Frank Tegtmeier

Spreading depression in human neocortical slices

Cortical spreading depression (CSD) occurrence has been suggested to be associated with seizures, migraine aura, head injury and brain ischemia-infarction. Only few studies identified CSD in human neocortical slices and no comprehensive study so far evaluated this phenomenon in human. Using the neocortical tissue excised for treatment of intractable epilepsy, we aimed to investigate CSD in human. CSD was induced by KCl injection and by modulating T-type Ca(2+) currents in incubated human neocortical tissues in an interphase mode. The DC-fluctuations were recorded by inserting microelectrodes into different cortical layers. Local injection of KCl triggered single CSD that propagated at 3.1+/-0.1 mm/min. Repetitive CSD also occurred spontaneously during long lasting application (5 h) of the T-type Ca(2+) channel blockers amiloride (50 microM) or NiCl(2) (10 microM) which was concomitant with a reversible extracellular potassium increase up to 50 mM. CSD could be blocked by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid in all cases. The results demonstrate that modulation of the Ca(2+) dynamics conditioned human neocortical slices and increased their susceptibility to generate CSD. Furthermore, these data indicate that glutamatergic pathway plays a role in CSD phenomenon in human.

Effects of the sodium channel blocker tetrodotoxin (TTX) on cellular ion homeostasis in rat brain subjected to complete ischemia

Anoxic depolarization (AD) and failure of the cellular ion homeostasis are suggested to play a key role in ischemia-induced neuronal death. Recent studies show that the blockade of Na+ influx significantly improved the neuronal outcome. In the present study, we investigated the effects of 10 microM tetrodotoxin (TTX) on ischemia-induced disturbances of ion homeostasis in the isolated perfused rat brain. TTX inhibited the spontaneous EEG activity, delayed the ischemia-induced tissue acidification, and significantly postponed the occurrence of AD by 65%. The [Ca2+]e elevation prior to AD was attenuated from 17.8% to 6% while the increase of the [Na+]e in this period was enhanced (from 2.9% to 7.3%). These findings implied that the ischemia-induced early cellular sodium load and the corresponding shrinkage of the extracellular space was counteracted by TTX. Our results suggest that the Na+ influx via voltage-dependent channels preceding complete breakdown of ion homeostasis is one major factor leading to cell depolarization. The massive Na+ influx coinciding with AD, however, may be mainly via non-selective cation channels or/and receptor-operated channels. Persistent Na+ influx deteriorates neuronal tissue integrity by favouring Ca2+ influx and edema formation. Blockade of ischemia-induced excessive Na+ influx is, therefore, a promising pharmacological approach for stroke treatment.

Ion Channel Involvement in Anoxic Depolarization Induced by Cardiac Arrest in Rat Brain

Anoxic depolarization (AD) and failure of ion homeostasis play an important role in ischemia-induced neuronal injury. In the present study, different drugs with known ion-channel-modulating properties were examined for their ability to interfere with cardiac-arrest-elicited AD and with the changes in the extracellular ion activity in rat brain. Our results indicate that only drugs primarily blocking membrane Na+ permeability (NBQX, R56865, and flunarizine) delayed the occurrence of AD, while compounds affecting cellular Ca2+ load (MK-801 and nimodipine) did not influence the latency time. The ischemia-induced [Na+]e reduction was attenuated by R56865. Blockade of the ATP-sensitive K+ channels with glibenclamide reduced the [K+]e increase upon ischemia, indicating an involvement of the KATP channels in ischemia-induced K+ efflux. The KATP channel opener cromakalim did not affect the AD or the [K+]e concentration. The ischemia-induced rapid decline of extracellular calcium was attenuated by receptor-operated Ca2+ channel blockers MK-801 and NBQX, but not by the voltage-operated Ca2+ channel blocker nimodipine, R56865, and flunarizine.

Eicosanoids in rat brain during ischemia and reperfusion--correlation to DC depolarization.

The effects of complete ischemia on cerebral arachidonic acid (AA) metabolism were investigated in the isolated perfused rat brain. During 12.5 min of ischemia, AA, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and 15-hydroxy-5,8,11,13-eicosatetraenoic acid increased 129-, 4-, and 10-fold, respectively, while subsequent reperfusion for 30 min resulted in normalized levels independently of the duration of preceding ischemia. Prostaglandin (PG) F2α, PGE2, PGD2, 6-keto-PGF1α, and thromboxane (Tx) B2 remained at preischemic levels during 12.5 min of complete ischemia. However, at the end of subsequent reperfusion for 30 min, the levels of the prostanoids PGF2α, PGE2, PGD2, 6-keto-PGF1α, and TxB2 increased according to the preceding ischemic time. The levels reached a maximum after 7.5 min of ischemia and were elevated by 7-, 14-, 48-, 3-, and 30-fold, respectively. A prolongation of ischemia of up to 12.5 min was not associated with further increases of prostanoids at the end of reperfusion. The mechanisms underlying the metabolism of eicosanoids are discussed in relation to the changes of cortical direct current potential.

