped CA1 pyramidal neurons in situ.

In hippocampal slices from rats, dialysis with rhodamine-123 (Rh-123) and/or fura-2 via the patch electrode allowed monitoring of mitochondrial potential (DeltaPsi) changes and intracellular Ca(2+) ([Ca(2+)](i)) of CA1 pyramidal neurons. Plasmalemmal depolarization to 0 mV caused a mean [Ca(2+)](i) rise of 300 nM and increased Rh-123 fluorescence signal (RFS) by

Diminished glutathione levels cause spontaneous and mitochondria-mediated cell death in neurons from trisomy 16 mice: a model of Down's syndrome.

It has been suggested that the increased neuronal death in cultures from trisomy 16 (Ts16) mice, a model of Down's syndrome, might result from a diminished concentration of reduced glutathione (GSH). In this study we used microfluorometric techniques to investigate the effect of GSH levels on neuronal survival in diploid and Ts16 cultures. Addition of the GSH precursors cysteine and cystine and the antioxidant tocopherol to the culture medium increased the GSH concentration up to 126.0% in diploid and up to 111.9% in Ts16 neurons. Moreover, we observed a reduced spontaneous neuronal death rate in diploid and Ts16 cultures. Following the application of 50-100 microM glutamate to culture medium, we found a GSH increase in the presence of cysteine, cystine, tocopherol, and cyclosporin A, an inhibitor of mitochondrial permeability transition (diploid, 105.8-110.8%; Ts16, 83.1-96.3%). However, only tocopherol and cyclosporin A had a protective effect on glutamate-induced neuronal death. The results suggest that reduced GSH levels affect the increase of a spontaneous and a mitochondria-mediated, cyclosporin A-sensitive type of neuronal cell death. Therefore, elevating intracellular GSH concentration may have neuroprotective effects in Down's syndrome and Alzheimer's disease.

Increased mitochondrial superoxide generation in neurons from trisomy 16 mice: a model of Down's syndrome.

Increased neuronal cell death in neurodegenerative diseases has been suggested to result from an increased mitochondrial generation of radical oxygen species (ROS). To test this hypothesis, we investigated superoxide formation in cultured hippocampal neurons from diploid and trisomy 16 mice (Ts16), a model of Down's syndrome. Microflurometric techniques were used to measure superoxide-induced oxidation rate of hydroethidine (HEt) to ethidium and reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) autofluorescence signal to monitor changes in neuronal energy metabolism. We found an increase in superoxide formation by more than 50% in Ts16 neurons in comparison with diploid control neurons. In the presence of the mitochondrial respiratory chain complex I inhibitor rotenone superoxide production was blocked in diploid neurons, but the increased superoxide generation in Ts16 neurons remained. Uncoupling of mitochondrial oxidative phosphorylation using carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused irreversible deficiency in the energy metabolism, monitored by NAD(P)H autofluorescence in Ts16 neurons, but not in diploid control neurons. These results suggest an increased basal generation of superoxide in Ts16 neurons, probably caused by a deficient complex I of mitochondrial electron transport chain, which leads to an impaired mitochondrial energy metabolism and finally neuronal cell death.

Intrinsic optical signal measurements reveal characteristic features during different forms of spontaneous neuronal hyperactivity associated with ECS shrinkage in vitro.

We induced three different forms of spontaneous synchronous hyperactivity in adult rat hippocampal-entorhinal cortex slices in order to investigate effects on the intrinsic optical signal and associated changes in the extracellular space (ECS) volume. Low-Mg2+ artificial cerebrospinal fluid (ACSF) and the addition of 4-aminopyridine induced synchronous hyperactivity resulting mainly from increased synaptic transmission, while low-Ca2+ ACSF induced hyperactivity in the absence of evoked synaptic transmission. In the two models of enhanced synaptic transmission, spontaneous activity lead to an immediate increase of light transmission. In contrast, a decrease of light transmission took place during low-Ca2+-induced hyperactivity. All three forms of synchronous neuronal hyperactivity were associated with a shrinkage of the ECS volume, as revealed by the tetraethylammonium signal, measured with ion-sensitive microelectrodes. This indicates that the change in the intrinsic optical signal is not simply related to a shrinkage in ECS volume. We conclude that different forms of spontaneous synchronous neuronal hyperactivity are associated with characteristic optical signals and that the direction of the change in intrinsic optical signal does not reflect ECS shrinkage alone.

Acute cell damage after low Mg2+-induced epileptiform activity in organotypic hippocampal slice cultures.

Upon perfusion with Mg2+-free artificial cerebrospinal fluid (ACSF) organotypic hippocampal slice cultures develop seizure-like events and tonic recurrent discharges in which areas CA3 and CA1 and, in contrast to acute slices, also the dentate gyrus (DG) participate. Using the fluorescent dye propidium iodide (PI) we show that sustained epileptic activity causes cell death in the DG and pyramidal cell layer particularly evident in the granule cell layer of the DG. This correlates with the decrease of the electrophysiological responses to hilar stimulation. Interestingly, perfusion with carbogenated serum-free ACSF also induces some cell death which is, however, mild compared with low magnesium treated slice cultures.