Hippocampal CA1 Neuronal Death Research Articles

Immediate and Delayed Treatments with Curcumin Prevents Forebrain Ischemia-Induced Neuronal Damage and Oxidative Insult in the Rat Hippocampus

Oxidative stress is believed to contribute to neurodegeneration following ischemic injury. The present study was undertaken to evaluate the possible antioxidant neuroprotective effect of curcumin (Cur) on neuronal death of hippocampal CA1 neurons following transient forebrain ischemia in rat. Treatment of Cur (200 mg/kg/day, i.p.) at three different times (immediately, 3 h and 24 h after ischemia) significantly (P<0.01) reduced neuronal damage 7 days after ischemia. Also, treatment of ischemic rats with Cur decreased the elevated levels of MDA and increased GSH contents, catalase and SOD activities to normal levels. In the in vitro, Cur was as potent as antioxidant (IC(50) = 1 microM) as butylated hydroxytoluene. The present study demonstrates that curcumin treatment attenuates forebrain ischemia-induced neuronal injury and oxidative stress in hippocampal tissue. Thus treatment with curcumin immediately or even delayed until 24 h may have the potential to be used as a protective agent in forebrain ischemic insult in human.

The CRH1 antagonist CP154,526 failed to alter ischemia-induced neurodegeneration and spatial memory deficits in rats but inhibited behavioral activity in the novel open field

Corticotropin-releasing hormone (CRH) has been implicated in ischemia-induced neurotoxicity, due in part to excitatory effects at the hippocampus, and the demonstrated neuroprotective effects of centrally administered, non-specific CRH antagonists. However, a number of issues remain to be clarified from these studies, including the relative contribution of CRH receptor subtypes, and the efficacy of these compounds to alter ischemia-induced behavioral impairments. In the current study, a highly selective, systemically administered CRH1 antagonist (CP154,526) failed to reverse global ischemia-induced cell death in hippocampal CA1 neurons or spatial memory impairments as assessed in the radial arm maze. Similarly, central administration of α-helical CRH failed to confer protection against ischemic damage. Interestingly, CRH1 antagonism reversed ischemia-induced hyperactivity in a novel open field, suggesting that modulation of this behavior is independent of effects on hippocampal CA1 cell loss. Failure of the current study to demonstrate neuroprotective effects of either the selective or non-selective CRH antagonists tested challenges the proposed neurotoxic role of CRH in global ischemia. These findings are discussed in relationship to recent findings reconsidering the participation of CRH in excitotoxic-mediated cellular damage.

Reproducible loss of CA1 neurons following carotid artery occlusion combined with halothane-induced hypotension

The 2-vessel occlusion approach to produce global ischemia in rats requires concomitant reduction of systemic blood pressure. We have utilized the hypotensive effect of halothane administrated by artificial respiration to prevent respiratory arrest and to ensure stable physiological conditions. Systemic blood pressure was reduced to 40–45 mm Hg by instant adjustments of the halothane concentration. Bilateral occlusion of the carotid arteries caused a profound and reproducible ischemia, as analyzed by laser-Doppler flowmetry. In the rats exposed to 11, 12, or 13 min of ischemia, 5% died and 5% developed seizures. The extent of neuronal death in CA1 was highly correlated to the duration of ischemia. Following 11 min of ischemia, CA1 neuronal cell death, as analyzed by Fluoro-Jade, was absent 1 day after injury, variable at day 4, and consistent at day 7. The numbers of cresyl violet- and NeuN-positive neurons at day 7 were 8% and 20% of control, respectively. OX42 immunoreactivity was low and variable at day 4, but pronounced at day 7. In conclusion, this rat global ischemia model is relatively simple to perform, has a low mortality, and produces a profound and highly reproducible delayed cell death of hippocampal CA1 neurons.