Tissue swelling and intracellular pH in the CA1 region of anoxic rat hippocampus

The fluorescent dye BCECF was used to simultaneously determine the intracellular pH (ratio 495 : 450 nm) and changes in relative tissue volume (fluorescence at the 450 nm isosbestic wavelength) in rat hippocampal slices. Anoxia in the presence of glucose caused tissue swelling and subsequent intracellular acidosis after a short and small transient alkaline peak. Reoxygenation reversed tissue swelling only partly and ended in persistent tissue swelling. The intracellular pH was initially further acidified before restoration to the normoxic intracellular pH occurred. Omitting glucose during anoxia caused similar but more marked changes of relative tissue volume. However, acidosis during anoxia was less marked and subsequently converted to alkalosis. Reoxygenation also caused initial acidification but the intracellular pH was not completely restored afterwards.

ANAEROBIC GLYCOLYSIS AND POSTANOXIC RECOVERY OF RESPIRATION OF RAT CORTICAL SYNAPTOSOMES ARE REDUCED BY SYNAPTOSOMAL SODIUM LOAD

Synaptosomes of rat cerebral cortex were used to study the effect of veratridine-induced Na+ load on postanoxic recovery of respiration and on aerobic and anaerobic ATP turnover, calculated from rates of oxygen consumption and lactate production. Non-stimulated synaptosomes: after onset of anoxia lactate synthesis of synaptosomes rose immediately from 0.8 to 17.7 nmol lactate/min/mg protein indicating an anaerobic ATP turnover of 17.7 nmol ATP/min/mg protein. This value accounts for 80% of ATP synthesized during oxygenated conditions and seems to cover the energetic demand of anoxic synaptosomes. This assumption was supported by linearity of lactate production throughout anoxia (90 min), by unaffected synaptosomal integrity and by complete recovery of postanoxic respiration after 90 min of anoxia. Stimulated synaptosomes: stimulation of oxygenated synaptosomes with 10(-5) mol/l veratridine enhanced ATP turnover 5-fold, due to activation of Na+/K+ ATPase, as a result of veratridine-induced Na+ influx. Consequently, if not limited in capacity, anaerobic ATP synthesis should be enhanced after addition of veratridine during anoxia. However, the opposite effect was observed. Veratridine reduced anaerobic glycolysis in a concentration-dependent manner. This inhibitory effect could be prevented by tetrodotoxin applied 5 min prior to veratridine. Inhibition of anaerobic glycolysis was independent of extrasynaptosomal glucose (1-30 mmol/l) and Ca2+ concentration (Ca(2+)-free and 1.2 mmol/l Ca2+). Veratridine stimulation of anoxic synaptosomes reduced also the recovery of postanoxic respiration. The data indicate that Na+ load inhibits anaerobic ATP synthesis, the only energy source during anaerobic conditions. To our knowledge, inhibition of anaerobic glycolysis due to increased Na+ influx has not been shown so far.

NiCl2 and amiloride induce spreading depression in guinea pig hippocampal slices

Spreading depressions (SD) occur in association with ischaemia, epilepsy and migraine. Intracellular calcium oscillations have been suggested to be involved in the generation and propagation of SD. The present study was performed to study the mechanism of conditioning guinea pig hippocampal slices by the T-type calcium channel blockers NiCl2 and amiloride. SD-like fluctuations of DC potential were recorded by inserting microelectrodes into the CA1 and CA3 regions. The SD occurrence was significantly greater with 10 µmol/l NiCl2 as well as with 25 and 50 µmol/l amiloride than with other concentrations of these substances. The concentration response curve was inversely U-shaped with the maximum repetition rates of SDs being achieved at 10 µmol/l NiCl2 as well as at 25 and 50 µmol/l amiloride. SD occurrence could be completely blocked by the NMDA antagonist APV (10 µmol/l) in all cases. These data demonstrate that modulation of the Ca2+ dynamics conditioned guinea pig hippocampal slices and increased their susceptibility to generate SD.

