Keiji Oguro

Estrogen Protects against Global Ischemia-Induced Neuronal Death and Prevents Activation of Apoptotic Signaling Cascades in the Hippocampal CA1

The importance of postmenopausal estrogen replacement therapy in affording protection against the selective and delayed neuronal death associated with cardiac arrest or cardiac surgery in women remains controversial. Here we report that exogenous estrogen at levels that are physiological for hormone replacement in postmenopausal women affords protection against global ischemia-induced neuronal death and prevents activation of apoptotic signaling cascades in the hippocampal CA1 of male gerbils. Global ischemia induced a marked increase in activated caspase-3 in CA1, evident at 6 hr after ischemia. Global ischemia induced a marked upregulation of the proapoptotic neurotrophin receptor p75(NTR) in CA1, evident at 48 hr. p75(NTR) expression was induced primarily in terminal deoxynucleotidyl transferase-mediated UTP nick-end labeling-positive cells, indicating expression in neurons undergoing apoptosis. Global ischemia also induced a marked downregulation of mRNA encoding the AMPA receptor GluR2 subunit in CA1. Caspase-3, p75(NTR), and GluR2 were not significantly changed in CA3 and dentate gyrus, indicating that the ischemia-induced changes in gene expression were cell specific. Exogenous estrogen attenuated the ischemia-induced increases in activated caspase-3 and blocked the increase in p75(NTR) in post-ischemic CA1 neurons but did not prevent ischemia-induced downregulation of GluR2. These findings demonstrate that long-term estrogen at physiological levels ameliorates ischemia-induced hippocampal injury and indicate that estrogen intervenes at the level of apoptotic signaling cascades to prevent onset of death in neurons otherwise destined to die.

Differential distribution of 68 Kd and 200 Kd neurofilament proteins in the gerbil hippocampus and their early distributional changes following transient forebrain ischemia

SummaryThe distribution of neurofilament (NF) proteins was studied immunohistochemically in the gerbil hippocampus with antibodies against NF68 (68 Kd molecular weight) and NF200 proteins, and changes in the distribution of NF proteins after transient ischemia were observed in order to investigate the temporal correlation between NF and delayed neuronal death. In the normal hippocampus, NF68-like immunoreactivity (NF68-LI) was densely distributed in nerve processes in CA2, CA3 and the hilus of the dentate gyrus but was less intense in CA1. In contrast, processes with NF200-LI appeared to be evenly distributed in CA1, CA2, CA3 and the dentate gyrus. Mongolian gerbils were subjected to transient ischemia for 5 min by bilateral carotid occlusion and subjected to immunohistochemistry 1, 2, 3 and 4 days after ischemia. Following transient ischemia, prior to neuronal cell death, the intensity of both NF68-LI and NF200-LI decreased in the whole hippocampal formation. This decrease was more obvious in the case of NF68-LI than NF200-LI. Four days after ischemia, when neuronal death of CA1 pyramidal cells had occurred, processes in CA1, particularly 68 Kd components, showed marked decreases in number and staining intensity, although processes in most layers of CA2, CA3 and the dentate gyrus appeared to be stained similarly to normal brain. Since NF68 protein is considered to be the major component of NF proteins and NF200 is an associated accessory protein, the current observations suggest that the poor distribution of NF68 in CA1 and the early loss of NF proteins may be closely related to selective vulnerability of CA1 pyramidal cells and delayed neuronal death.

Disturbance of hippocampal long-term potentiation after transient ischemia in GFAP deficient mice

GFAP (glial fibrillary acidic protein) is an intermediate filament protein found exclusively in the astrocytes of the central nervous system. We studied the role of GFAP in the neuronal degeneration in the hippocampus after transient ischemia using knockout mice. Wild‐type C57 Black/6 (GFAP+/+) mice and mutant (GFAP−/−) mice were subjected to occlusion of both carotid arteries for 5–15 min. Hippocampal slices were prepared 3 days after reperfusion and the field excitatory postsynaptic potentials (fEPSP) in the CA1 were recorded. High frequency stimulation induced robust long‐term potentiation (LTP) in GFAP−/−, as in GFAP+/+ mice. After ischemia, however, the LTP in GFAP−/− was significantly depressed. Similarly, paired pulse facilitation (PPF) displayed little difference between GFAP+/+ and GFAP−/−, but after ischemia, the PPF in GFAP−/− showed a depression. Histological study revealed that loss of CA1 and CA3 pyramidal neurons after ischemia was marked in GFAP−/−. MAP2 (dendritic) immunostaining in the post‐ischemic hippocampus showed little difference but NF200 (axonal) immunoreactivity was reduced in GFAP−/−. S100β (glial) immunoreactivity was similar in the post‐ischemic hippocampus of the GFAP+/+ and GFAP−/−, indicating that reactive astrocytosis did not require GFAP. Our results suggest that GFAP has an important role in astrocyte‐neural interactions and that ischemic insult impairs LTP and accelerates neuronal death.

