Late calcium EDTA rescues hippocampal CA1 neurons from global ischemia-induced death.

Transient global ischemia induces a delayed rise in intracellular Zn2+, which may be mediated via glutamate receptor 2 (GluR2)-lacking AMPA receptors (AMPARs), and selective, delayed death of hippocampal CA1 neurons. The molecular mechanisms underlying Zn2+ toxicity in vivo are not well delineated. Here we show the striking finding that intraventricular injection of the high-affinity Zn2+ chelator calcium EDTA (CaEDTA) at 30 min before ischemia (early CaEDTA) or at 48-60 hr (late CaEDTA), but not 3-6 hr, after ischemia, afforded robust protection of CA1 neurons in approximately 50% (late CaEDTA) to 75% (early CaEDTA) of animals. We also show that Zn2+ acts via temporally distinct mechanisms to promote neuronal death. Early CaEDTA attenuated ischemia-induced GluR2 mRNA and protein downregulation (and, by inference, formation of Zn2+-permeable AMPARs), the delayed rise in Zn2+, and neuronal death. These findings suggest that Zn2+ acts at step(s) upstream from GluR2 gene downregulation and implicate Zn2+ in transcriptional regulation and/or GluR2 mRNA stability. Early CaEDTA also blocked mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein-binding protein with low pI), caspase-3 activity (but not procaspase-3 cleavage), p75NTR induction, and DNA fragmentation. These findings indicate that CaEDTA preserves the functional integrity of the mitochondrial outer membrane and arrests the caspase death cascade. Late injection of CaEDTA at a time when GluR2 is downregulated and caspase is activated inhibited the delayed rise in Zn2+, p75NTR induction, DNA fragmentation, and cell death. The finding of neuroprotection by late CaEDTA administration has striking implications for intervention in the delayed neuronal death associated with global ischemia.

Ischemic preconditioning: neuronal survival in the face of caspase-3 activation.

Apoptosis is an evolutionarily conserved process critical to tissue development and tissue homeostasis in eukaryotic organisms and, when dysregulated, causes inappropriate cell death. Global ischemia is a neuronal insult that induces delayed cell death with many features of apoptosis. Ischemic preconditioning affords robust protection of CA1 neurons against a subsequent severe ischemic challenge. The molecular mechanisms underlying ischemic tolerance are unclear. Here we show that ischemia induces pronounced caspase-3 activity in naive neurons that die and in preconditioned neurons that survive. Preconditioning intervenes downstream of proteolytic processing and activation of caspase-3 (a protease implicated in the execution of apoptosis) and upstream of the caspase-3 target caspase-activated DNase (CAD, a deoxyribonuclease that catalyzes DNA fragmentation) to arrest neuronal death. We further show that global ischemia promotes expression of the pro-survival inhibitor-of-apoptosis (IAP) family member cIAP, but unleashes Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI), a factor that neutralizes the protective actions of IAPs and promotes neuronal death. Preconditioning blocks the mitochondrial release of Smac/DIABLO, but not the ischemia-induced upregulation of IAPs. In the absence of Smac/DIABLO, cIAP halts the caspase death cascade and arrests neuronal death. These findings suggest that preconditioning preserves the integrity of the mitochondrial membrane, enabling neurons to survive in the face of caspase activation.

Ischemic insults derepress the gene silencer REST in neurons destined to die.

A subset of genes implicated in genetic and acquired neurological disorders encode proteins essential to neural patterning and neurogenesis. The gene silencing transcription factor neuronal repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF) plays a critical role in elaboration of the neuronal phenotype. In neural progenitor and non-neural cells, REST acts by repression of a subset of neural genes important to synaptic plasticity and synaptic remodeling, including the AMPA receptor (AMPAR) subunit GluR2. Here we show that global ischemia triggers REST mRNA and protein expression. REST suppresses GluR2 promoter activity and gene expression in neurons destined to die. Because the GluR2 subunit governs AMPAR Ca2+ permeability, these changes are expected to have profound effects on neuronal survival. In keeping with this concept, acute knockdown of the REST gene by antisense administration prevents GluR2 suppression and rescues post-ischemic neurons from ischemia-induced cell death in an in vitro model. To our knowledge, our study represents the first example of ischemia-induced induction of a master transcriptional regulator gene and its protein expression critical to neural differentiation and patterning in adult neurons. Derepression of REST is likely to be an important mechanism of insult-induced neuronal death.

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."

Ischemic preconditioning acts upstream of GluR2 down-regulation to afford neuroprotection in the hippocampal CA1.

Animals subjected to sublethal transient global ischemia (ischemic preconditioning) exhibit neuroprotection against subsequent global ischemia-induced neuronal death in the hippocampal CA1 (ischemic tolerance). The molecular mechanisms underlying ischemic tolerance are unclear. Here we report that ischemic preconditioning induced a small, transient down-regulation of GluR2 mRNA expression and greatly attenuated subsequent ischemia-induced GluR2 mRNA and protein down-regulation and neuronal death. Ischemic preconditioning and GluR2 antisense knockdown acted synergistically to increase cell death. Sublethal antisense knockdown did not protect against subsequent ischemic insults or antisense knockdown. These findings indicate that ischemic preconditioning acts at step(s) upstream from suppression of GluR2 gene expression to afford neuroprotection and implicate transcriptional regulation of GluR2 expression in the adaptive mechanisms associated with ischemic tolerance.

Leptomeningeal dissemination of pilocytic astrocytoma via hematoma in a child. Case report.

A case of recurrent pilocytic astrocytoma with leptomeningeal dissemination (LMD) is described. A cerebellar tumor was diagnosed in a 3-year-old boy, in whom resection was performed. When the boy was 6 years of age, recurrence was treated with surgery and local radiotherapy. At age 13 years, scoliosis was present, but the patient was asymptomatic. Twelve years after initial surgery LMD was demonstrated in the lumbar spinal region without recurrence of the original tumor. This tumor also was subtotally removed. During the procedure, a hematoma was observed adjacent to the tumor, but the border was clear. Histological examination of the spinal cord tumor showed features similar to those of the original tumor. There were no tumor cells in the hematoma. The MIB-1 labeling index indicated no malignant change compared with the previous samples. Radiotherapy was performed after the surgery. The importance of early diagnosis and management of scoliosis is emphasized, and the peculiar pattern of dissemination of the pilocytic astrocytoma and its treatment are reviewed.

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(-/-). S100beta (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.