Functional Proteins Involved in Regulation of Intracellular Ca2+ for Drug Development: Role of Calcium/Calmodulin-Dependent Protein Kinases in Ischemic Neuronal Death

Excessive elevation of intracellular calcium level seems to be a trigger of ischemic neuronal injury. Calcium/calmodulin (CaM)-dependent protein kinase kinase (CaM-KK) is an upstream kinase for CaM kinase IV (CaM-KIV) that was reported to prevent apoptosis through phosphorylation of CREB (cyclic AMP responsive element-binding protein). We here observed that CaM-KK could directly activate Akt, thereby preventing apoptosis in cultured cells. Then we examined changes in Akt and CaM-KIV activities in gerbil forebrain ischemia. In 5-minischemia-caused delayed neuronal death in hippocampal CA1 neurons, Akt and CaM-KIV activities were decreased after reperfusion. On the other hand, during induction of ischemic tolerance, Akt activity gradually and persistently increased in the CA1 neurons with transient increase in CREB phosphorylation. Inhibition of Akt activity with wortmannin or CREB-DNA binding with CRE-decoy injection resulted in failure of generation of ischemic tolerance. These results indicated activation of Akt and CaM-KIV play important roles in induction of the ischemic tolerance. Activation of CaM-KK may provide a new strategy for overcoming the ischemic stress.

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.

In vivo influence of ceramide accumulation induced by treatment with a glucosylceramide synthase inhibitor on ischemic neuronal cell death

It has been shown that exogenous ceramide induces delayed neuronal death (DND) of cultured hippocampal neurons. To evaluate the role of endogenous ceramide in ischemic DND, the glucosylceramide synthase inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( d-PDMP), was used to generate ceramide in gerbil hippocampi in vivo. The trimethylsilylated derivatives of ceramide were analyzed directly by gas chromatography mass spectrometry, after separation with high-performance thin-layer chromatography. The ceramide compositions in vehicle hippocampus consisted mainly of C18:0 fatty acyl sphingosine (87.9%), with C16:0 and C20:0 ceramides being minor components (7.1% and 5.1%, respectively). Ceramide level in the hippocampi from gerbils subjected to d-PDMP treatment was 1.5-fold higher than those from vehicle-treated gerbils. In spite of the accumulation of ceramide observed in the d-PDMP group, the histological studies did not reveal any ischemic neuronal death in hippocampal CA1 neurons with the gerbils that had been subjected to a sham operation (2-min sublethal ischemia). These results suggest that the ceramide accumulation induced by blocking the de novo synthesis of glucosylceramide with d-PDMP may be independent of the metabolic pathway underlying ischemic DND.

Zinc released from metallothionein-iii may contribute to hippocampal CA1 and thalamic neuronal death following acute brain injury

Vesicular zinc was initially considered the sole source of toxic intraneuronal zinc accumulation in response to acute brain injury, but recent evidence suggests that additional sources also exist. Because metallothioneins (MTs) can bind and release zinc, we examined the possibility that the brain-specific form, MT-III, is such a zinc source. After kainate-induced seizures, cytoplasmic zinc accumulation and neuronal death in the hippocampal CA1 region and the thalamus were substantially lower in Mt3-null mice than in wild-type mice. Furthermore, compared with zinc transporter 3 ( Znt3)-null mice, Znt3/Mt3 double-null mice exhibited further reductions in neuronal death in CA1 following kainate-induced seizures. Similar reductions in zinc accumulation and neuronal death in hippocampal CA1 and the dentate gyrus in Mt3-null mice were observed in a sodium nitroprusside model of acute brain injury. In contrast to CA1, more neuronal death occurred after kainate-induced seizures in CA3 of Mt3-null mice. These results suggest that intracellular zinc release from MT-III may contribute substantially to zinc-mediated neuronal death in certain brain areas, including the hippocampal CA1 region and the thalamus.

Neuroprotective effect of postischemic administration of sodium orthovanadate in rats with transient middle cerebral artery occlusion.

Orthovanadate is a competitive inhibitor of protein tyrosine phosphatases. Some of its reported biologic effects are its insulin mimetic property and its activation of phosphoinositide 3-kinase and extracellular-signal regulated kinase (ERK). The authors previously reported its neuroprotective effect on delayed neuronal death of gerbil hippocampal CA1 neurons via Akt and ERK activation after transient forebrain ischemia. In the present study, the neuroprotective effect of postischemic intraperitoneal administration of sodium orthovanadate (2 l/kg of 50-mmol/l sodium orthovanadate in saline) was investigated in rats with transient middle cerebral artery occlusion. Ischemic neuronal injury was evaluated 1 day and 28 days after ischemia. The neuroprotective effect of orthovanadate was significant in the cortex but not the caudate putamen (ischemic core) at both 1 and 28 days after ischemia. In orthovanadate group, the activities of Akt and ERK were maintained after reperfusion; they were decreased in saline group. Blood glucose level decreased but within normal range. Regional cerebral blood flow was lower than that of saline group only at 0 hours after reperfusion. These data suggest that orthovanadate has neuroprotective effects in rats with transient middle cerebral artery occlusion and that these effects are mediated by Akt and ERK activation. Furthermore, low blood glucose levels and gradual recovery of regional cerebral blood flow may contribute to neuroprotection.

