Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain.

In rats ischemia of the forebrain induced by a 30-minute occlusion of the carotid artery, followed by 120 minutes of arterial reperfusion, produced ischemic lesions of selectively vulnerable pyramidal cells in both hippocampi. Focal microinfusion into the dorsal hippocampus of 2-amino-7-phosphonoheptanoic acid, an antagonist of excitation at the N-methyl-D-aspartate-preferring receptor, before ischemia was induced protected against the development of ischemic damage. It is proposed that excitatory neurotransmission plays an important role in selective neuronal loss due to cerebral ischemia.

Microarray profile of seizure damage-refractory hippocampal CA3 in a mouse model of epileptic preconditioning.

A neuroprotected state can be acquired by preconditioning brain with a stimulus that is subthreshold for damage (tolerance). Acquisition of tolerance involves coordinate, bi-directional changes to gene expression levels and the re-programmed phenotype is determined by the preconditioning stimulus. While best studied in ischemic brain there is evidence brief seizures can confer tolerance against prolonged seizures (status epilepticus). Presently, we developed a model of epileptic preconditioning in mice and used microarrays to gain insight into the transcriptional phenotype within the target hippocampus at the time tolerance had been acquired. Epileptic tolerance was induced by an episode of non-damaging seizures in adult C57Bl/6 mice using a systemic injection of kainic acid. Neuron and DNA damage-positive cell counts 24 h after status epilepticus induced by intraamygdala microinjection of kainic acid revealed preconditioning given 24 h prior reduced CA3 neuronal death by approximately 45% compared with non-tolerant seizure mice. Microarray analysis of over 39,000 transcripts (Affymetrix 430 2.0 chip) from microdissected CA3 subfields was undertaken at the point at which tolerance was acquired. Results revealed a unique profile of small numbers of equivalently up- and down-regulated genes with biological functions that included transport and localization, ubiquitin metabolism, apoptosis and cell cycle control. Select microarray findings were validated post hoc by real-time polymerase chain reaction and Western blotting. The present study defines a paradigm for inducing epileptic preconditioning in mice and first insight into the global transcriptome of the seizure-damage refractory brain.

Conjugated linoleic acid impairs endothelial function.

OBJECTIVE: To determine the effect of dietary supplementation with conjugated linoleic acid (CLA) on body mass index (BMI), body fat distribution, endothelial function, and markers of cardiovascular risk. METHODS AND RESULTS: Forty healthy volunteers with BMI >27 kg/m2 were randomized to receive a CLA isomeric mixture or olive oil in a 12-week double-blind study. Subcutaneous body fat and abdominal/hepatic fat content were assessed using skin-fold thicknesses and computed tomography scanning, respectively. Endothelial function was assessed by brachial artery flow-mediated dilatation (FMD). Plasma isoprostanes were measured as an index of oxidative stress. CLA supplementation did not result in a significant change in BMI index or total body fat. There was a significant decrease in limb (-7.8 mm, P<0.001), but not torso skin-fold thicknesses or abdominal or liver fat content. Brachial artery FMD declined (-1.3%, P=0.013), and plasma F2-isoprostanes increased (+91 pg/mL, P=0.042). CONCLUSIONS: A CLA isomeric mixture had at most modest effects on adiposity and worsened endothelial function. On the basis of these results, the use of the isomeric mixture of CLA as an aid to weight loss cannot be recommended.

Activation of the caspase 8 pathway mediates seizure-induced cell death in cultured hippocampal neurons.

In response to harmful stresses, cells induce programmed cell death (PCD) or apoptosis. Seizures can induce neural damage and activate biochemical pathways associated with PCD. Since seizures trigger intracellular calcium overload, it has been presumed that the intrinsic cell death pathway mediated by mitochondrial dysfunction would modulate cell death following seizures. However, previous work suggests that the extrinsic cell death pathway may initiate the damage program. Here we investigate intrinsic versus extrinsic cell death pathway activation using caspase cleavage as a marker for activation of these pathways in a rat in vitro model of seizures. Hippocampal cells, chronically treated with kynurenic acid, had kynurenic acid withdrawn to induce seizure-like activity for 40 min. Subjecting rat hippocampal cultures to seizures increased cell death and apoptosis-like DNA fragmentation using TUNEL staining. Seizure-induced cell death was blocked by both MK801 (10 microM) and CNQX (40 microM), which suggests multiple glutamate receptors regulate seizure-induced cell death. Cleavage of the initiator caspases, caspase 8 and 12 were increased 4h following seizure, and cleavage of the quintessential executioner caspase, caspase 3 was increased 4h following seizure. In contrast, caspase 9 cleavage only increased 24h following seizure. Using an affinity labeling approach to trap activated caspases in situ, we show that caspase 8 is the apical caspase activated following seizures. Finally, we show that the caspase 8 inhibitor Ac-IETD-CHO was more effective at blocking seizure-induced cell death than the caspase 9 inhibitor Ac-LEHD-CHO. Taken together, our data suggests the extrinsic cell death pathway-associated caspase 8 is activated following seizures in vitro.

Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures.

Nuclear changes, including internucleosomal DNA fragmentation, are characteristic features of neuronal apoptosis resulting from transient cerebral ischemia and related brain insults for which the molecular mechanism has not been elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding caspase-activated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kDa nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the induction of internucleosomal DNA fragmentation in the hippocampus in a rat model of transient global ischemia and in primary neuronal cultures under ischemia-like conditions. At 8-72 hr after ischemia, CAD/DFF40 mRNA and protein were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 formed a heterodimeric complex in the nucleus with its natural inhibitor CAD (ICAD) and was activated after ischemia in a delayed manner (>24 hr) by caspase-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inhibited by purified ICAD protein. These results strongly suggest that CAD/DFF40 is the endogenous endonuclease that mediates caspase-3-dependent internucleosomal DNA degradation and related nuclear alterations in ischemic neurons.

Seizure-like activity leads to the release of BAD from 14-3-3 protein and cell death in hippocampal neurons in vitro.

Seizure-induced neuronal death may involve engagement of the BCL-2 family of apoptosis-regulating proteins. In the present study we examined the activation of proapoptotic BAD in cultured hippocampal neurons following seizures induced by removal of chronic glutamatergic transmission blockade. Kynurenic acid withdrawal elicited an increase in seizure-like electrical activity, which was inhibited by blockers of AMPA (CNQX) and NMDA (MK801 and AP5) receptor function. However, only NMDA receptor antagonists inhibited calcium entry as assessed by fura-2, and cell death of hippocampal neurons. Seizures increased proteolysis of caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) of cells. Seizure-like activity induced dephosphorylation of BAD and the disruption of its constitutive interaction with 14-3-3 proteins. In turn, BAD dimerized with antiapoptotic BCL-Xl after seizures. However, the absence of neuroprotective effects of pathway intervention suggests that BAD may perform a reinforcement rather than instigator role in cell death following seizures in vitro.

Formation of the Apaf-1/cytochrome c complex precedes activation of caspase-9 during seizure-induced neuronal death.

In this study we examine the in vivo formation of the Apaf-1/cytochrome c complex and activation of caspase-9 following limbic seizures in the rat. Seizures were elicited by unilateral intraamygdala microinjection of kainic acid to induce death of CA3 neurons within the hippocampus of the rat. Apaf-1 was found to interact with cytochrome c within the injured hippocampus 0-24 h following seizures by co-immunoprecipitation analysis and immunohistochemistry demonstrated Apaf-1/cytochrome c co-localization. Cleavage of caspase-9 was detected approximately 4 h following seizure cessation within ipsilateral hippocampus and was accompanied by increased cleavage of the substrate Leu-Glu-His-Asp-p-nitroanilide (LEHDpNA) and subsequent strong caspase-9 immunoreactivity within neurons exhibiting DNA fragmentation. Finally, intracerebral infusion of z-LEHD-fluoromethyl ketone increased numbers of surviving CA3 neurons. These data suggest seizures induce formation of the Apaf-1/cytochrome c complex prior to caspase-9 activation and caspase-9 may be a potential therapeutic target in the treatment of brain injury associated with seizures.

Deep prepiriform cortex modulates neuronal cell death in global ischemia.

Deep prepiriform cortex has an important role in modulating neurotransmission during limbic seizures. We used pharmacologic blockade of non-N-methyl-D-aspartate (NMDA) receptors to study excitatory circuitry from the deep prepiriform cortex to the hippocampus during global ischemia in rat. NBQX, a potent non-NMDA glutamate receptor antagonist, was microinjected stereotactically into the deep prepiriform cortex before global ischemia for 10 min. Neuronal cell death in the hippocampus was evaluated quantitatively 72 h after ischemia. The NBQX-injected rats had a greater number of surviving cells in CA1 sector of hippocampus than did saline-injected controls or rats that received NBQX injections 1 mm from the target. Thus, excitatory amino acid-mediated circuitry emanating from deep prepiriform cortex modulates ischemic neuronal injury in the hippocampus.

Acidosis causes failure of astrocyte glutamate uptake during hypoxia.

