Hisato Yanase

Origin of intracellular Ca2+ elevation induced by in vitro ischemia-like condition in hippocampal slices

Microfluorometry was used to investigate the origin of intracellular Ca2+ ([Ca2+]i) elevation in field CA1 of gerbil hippocampal slices perfused with a glucose-free physiological medium equilibrated with a 95% N2/5% CO2 gas mixture (standard in vitro ischemia-like condition). Large [Ca2+]i elevation was detected about 4 min after the beginning of standard in vitro ischemia-like condition, which was accompanied with a negative shift of extracellular DC potential. When slices were perfused with Ca(2+)-free in vitro ischemia-like medium, large [Ca2+]i elevation was observed about 3.5 min after the beginning of Ca(2+)-free in vitro ischemia-like condition, however, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the standard in vitro ischemia-like medium that contained Ca2+. When slices were perfused with the Ca(2+)-free in vitro ischemia-like medium that contained dantrolene (50 microM) which is known to prevent Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores, the increase in [Ca2+]i was more gradual and of a lesser extent compared with that detected in the slices perfused with the Ca(2+)-free in vitro ischemia-like medium that did not contain dantrolene. These results indicate that large [Ca2+]i elevation induced by in vitro ischemia-like condition in field CA1 of the hippocampus was caused by both Ca2+ influx from extracellular space and Ca2+ release from intracellular Ca2+ stores, and that a part of the Ca2+ release was due to Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores.

Changes in Intracellular Ca2+ and Energy Levels During In Vitro Ischemia in the Gerbil Hippocampal Slice

Abstract: The time course of the decline in energy levels during an in vitro ischemia‐like condition was compared with changes in intracellular Ca2+ concentration ([Ca2+]i) in subregions of the gerbil hippocampal slice [CA1, CA3, and the inner and outer portions of the dentate gyrus (DG)]. Hippocampal transverse slices were loaded with a fluorescent indicator, rhod‐2. During the on‐line monitoring of [Ca2+]i, the slices were perfused with an in vitro ischemia‐like medium (33°C). The slices were collected at several experimental time points, frozen, dried, and dissected into subregions. The contents of adenine nucleotides (ATP, ADP, and AMP) and phosphocreatine (PCr) were measured by HPLC methods. Region‐specific and acute [Ca2+]i elevations were observed in CA1 ∼4 min after onset of the in vitro ischemia‐like condition and also in the inner portion of the DG with a delay of 10–40 s. The change in ATP levels was related to the increase in [Ca2+]i. ATP levels in all subregions gradually decreased before the acute [Ca2+]i elevation. Concomitant with the acute [Ca2+]i elevation in CA1 and the inner portion of the DG, ATP levels in the subregions rapidly decreased, whereas declines in levels of high‐energy‐charge phosphates were gradual in CA3 and the outer portion of the DG, in which the remarkable [Ca2+]i elevation was not observed. These results suggest that ATP depletion observed in CA1 and the inner portion of the DG is due to the region‐specific increase in [Ca2+]i, which activates a Ca2+‐ATP‐driven pump and produces a subsequent fall in neuronal ATP content.

Mild hypothermia-a revived countermeasure against ischemic neuronal damages

Although hypothermia as a means of cerebral protection against and resuscitation from ischemic damage has a history of approximately six decades, extensive studies, both in basic and clinical fields, on the mechanisms, effects and methods of mild hypothermia at temperatures no less than 31 degrees C have started only in the last decade. In experiments on rodents, hypothermia in the postischemic period that is introduced up to several hours after reperfusion and is maintained for one day followed by a slow rewarming, significantly protects hippocampal neurons against damage. The mode of action of hypothermia is apparently non-specific and multi-focal in widely progressing cascade reactions in ischemic cells; namely, suppressing: (1) glutamate surge followed by; (2) intraneuronal calcium mobilization; (3) sustained activation of glutamate receptors; (4) dysfunction of blood brain barrier; (5) proliferation of microglial cells; and (6) production of superoxide anions and nitric oxide. In addition, mild hypothermia modulates processes in ischemic condition at the level of cell nucleus, such as the binding of transcription factor AP-1 to DNA, and ameliorates the depression of protein synthesis. This non-specific and widely affecting manner might explain why hypothermia is superior to any medicine developed. Recent clinical trials of mild hypothermia in various individual institutions have revealed significantly beneficial outcomes in some cases, along with an accumulation of practical knowledge of techniques and treatments. Large scale randomized studies involving multiple institutions as well as exchange of informations and ideas are needed for further development of hypothermia treatment.