Overexpression of Ca 2+-permeable AMPA receptor promotes delayed cell death of hippocampal CA1 neurons following transient forebrain ischemia

To examine the role of Ca 2+ entry through AMPA receptors in the pathogenesis of the ischemia-induced cell death of hippocampal neurons, we delivered cDNA of Q/R site-unedited form (GluR2Q) of AMPA receptor subunit GluR2 in the hippocampus by using an HVJ-liposome-mediated gene transfer technique. Two days prior to transient forebrain ischemia, we injected an HVJ-liposome containing cDNA of the GluR2Q-myc fusion gene into a rat unilateral hippocampus. In the absence of ischemic insult, overexpression of Ca 2+-permeable GluR2Q did not cause any neurodegeneration in the cDNA-injected hippocampus. In ischemic rats, overexpression of Ca 2+-permeable GluR2Q markedly promoted ischemic cell death of CA1 pyramidal neurons, while complete rescue of CA1 pyramidal neurons from ischemic damage occurred in the hippocampal hemisphere opposite the GluR2Q expression. Overexpression of the Q/R-site edited form (GluR2R) of subunit GluR2 did not affect the ischemia-induced damage of CA1 pyramidal neurons. From these results, we suggest that the Ca 2+-permeability of AMPA receptors does not have a direct contribution to glutamate receptor-mediated neurotoxicity but has a promotive action in the evolution of ischemia-induced neurodegeneration of vulnerable neurons.

Neuroprotective efficacy of nimesulide against hippocampal neuronal damage following transient forebrain ischemia

Cyclooxygenase-2 is involved in the inflammatory component of the ischemic cascade, playing an important role in the delayed progression of the brain damage. The present study evaluated the pharmacological effects of the selective cyclooxygenase-2 inhibitor nimesulide on delayed neuronal death of hippocampal CA1 neurons following transient global cerebral ischemia in gerbils. Administration of therapeutically relevant doses of nimesulide (3, 6 and 12 mg/kg; i.p.) 30 min before ischemia and at 6, 12, 24, 48 and 72 h after ischemia significantly (P<0.01) reduced hippocampal neuronal damage. Treatment with a single dose of nimesulide given 30 min before ischemia also resulted in a significant increase in the number of healthy neurons in the hippocampal CA1 sector 7 days after ischemia. Of interest is the finding that nimesulide rescued CA1 pyramidal neurons from ischemic death even when treatment was delayed until 24 h after ischemia (34±9% protection). Neuroprotective effect of nimesulide is still evident 30 days after the ischemic episode, providing the first experimental evidence that cyclooxygenase-2 inhibitors confer a long-lasting neuroprotection. Oral administration of nimesulide was also able to significantly reduce brain damage, suggesting that protective effects are independent of the route of administration. The present study confirms the ability of cyclooxygenase-2 inhibitors to reduce brain damage induced by cerebral ischemia and indicates that nimesulide can provide protection when administered for up to 24 h post-ischemia.

Ischemia and Ischemic Tolerance Induction Differentially Regulate Protein Expression of GluR1, GluR2, and AMPA Receptor Binding Protein in the Gerbil Hippocampus