Lactate and Postischemic Recovery of Energy Metabolism and Electrical Activity in the Isolated Perfused Rat Brain

&NA; The aim of the present study was to evaluate whether lactate can maintain the energy metabolism and electrical activity of isolated perfused rat brain in the absence of glucose. To exhaust cerebral glucose stores and simultaneously raise endogenous lactate, complete ischemia was induced. After ischemia, when a glucose‐free perfusate was supplied, restoration of interstitial potassium (Symbol.), cortical discontinuous current (DC) potential, electroencephalogram (EEG) activity, and ATP and phosphocreatine (PCr) was not significantly different from postischemic recovery findings when a glucosecontaining perfusate was used. In the group receiving glucose‐free perfusate, postischemic application of 1 mM iodoacetic acid did not inhibit the recovery of electrical activity,Symbol., or DC potential. After recovery of Symbol. in glucose‐free reperfusion, a 20‐30‐Hz EEG pattern appeared and was maintained for about 20 min followed by disappearance of spontaneous electrical activity. An abrupt increase of Symbol., a steep negative DC shift, and a substantial decrease of ATP and PCr occurred after about 22 min of reperfusion. During the first 5 min of glucose‐free reperfusion, consumption of lactate was significantly higher (0.89 &mgr;mol/g wet weight/min) than during reperfusion with medium containing glucose (0.41 &mgr;mol/g ww/min). Increasing amounts of tissue lactate prolonged maintenance of electrical function in glucose‐free reperfusion. This correlation could not be found for free fatty acids. In conclusion, after a few minutes of ischemia, the brain is able to recover cellular ion transport and electrical activity without a supply of glucose, preferentially by combustion of lactate accumulated in brain tissue. This mechanism is only useful during a limited time period until the lactate accumulated during ischemia is combusted. Symbol. no caption available.

CRYOPRESERVATION OF FRESHLY ISOLATED SYNAPTOSOMES PREPARED FROM THE CEREBRAL-CORTEX OF RATS

In the present study, we established a cryopreservation method for freshly isolated synaptosomes prepared from the cerebral cortex of rats. Freshly prepared synaptosomes were either shock-frozen or frozen under temperature-controlled conditions using a programmable temperature controller. Each group was resuspended in iso-osmotic or hyperosmotic sucrose buffer prior to freezing, resulting in 4 different preservation protocols. The viability of the frozen synaptosomes was estimated by the recovery of basal and stimulated respiration after short-term storage (1 h) in liquid nitrogen. With regard to basal, FCCP- and veratridine-induced respiration, best recovery revealed controlled-frozen synaptosomes resuspended in iso-osmotic sucrose buffer (con/iso group). Basal respiration of this group recovered completely, whereas veratridine- and FCCP-induced oxygen uptake was decreased to 87.7% and 82.4% of control, respectively. Further investigations performed with the con/iso group revealed complete recovery of anaerobic and aerobic lactate synthesis, and unaffected synaptosomal integrity, as judged by the amount of released L-lactate dehydrogenase before and after the cryopreservation procedure. Long-term storage of the con/iso group in liquid nitrogen up to 88 days did not have any influence on synaptosomal viability, as evaluated by the recovery of anaerobic lactate production and synaptosomal respiration. Therefore, based on the results of respiration, synaptosomal integrity, and lactate synthesis, metabolically active synaptosomes could be obtained after cryopreservation and storage in liquid nitrogen for at least 88 days.

DOSE-DEPENDENT EFFECTS OF TETRAETHYLAMMONIUM ON CIRCLING SPREADING DEPRESSIONS IN CHICKEN RETINA

The K+‐channel blocker tetraethylammonium (TEA) was applied to study its effects in self‐sustaining, circling spreading depression in the chicken retina (RSD). Extracellular K+ (Ke+) activities and the direct‐current (DC) signal were recorded using doublebarrelled microelectrodes. Superfusion of TEA‐concentrations of 10 to 250 μM for 4 to 7 min reduced the RSD‐associated DC amplitude (by 9 to 45%) and the maximum of the Ke+ concentration (by 9 to 34%) in a dose‐dependent manner. Propagation velocity of the RSD was lowered by 24%. At concentrations higher than 250 μM TEA (0.5 to 10 mM), the propagation was slowed by more than 60%, after which the RSD disappeared. Recovery upon reperfusion with Ringer was immediate. These observations illustrate: 1) TEA affects the Ke+ changes during RSD at very low concentrations. 2) The reduced Ke+ transients are accompanied by a reduction of the DC shifts. 3) These changes of the electrical properties of the RSDs are paralleled by a reduction of the propagation velocity. 4) The effects of TEA are reversible. 5) The changes of these parameters occur dose‐dependently. These data suggest a close relationship between the amplitudes of the ionic/electric changes during RSDs and the mechanisms of propagation.