Rho kinase inhibition protects CA1 cells in organotypic hippocampal slices during in vitro ischemia

Simultaneous recordings of intracellular Ca2+([Ca2+]i) signal and extracellular DC potential were obtained from the CA1 region in 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)-ethane- N, N, N′, N′-tetraacetic acid penta-acetoxymethyl ester (Fura-2/AM)-loaded rat hippocampal slices. Superfusion with oxygen- and glucose-deprived medium (in vitro ischemia) for 5–6 min produced a rapid rise of the [Ca2+]i level in the stratum radiatum (rising phase of the [Ca2+]i signal), which occurred simultaneously with a rapid negative DC potential (rapid negative potential). When oxygen and glucose were reintroduced, the increased [Ca2+]i signal diminished rapidly (falling phase of the [Ca2+]i signal) during the generation of a slow negative DC potential (slow negative potential), which occurred within 1 min from the onset of the reintroduction. Thereafter, the [Ca2+]i signal partially and the slow negative potential completely returned to the preexposure level approximately 6 min after the reintroduction. The changes in [Ca2+]i signal during and after in vitro ischemia were very similar to the changes in the membrane potential of glial cells. The rising and falling phases of [Ca2+]i signal corresponded to the rapid depolarization and a depolarizing hump, respectively, in the repolarizing phase of glial cells. A prolonged application of in vitro ischemia or a reintroduction of either glucose or oxygen suppressed the falling phase after ischemic exposure. The application of ouabain (30 μM) generated both a rapid negative potential and a rapid elevation of [Ca2+]i, but no slow negative potential or rapid reduction in [Ca2+]i were observed. When oxygen and glucose were reintroduced to slices in the Na+-free or ouabain- or Ni2+-containing medium, the falling phase was suppressed. The falling phase was significantly accelerated in Ca2+- and Mg2+-free with EGTA-containing medium. In contrast, the falling phase was significantly slower in the Ca2+-free with high Mg2+- and EGTA-containing medium. The falling phase of the [Ca2+]isignal after ischemic exposure is thus considered to be primarily dependent on the reactivation of Na+, K+-ATPases, while the extrusion of cytosolic Ca2+ via the forward-mode operation of Na+/Ca2+ exchangers in glial cells is thought to be directly involved in the rapid reduction of [Ca2+]i after ischemic exposure.

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

Brain Research ◽

10.1016/0006-8993(93)91700-3 ◽

1993 ◽

Vol 601 (1-2) ◽

pp. 103-110 ◽

Cited By ~ 93

Author(s):

Akira Mitani ◽

Hisato Yanase ◽

Kimiko Sakai ◽

Youseke Wake ◽

Kiyoshi Kataoka

Keyword(s):

Hippocampal Slices ◽

In Vitro Ischemia

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Diazepam Promotes ATP Recovery and Prevents Cytochrome c Release in Hippocampal Slices After In Vitro Ischemia

Journal of Neurochemistry ◽

10.1046/j.1471-4159.2000.0751242.x ◽

2002 ◽

Vol 75 (3) ◽

pp. 1242-1249 ◽

Cited By ~ 51

Author(s):

Francesca Galeffi ◽

Shamim Sinnar ◽

Rochelle D. Schwartz-Bloom

Keyword(s):

Cytochrome C ◽

Hippocampal Slices ◽

Cytochrome C Release ◽

In Vitro Ischemia

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In vitro ischemia-induced intracellular Ca 2+ elevation in cerebellar slices: a comparative study with the values found in hippocampal slices

Acta Neuropathologica ◽

10.1007/s004010050208 ◽

1994 ◽

Vol 89 (1) ◽

pp. 2-7 ◽

Cited By ~ 1

Author(s):

A. Mitani ◽

Hisato Yanase ◽

Shigeru Namba ◽

Masachika Shudo ◽

Kiyoshi Kataoka

Keyword(s):

Comparative Study ◽

Hippocampal Slices ◽

In Vitro Ischemia ◽

Intracellular Ca

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Anesthetics and Mild Hypothermia Similarly Prevent Hippocampal Neuron Death in an In Vitro Model of Cerebral Ischemia

Anesthesiology ◽

10.1097/00000542-200005000-00024 ◽

2000 ◽

Vol 92 (5) ◽

pp. 1343-1349 ◽

Cited By ~ 75

Author(s):

Robert Popovic ◽

Richard Liniger ◽

Philip E. Bickler

Keyword(s):

Cerebral Ischemia ◽

Mild Hypothermia ◽

Cell Damage ◽

Hippocampal Slices ◽

Cell Loss ◽

Glutamate Excitotoxicity ◽

Neuron Death ◽

In Vitro Ischemia ◽

Aspartate Receptor

Background General anesthetics reduce neuron loss following focal cerebral ischemia in rodents. The relative efficacy of this action among different anesthetics clinically used for neuroprotection is uncertain. In addition, it remains unclear how anesthetics compare to neuroprotection afforded by mild hypothermia. This study was performed to evaluate the comparative effects of isoflurane, sodium pentothal, and mild hypothermia in a hippocampal slice model of cerebral ischemia and to determine if the mechanism of neuroprotection of isoflurane involves inhibition of glutamate excitotoxicity. Methods Survival and morphology of CA1, CA3, and dentate gyrus neurons in rat hippocampal slices were examined after 10 or 20 min of combined oxygen-glucose deprivation (in vitro ischemia) followed by a 5-h recovery period. Results 10 or 20 min in vitro ischemia at 37 degrees C killed 35-40% of neurons in CA1 (P < 0.001), 6% in CA3 (not significant) and 18% in dentate (P < 0.05). Isoflurane (0.7 and 2.0%, approximately 0.45 and 1.5 minimum alveolar concentration), pentothal (50 microm, approximately 1 minimum alveolar concentration equivalent) and mild hypothermia (34 degrees C) all reduced CA1 cell loss and morphologic damage to similar degrees in 10- and 20-min periods of ischemia (P < 0.001). The noncompetitive N-methyl-D-aspartate antagonist MK-801 prevented cell damage, showing that N-methyl-D-aspartate receptor activation is an important mechanism of injury in this model. Glutamate (1 mm) produced cell loss similar to in vitro ischemia. Isoflurane (2%) prevented cell damage from glutamate exposure. Conclusions In hippocampal slices, neuron death from simulated ischemia was predominately due to activation of glutamate receptors. Isoflurane, sodium pentothal, an N-methyl-D-aspartate receptor antagonist, and mild hypothermia prevented cell death to similar degrees. For isoflurane, the mechanism appears to involve attenuation of glutamate excitotoxicity.

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