Postischemic delayed neuronal death (DND) of hippocampal CA1 neurons has been suggested to occur as a result of formation of calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors lacking the GluR2 subunit (GluR2 hypothesis). DND can be prevented by a short tolerance-inducing ischemic period. The present study was designed to assess whether postischemic protein levels of GluR2 predict neuronal death. Additionally, the role of AMPA receptor binding protein (ABP) was investigated with respect to neuronal death or survival. Postischemic protein expression of GluR1, GluR2, and ABP was analyzed in 3 experimental paradigms of transient global ischemia with the use of subunit-specific antisera and semiquantitative densitometric evaluation. Gerbils were subjected (1) to a 5-minute ischemic period resulting in DND of CA1 neurons; (2) to a 2.5-minute period of ischemia mediating tolerance induction; and (3) to 5 minutes of ischemia in the ischemia-tolerant state (2.5+5 minutes of ischemia 4 days apart). The major finding was that GluR2 protein levels were significantly downregulated in neuronal subpopulations destined to survive, ie, in CA1 principal neurons after ischemic tolerance induction and in the ischemia-tolerant state, as well as in CA3 neurons after a 5-minute period of ischemia. ABP expression remained unaffected. Our results modify the GluR2 hypothesis in that postischemic GluR2 reduction also occurs in hippocampal CA1 and CA3 principal neurons without subsequent neuronal death. ABP is obviously not involved in mechanisms of DND or ischemic tolerance induction.

Overexpression of Copper/Zinc Superoxide Dismutase in Transgenic Rats Protects Vulnerable Neurons against Ischemic Damage by Blocking the Mitochondrial Pathway of Caspase Activation

Mitochondria are known to be involved in the early stage of apoptosis by releasing cytochrome c, caspase-9, and the second mitochondria-derived activator of caspases (Smac). We have reported that overexpression of copper/zinc superoxide dismutase (SOD1) reduced superoxide production and ameliorated neuronal injury in the hippocampal CA1 subregion after global ischemia. However, the role of oxygen free radicals produced after ischemia/reperfusion in the mitochondrial signaling pathway has not been clarified. Five minutes of global ischemia was induced in male SOD1-transgenic (Tg) and wild-type (Wt) littermate rats. Cytosolic expression of cytochrome c and Smac and activation of caspases were evaluated by immunohistochemistry, Western blot, and caspase activity assay. Apoptotic cell death was characterized by DNA nick end and single-stranded DNA labeling. In the Wt animals, early superoxide production, mitochondrial release of cytochrome c, Smac, and cleaved caspase-9 were observed after ischemia. Active caspase-3 was subsequently increased, and 85% of the hippocampal CA1 neurons showed apoptotic DNA damage 3 d after ischemia. Tg animals showed less superoxide production and cytochrome c and Smac release. Subsequent active caspase-3 expression was not evident, and only 45% of the neurons showed apoptotic DNA damage. A caspase-3 inhibitor (N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone) reduced cell death only in Wt animals. These results suggest that overexpression of SOD1 reduced oxidative stress, thereby attenuating the mitochondrial release of cytochrome c and Smac, resulting in less caspase activation and apoptotic cell death. Oxygen free radicals may play a pivotal role in the mitochondrial signaling pathway of apoptotic cell death in hippocampal CA1 neurons after global ischemia.

Gene therapy for preventing neuronal death using hepatocyte growth factor: in vivo gene transfer of HGF to subarachnoid space prevents delayed neuronal death in gerbil hippocampal CA1 neurons.

To develop a novel strategy to prevent delayed neuronal death (DND) following transient occlusion of arteries, the gene of hepatocyte growth factor (HGF), a novel neurotrophic factor, was transfected into the subarachnoid space of gerbils after transient forebrain ischemia. Importantly, transfection of HGF gene into the subarachnoid space prevented DND, accompanied by a significant increase in HGF in the cerebrospinal fluid. Prevention of DND by HGF is due to the inhibition of apoptosis through the blockade of bax translocation from the cytoplasm to the nucleus. HGF gene transfer into the subarachnoid space may provide a new therapeutic strategy for cerebrovascular disease.

Dantrolene ameliorates delayed cell death and concomitant DNA fragmentation in the rat hippocampal CA1 neurons subjected to mild ischemia

The present study investigated whether dantrolene, which inhibits the Ca 2+ release from the intracellular Ca 2+ store sites, reduced nuclear DNA fragmentation and produced neuronal protection in a model of global forebrain ischemia. Male Wistar rats were subjected to four-vessel occlusion (4VO) for 5 min and then infused continuously with dantrolene or vehicle into the cerebral ventricle for 3 days. The intact rats did not undergo any intervention. The number of viable and DNA nick-end-labeled neurons in the hippocampal CA1 were evaluated 4 days after the ischemia. The number of viable neurons in the dantrolene-treated rats was significantly higher than that in the vehicle-treated rats and lower than that in the intact animals ( P<0.01 and <0.05, respectively). The number of DNA nick-end-labeled nuclei was significantly lower in dantrolene-treated rats compared with the vehicle-treated animals ( P<0.0001). No nick-end labeling was observed in the intact animals. A linear correlation was found between the number of viable cells and nick-end labeled nuclei in the CA1 ( r=0.91, P<0.0001). These results suggest that the postischemic intraventricular dantrolene is effective in precluding neuronal death and concomitant nuclear DNA fragmentation following transient global ischemia.