Reduced calcium elevation in hippocampal Ca1 neurons of ischemia-tolerant gerbils

TRANSIENT forebrain ischemia causes selective neuronal death in the hippocampal CA1 neurons. A short sublethal ischemic episode preceding ischemia of longer duration is known to increase tolerance against cell death. The mechanisms of this ischemic tolerance are still poorly understood. Here we show, using Ca2+ imaging, that intracellular calcium ([Ca2+]i) elevation in CA1 neurons after an anoxic-aglycemic episode is markedly inhibited in the ischemia-tolerant gerbil. The hippocampus of gerbils which did not acquire tolerance showed a high [Ca2+]i elevation during the anoxicaglycemic episode, similar to controls. Since hypoxia/ischemia-induced neurodegeneration can be triggered by cytoplasmic Ca2+ overload, the tolerant gerbil may regulate calcium and keep [Ca2+]i below the critical level for initiating neuronal death. NeuroReport 9: 1875–1878

Single glutamate channels in CA1 pyramidal neurones after transient ischaemia

Patch clamp recordings were made from CA1 pyramidal neurones to study changes in the glutamate receptor subtypes in the gerbil hippocampus after transient ischaemia. In whole-cell recordings, the maximum chord conductances of AMPA currents in ischaemic neurones were increased over those of control neurones but NMDA-induced currents in the ischaemic neurones were smaller than the control. In AMPA-activated single channel currents, an open time histogram of the control neurones was well fitted by a single exponential function whereas in the ischaemic patches it was fitted by a double exponential function, indicating that currents consisted of at least two kinetically different types. These functional changes of the glutamate receptor channels may contribute to the abnormalities of the excitatory synaptic currents recorded in post-ischaemic CA1 neurones.

Upregulation of GluR2 decreases intracellular Ca2+ following ischemia in developing gerbils.

Developing animals are known to be resistant to cerebral ischemia. To investigate the mechanisms by which developing animals exhibit ischemic resistance, we examined the changes in intracellular calcium ([Ca2+]i) after oxygen-glucose deprivation (OGD) using hippocampal slices from gerbils. We found that increases of [Ca2+]i in hippocampal CA1 neurons is significantly less after OGD in developing gerbils than in adults. Western blot analysis of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) receptors (AMPARs) showed that GluR2 expression, but not that of the other AMPARs is significantly higher in developing gerbils than in adults. Expression of the anti-apoptotic proteins such as HSP70, Bcl-XL, and plasma membrane Ca2+-ATPase type1 (PMCA1) are not higher in the developing gerbils than in adults. These results suggest that the higher expression of GluR2 is important for the smaller increases in [Ca2+]i and enhanced resistance to ischemia-induced neuronal damage in developing animals.

Differential effects of novel wasp toxin on rat hippocampal interneurons

We studied the effects of a wasp toxin beta-pompilidotoxin (beta-PMTX) on rat hippocampal CA1 interneurons by the current-clamp technique. The firing patterns of pyramidal neurons and pyramidale interneurons were not affected by beta-PMTX, but in oriens and radiatum interneurons, beta-PMTX converted the action potentials to prolonged depolarizing potentials by slowing the inactivation of Na(+) channels. In lacunosum moleculare interneurons, beta-PMTX induced initial bursting spikes followed by block of succeeding spikes. Comparison of beta-PMTX with a sea anemone toxin, ATX II, revealed that ATX II altered the firing properties of pyramidal neurons and pyramidale interneurons that were unchanged by beta-PMTX. Our results suggest that beta-PMTX modulates Na(+) currents in CA1 interneurons differently in various CA1 neurons and the toxin is useful to classify Na(+) channel subtypes.

Spontaneous excitatory postsynaptic currents in hippocampal CA1 pyramidal neurons of the gerbil after transient ischemia

The changes in the spontaneous excitatory postsynaptic currents (sEPSCs) after transient cerebral ischemia were studied using whole-cell recording from CA1 pyramidal neurons in the gerbil. In neurons recorded 1-2 days after ischemia, sEPSCs had a slowed time course with the decay time constant fitted by a single exponential and it progressively increased after ischemia. Frequency and amplitude distribution of sEPSCs in ischemic neurons were not significantly different from those in the control neurons. The results support the view that abnormal non-N-methyl-D-aspartic acid currents originate at the degenerated postsynaptic site, unrelated to the presynaptic releasing mechanisms.

Ischemic preconditioning decreases intracellular zinc accumulation induced by oxygen-glucose deprivation in gerbil hippocampal CA1 neurons

In normal gerbils, intracellular zinc ions ([Zn2+]i) and calcium ions ([Ca2+]i) accumulate in hippocampal CA1 neurons after global ischemia. We examined whether ischemic preconditioning modifies these changes in gerbil hippocampal slices. In normal slices, large increases in [Zn2+]i and [Ca2+]i were observed in the stratum radiatum of the CA1 area after oxygen-glucose deprivation. In preconditioned slices, there were significantly decreased peak levels of [Zn2+]i and [Ca2+]i in CA1. However, there were no differences in the peak levels of these ions in CA3 and dentate gyrus. These results suggest that modified [Zn2+]i and [Ca2+]i accumulation after an ischemic insult might be important for the mechanisms of ischemic tolerance induced by preconditioning.

Germ Cell Tumors originating in the Basal Ganglia and Thalamus

Two patients with germ cell tumors of the basal ganglia and the thalamus are described. A 10year-old boy developed left hemiparesis and precocious puberty. Computed tomography (CT) of the brain revealed a small lesion in the right basal ganglia, which appeared to be rapidly growing. Elevation of human chorionic gonadotropin (HCG) in both serum and cerebrospinal fluid was compatible with germ cell tumor. A craniotomy was performed and the tumor was partially removed. Histological examination verified a mixed-type germ cell tumor composed of embryonal carcinoma, choriocarcinoma, and germinoma. An 11-year-old boy complained of right hemiparesis, and a CT scan showed a well-enhanced, cystic tumor in the left thalamic region. The tumor was partially resected and was diagnosed histologically as germinoma. A high level of HCG was found in the contents of the cyst. HCG and alpha-fetoprotein, which are so-called tumor markers, decreased over the course of treatment in both cases, and seemed to be indicators of the effectiveness of treatment. However, the authors feel that the differential diagnosis of germ cell tumors cannot be based solely on the level of these tumor markers. The characteristic features of germ cell tumors of the basal ganglia and thalamus are discussed.