Protective effect of γ-glutamylethylamide (theanine) on ischemic delayed neuronal death in gerbils

We examined the protective effect of γ-glutamylethylamide (theanine) on ischemic delayed neuronal death in field CA1 of the gerbil hippocampus. One microliter of theanine from each three concentrations (50, 125 and 500 μM) was administered through the lateral ventricle 30 min before ischemia. Transient forebrain ischemia was induced by bilateral occlusion of the common carotid arteries for 3 min under careful control of brain temperature at approximately 37°C. Seven days after ischemia, the number of intact CA1 neurons in the hippocampus was assessed. Ischemia-induced neuronal death in hippocampal CA1 region was significantly prevented in a dose-dependent manner in the theanine-pretreated groups. These findings indicate that theanine might be useful clinically for preventing ischemic neuronal damage.

Continuous monitoring and regulating of brain temperature in the conscious and freely moving ischemic gerbil: Effect of MK‐801 on delayed neuronal death in hippocampal CA1

Many glutamate antagonists have been reported to have a neuroprotective effect against ischemic brain damage; however, some of them have been also reported to induce hypothermia that confers remarkable neuroprotection against the damage. In order to avoid the confounding effects of hypothermia, we assembled a telemeter‐based brain temperature control system that allows continuous monitoring and regulating of brain temperature during an ischemic insult and in the post‐ischemic period in conscious and freely moving animals. Experiments were performed in gerbils that were subjected to administration of MK‐801 (3, 5, and 10 mg/kg) and/or to 5‐min ischemia. The system monitored continuous changes in brain temperature and regulated brain temperature at normothermic levels, revealing that a neuroprotective effect of 3 mg/kg MK‐801 against ischemia‐induced delayed hippocampal CA1 neuronal death was mainly due to hypothermia, whereas a high dose of MK‐801 (5 and 10 mg/kg) produced a neuroprotective effect even when the brain temperature was maintained at normothermic levels. These results indicate that this system is very useful to test potential antiischemic agents, especially when the agents have hypothermic side effects. J. Neurosci. Res. 47:440–448, 1997.

CNS-specific prostacyclin ligands as neuronal survival-promoting factors in the brain

Prostacyclin (PGI2) is a critical regulator of the cardiovascular system, via dilatation of vascular smooth muscle and inhibition of platelet aggregation (Moncada, S. 1982, Br. J. Pharmacol., 76, 3). Our previous studies demonstrated that a novel subtype of PGI2 receptor, which is clearly distinct from a peripheral subtype in terms of ligand specificity, is expressed in the rostral region of the brain, e.g. cerebral cortex, hippocampus, thalamus and striatum, and that (15r)‐16‐m‐17,18,19,20‐tetranorisocarbacyclin (15r‐TIC) and 15‐deoxy‐16‐m‐17,18,19,20‐tetranorisocarbacyclin (15‐deoxy‐TIC) specifically bind to the central nervous system (CNS)‐specific PGI2 receptor. Here, we report that these CNS‐specific PGI2 receptor ligands, including PGI2 itself, prevented the neuronal death. They prevented apoptotic cell death of hippocampal neurons induced by high (50%) oxygen atmosphere, xanthine + xanthine oxidase, and serum deprivation. IC50s for neuronal death were ∼ 30 and 300 nm for 15‐deoxy‐TIC and 15r‐TIC, respectively, which well correlated with the binding potency for the CNS‐specific PGI2 receptor. 6‐Keto‐PGF1α (a stable metabolite of PGI2), peripheral nervous system‐specific PGI2 ligands and other prostaglandins (PGs) than PGI2 did not show such neuroprotective effects. In vivo, 15r‐TIC protected CA1 pyramidal neurons against ischaemic damage in gerbils. These results indicate that CNS‐specific PGI2 ligands have neuronal survival‐promoting activity both in vitro and in vivo, and may represent a new type of therapeutic drug for neurodegeneration.

Lidocaine suppresses the anoxic depolarization and reduces the increase in the intracellular Ca2+ concentration in gerbil hippocampal neurons

Background: The movement of ions, particularly Ca2+, across the plasma membrane of neurons is regarded as an initial element of the development of ischemic neuronal damage. Because the mechanism by which lidocaine protects neurons against ischemia is unclear, the effects of lidocaine on the ischemia‐induced membrane depolarization, histologic outcome, and the change in the intracellular Ca2+ concentration in the gerbil hippocampus were studied. Methods: The changes in the direct‐current potential shift in the hippocampal CA1 area produced by transient forebrain ischemia for 4 min were compared in animals given lidocaine (0.8 micro mol administered intracerebroventricularly) 10 min before ischemia and those given saline. The histologic outcome was evaluated 7 days after ischemia by assessing delayed neuronal death in hippocampal CA1 pyramidal cells in these animals. In a second study, hypoxia‐induced intracellular Ca2+ increases were evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effects of lidocaine (10, 50, and 100 micro Meter) on the Ca2+ accumulation were examined. In addition, the effect of lidocaine (100 micro Meter) drug perfusion with a Ca2+ ‐free ischemia‐like medium was investigated. Results: The preischemic administration of lidocaine delayed the onset of the ischemia‐induced membrane depolarization (anoxic depolarization) and reduced its maximal amplitude. The histologic outcome was improved by the preischemic treatment with lidocaine. The in vitro hypoxia‐induced increase in the intracellular concentration of Ca2+ was suppressed by the perfusion with lidocaine‐containing mediums (50 and 100 micro Meter), regarding the initiation and the extent of the increase. The hypoxia‐induced intracellular Ca2+ elevation in the Ca2+ ‐free condition was similar to that in the Ca2+ ‐containing condition. Perfusion with lidocaine (100 micro Meter) inhibited this elevation in the Ca2+ ‐free condition. Conclusions: Lidocaine helps protect neurons from ischemia by suppressing the direct‐current potential shift, by inhibiting the release of Ca2+ from the intracellular Ca2+ stores, and by inhibiting the influx from the extracellular space.