Both caspase-dependent and caspase-independent pathways may be involved in hippocampal CA1 neuronal death because of loss of cytochrome c From mitochondria in a rat forebrain ischemia model.

In a rat forebrain ischemia model, the authors examined whether loss of cytochrome c from mitochondria correlates with ischemic hippocampal CA1 neuronal death and how cytochrome c release may shape neuronal death. Forebrain ischemia was induced by bilateral common carotid artery occlusion with simultaneous hypotension for 10 minutes. After reperfusion, an early rapid depletion of mitochondrial cytochrome c and a late phase of diffuse redistribution of cytochrome c occurred in the hippocampal CA1 region, but not in the dentate gyrus and CA3 regions. Intracerebroventricular administration of Z-DEVD-FMK, a relatively selective caspase-3 inhibitor, provided limited but significant protection against ischemic neuronal damage on day 7 after reperfusion. Treatment with 3 minutes of ischemia (ischemic preconditioning) 48 hours before the 10-minute ischemia attenuated both the early and late phases of cytochrome c redistribution. In another subset of animals treated with cycloheximide, a general protein synthesis inhibitor, the late phase of cytochrome c redistribution was inhibited, whereas most hippocampal CA1 neurons never regained mitochondrial cytochrome c. Examination of neuronal survival revealed that ischemic preconditioning prevents, whereas cycloheximide only delays, ischemic hippocampal CA1 neuronal death. DNA fragmentation detected by terminal deoxytransferase-mediated dUTP-nick end labeling (TUNEL) in situ was largely attenuated by ischemic preconditioning and moderately reduced by cycloheximide. These results indicate that the loss of cytochrome c from mitochondria correlates with hippocampal CA1 neuronal death after transient cerebral ischemia in relation to both caspase-dependent and -independent pathways. The amount of mitochondrial cytochrome c regained may determine whether ischemic hippocampal CA1 neurons survive or succumb to late-phase death.

Metabotropic glutamate receptor subtypes are differentially expressed after transient cerebral ischemia without, during and after tolerance induction in the gerbil hippocampus

Postischemic delayed neuronal death (DND) of hippocampal CA1 neurons can be prevented by a preconditioning sublethal ischemic stimulus. To check for possible participation of metabotropic glutamate receptors (mGluRs) in neuronal death or survival, we analyzed postischemic protein expression of subtypes 1b and 5 of group I mGluRs, which are thought to exert neurotoxic effects after pathological activation due to ischemia, and subtypes 2 and 3 of group II mGluRs, which in contrast are thought to be neuroprotective in this state, respectively. Therefore, three groups of gerbils with reperfusion intervals between 8 h and 4 days ( n=5 each) were investigated: one group was subjected to 5 min ischemia, resulting in DND of CA1 neurons, a second group to a tolerance inducing 2.5 min period of ischemia and a third group to 5 min ischemia after prior tolerance induction. The major finding was a transient postischemic reduction of mGluR1b and 5 expression in the ischemic tolerant CA1 subfield at 8 h. This downregulation of neurotoxic mGluRs may indicate a contribution to the survival of highly vulnerable CA1 neurons in the ischemic tolerant state.

T Mitochondrial Release of CytochromecCorresponds to the Selective Vulnerability of Hippocampal CA1 Neurons in Rats after Transient Global Cerebral Ischemia

Release of cytochrome c from mitochondria to the cytosol is a critical step in apoptotic cell death after focal cerebral ischemia. The relationship among cytochrome c release, selective vulnerability, and delayed death of hippocampal CA1 neurons after transient global ischemia was examined. Global ischemia was induced by 10 min of bilateral common carotid artery occlusion and hypotension in rats. Cytosolic expression of cytochrome c was evaluated by immunohistochemistry and Western blotting. Apoptosis after global ischemia was also characterized by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling (TUNEL) staining and DNA gel electrophoresis. Immunohistochemistry showed cytosolic cytochrome c-positive cells exclusively in the CA1 subregion of the hippocampus as early as 2 hr after ischemia. Double fluorescent immunostaining confirmed that CA1 neurons and a small number of astrocytes expressed cytochrome c. Western blot analysis revealed a band (15 kDa) of cytochrome c in the cytosolic fraction and a corresponding decrease in the mitochondrial fraction. A significant number of TUNEL-positive cells appeared only in the CA1 pyramidal cell layer of the hippocampus, and DNA gel electrophoresis showed a significant amount of DNA fragmentation 3-5 d after ischemia. Our data provide the first evidence that cytochrome c was released to the cytosol from mitochondria in CA1 neurons after global ischemia and that the release preceded DNA fragmentation. These findings suggest cytochrome c involvement in the delayed death of hippocampal CA1 neurons in rats after transient global ischemia.

Protective Effect of Prostaglandin I2 Analogs on Ischemic Delayed Neuronal Damage in Gerbils

We found a novel subtype of prostaglandin (PG) I2 receptor (IP2) expressed in the central nervous system. Recently we have demonstrated that (15R)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (15R-TIC) and 15-deoxy-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (15-deoxy-TIC), IP2-specific ligands, significantly prevented high (50%) oxygen-induced apoptotic neuronal death in cultured hippocampal neurons. We report here a potent neuroprotective effect of such analogs on delayed neuronal death of hippocampal CA1 neurons following transient ischemia for 3 min in gerbils. (15S)-16-m-tolyl-17,18,19,20-tetranorisocarbacyclin (15S-TIC), which nonselectively acts both on the PGI2 receptor expressed in the peripheral tissue (IP1) and on IP2, also showed a neuroprotective effect on such an ischemic model at higher doses than those for 15R-TIC and 15-deoxy-TIC. These PGI2 analogs did not affect brain temperature, indicating that the agents showed the neuroprotective effect not by a hypothermic effect, but rather by the direct action on neurons.

Inhibition of caspases prevents cell death of hippocampal CA1 neurons, but not impairment of hippocampal long-term potentiation following global ischemia

An essential role for caspases in programmed neuronal cell death has been demonstrated in various in vitro studies, and synthetic caspase inhibitors have recently been shown to prevent neuronal cell loss in animal models of focal cerebral ischemia and traumatic brain injury, respectively. The therapeutic utility of caspase inhibitors, however, will depend on preservation of both structural and functional integrity of neurons under stressful conditions. The present study demonstrates that expression and proteolytic activity of caspase-3 is up-regulated in the rat hippocampus after transient forebrain ischemia. Continuous i.c.v. infusion of the caspase inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone significantly attenuated caspase-3-like enzymatic activity, and blocked delayed cell loss of hippocampal CA1 neurons after ischemia. Administration of N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, however, did not prevent impairment of induction of long-term potentiation in post-ischemic CA1 cells, suggesting that caspase inhibition alone does not preserve neuronal functional plasticity.

Metyrapone alleviates ischemic neuronal damage in the gerbil hippocampus

Transient forebrain ischemia was induced in gerbils, and the effect of a pre-ischemic treatment with metyrapone (100 mg/kg) on delayed neuronal death in hippocampal CA1 neurons was evaluated. The effect of metyrapone on the ischemic release of amino acids in the CA1 region was also examined by microdialysis. Hippocampal slices were used for the evaluation of the hypoxia-induced intracellular Ca 2+ increase by microfluorometry. The metyrapone treatment morphologically improved the damage provoked by 3 min of ischemia, although it did not alleviate the damage by 5 min. Ischemia for 3 min produced a 306% increase in the glutamate concentration in perfusates, and metyrapone suppressed the peak value to 42% of that in the control group. The extent of the increase in fluorescence intensity by intracellular Ca 2+ was lower by 16% in slices from metyrapone-treated animals than in controls 600 s after induction of hypoxia. The removal of Ca 2+ from the perfusion medium suppressed the hypoxic Ca 2+ increase, and the increase was further reduced in slices pretreated with metyrapone. The increase in the level of endogenous glucocorticoids, which occurs in cerebral ischemia, may aggravate ischemic neuronal damage.