Postischemic Enhancements of N-Methyl-D-Aspartic Acid (NMDA) and Non-NMDA Receptor-Mediated Responses in Hippocampal CA1 Pyramidal Neurons

Glutamate receptor-mediated responses were investigated by using a whole-cell recording and an intracellular calcium ion ([Ca2+]i) imaging in gerbil postischemic hippocampal slices prepared at 1, 3, 6, 9, 12, and 24 hours after 5-minute ischemia. Bath application of N-methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate showed that NMDA-, AMPA- and kainate-induced currents were enhanced in postischemic CA1 pyramidal neurons at 1 to 12 hours after 5-minute ischemia. NMDA and non-NMDA receptor-mediated excitatory postsynaptic currents (EPSC) were examined in postischemic CA1 pyramidal neurons at 3 hours after 5-minute ischemia to confirm whether synaptic responses are enhanced in the postischemic CA1 pyramidal neurons. The amplitudes of NMDA- and non-NMDA-receptor-mediated EPSC were enhanced in the postischemic CA1 pyramidal neurons. NMDA-, AMPA-, and kainate-induced [Ca2+]i elevations were also examined to determine whether the enhancement of currents is accompanied by the enhancement of [Ca2+]i elevation. The enhancements of NMDA-, AMPA-, and kainate-induced [Ca2+]i elevations were shown in the postischemic CA1. These results indicate that NMDA and non-NMDA receptor-mediated responses are persistently enhanced in the CA1 pyramidal neurons 1 to 12 hours after transient ischemia, and suggest that the enhancement of glutamate receptor-mediated responses may act as one of crucial factors in the pathologic mechanism responsible for leading postischemic CA1 pyramidal neurons to irreversible neuronal injury.

Acidic fibroblast growth factor delays in vitro ischemia-induced intracellular calcium elevation in gerbil hippocampal slices: a sign of neuroprotection

Using microfluorometry, effects of acidic fibroblast growth factor (aFGF) on the in vitro ischemia-induced intracellular calcium elevation were investigated in gerbil hippocampal slices at 35 degrees C. When slices were superfused with hypoxic and glucose-free medium, the mean latency of the in vitro ischemia-induced calcium elevation was 209 +/- 51 s. The addition of aFGF in medium (25 micrograms/l) delayed the calcium elevation throughout the experiments: the mean latency was 541 +/- 94 s. This retardation in calcium elevation may be indicative of neuroprotective nature of aFGF.

In vitro ischemia-induced intracellular Ca2+ elevation in cerebellar slices: a comparative study with the values found in hippocampal slices

Changes in levels of intracellular calcium ion ([Ca2+]i) induced by in vitro ischemic conditions in gerbil cerebellar and hippocampal slices were investigated using a calcium imaging system and electron microscopy. When the cerebellar slice was perfused with a glucose-free physiological medium equilibrated with a 95% N2/5% CO2 gas mixture (in vitro ischemic medium), a large [Ca2+]i elevation was region-specifically induced in the molecular laver of the cerebellar cortex (a dendritic field of Purkinje cells). When the hippocampal slice was perfused with in vitro ischemic medium, a large [Ca2+]i elevation was region-specifically induced in CA1 field of the hippocampal slices. Electron microscopic examinations showed that the large [Ca2+]i elevations occurred in Purkinje cells and CA1 pyramidal neurons. To isolate Ca2+ release from intracellular Ca2+ store sites, the slices were perfused with Ca2+-free in vitro ischemic medium. the increases in [Ca2+]i in both cerebellar and hippocampal slices were significantly lower than those observed in the slices perfused with the Ca2+-containing in vitro ischemic medium. However, the suppression of the [Ca2+]i-elevation in the molecular layer of the cerebellar slices was smaller than that in the CA1 field of the hippocampal slices. These results reinforce the hypothesis that calcium plays a pivotal role in the development of ischemia-induced neuronal death, and suggest that Ca2+ release from intracellular Ca2+ store sites may play an important role in the ischemia-induced [Ca2+]i elevation in Purkinje cells.