Failure of glutamate uptake during ischemia can lead to neurotoxic accumulations of glutamate in brain extracellular space. Hypoxia and acidosis are metabolic consequences of ischemia that may individually or in combination impair glutamate uptake. We used primary rat astrocyte cultures to study the effects of acidosis, chemical hypoxia, and the combination of acidosis plus chemical hypoxia on glutamate uptake. Chemical hypoxia alone reduced uptake by 35-45%. Reduction in pH from 7.4 to 5.8 also caused a significant but incomplete inhibition of glutamate uptake, and this effect was more pronounced in medium buffered with CO2/bicarbonate. However, the combination of chemical hypoxia plus acidosis reduced glutamate uptake to below 10% of controls. Astrocyte ATP levels, like glutamate uptake, were significantly reduced by chemical hypoxia and further reduced by the combination of hypoxia plus acidosis. Acidosis under normoxic conditions had no significant effect on astrocyte ATP levels. These results suggest two mechanisms by which acidosis may contribute to failure of astrocyte glutamate uptake during ischemia: Acidosis may act in concert with hypoxia to cause ATP depletion, and acidosis may also have direct effects on glutamate transporters unrelated to effects on cellular ATP levels. pH effects on glutamate uptake may be an important factor affecting neuronal survival during incomplete ischemia.

Sequential metabolic changes in rat brain following middle cerebral artery occlusion: a 2-deoxyglucose study.

The distribution and time course of altered cerebral metabolism following permanent focal ischemia was studied in rat using the 2-deoxyglucose (2DG) technique. Increased 2DG uptake preceded decreased 2DG uptake and infarction in the caudate putamen and cortex. Decreased 2DG uptake without infarction was observed for 72 h in thalamus and for 24 h in hippocampus (areas remote from the ischemic zones). This study supports the concept of cell excitation as a pathophysiologic process in permanent focal ischemia. The time course of increased metabolism may demarcate the time window of opportunity for the previously demonstrated attenuation of stroke size with inhibition of cell excitation by pharmacologic blockade of excitatory amino acid neurotransmission.

Stress proteins and tolerance to focal cerebral ischemia.

Stress proteins are induced after a variety of neuronal injuries. The inducible 72-kDa heat shock protein (hsp70) is a stress protein that protects neurons from glutamate toxicity in vitro. Hsp70 has also been proposed to underlie the phenomenon of ischemic tolerance whereby brief sublethal intervals of global ischemia protect the hippocampus from subsequent lethal prolonged ischemia. To determine if the phenomenon of tolerance occurs in cortex after focal ischemia, the rat middle cerebral artery (MCA) was occluded by the suture method. Three 10-min intervals of transient ischemia (3 x 10-isc) separated by 45-min periods of reperfusion made up the most effective paradigm of preconditioning ischemia studied, and substantially reduced the volume of infarction 72 h after subsequent 100-min MCA occlusion. This approach induced protection if the interval between the 3 x 10-isc and the 100-min ischemia was 2, 3, or 5 days but not 1 or 7 days. Three 10-min intervals of transient ischemia alone produced minimal histological changes in the cortex at 72 h. Moreover, there were no significant changes in regional cerebral blood flow in the tolerant regions at 72 h after 3 x 10-isc before or during MCA occlusion. To explore the role of stress proteins in the induction of tolerance, expression of hsp70 and the glucose-regulated proteins grp75 and grp78 were studied. Samples from tolerant regions of the brain that had undergone preconditioning ischemia were evaluated at 1, 2, 3, 5, 7, and 14 days after 3 x 10-isc by Western blot analysis. The time course of hsp70 expression most closely correlated with tolerance. Hsp70 protein expression increased during times when tolerance was present (at 2-5 days) but did not increase thereafter (at 7 and 14 days). However, hsp70 was also increased before tolerance was present (at 1 day). Immunocytochemistry showed that hsp70 protein was expressed in neurons in the tolerant regions 24 h after 3 x 10-isc and was expressed in both neurons and glia after 72 h. Although immunocytochemistry suggested that there was increased neuronal expression of grp75 and grp78, no significant differences were found in protein expression as determined by Western blot before (at 1 day), during (at 2-5 days), and after (at 7 days and thereafter) tolerance. Thus, the time course of grp75 and grp78 expression did not correlate with that of tolerance. This model of ischemic tolerance is a useful method by which mechanisms of endogenous neuroprotection may be explored.

Limiting ischemic injury by inhibition of excitatory amino acid release.

Excitatory amino acids (EAAs) are important mediators of ischemic injury in stroke. N-Methyl-D-aspartate (NMDA) receptor antagonists have been shown to be very effective neuroprotective agents in animal models of stroke, but may have unacceptable toxicity for human use. An alternative approach is to inhibit the release of EAAs during stroke. BW1003C87 [5-(2,3,5-trichlorophenyl)-2,4-diaminopyrimidine], a drug that inhibits veratrine-induced release of the EAA glutamate in vitro, was tested in a rat model of proximal middle cerebral artery (MCA) occlusion. BW1003C87 significantly decreased ischemia-induced glutamate release in brain when given either 5 min before or 15 min following permanent MCA occlusion. Pretreated and posttreated rats had smaller infarct volumes and preserved glucose metabolism in the ischemic cortex at 24 h after MCA occlusion. BW1003C87 did not induce heat shock protein in the cingulate or retrosplenial cortex, suggesting that it does not injure neurons in these regions as do NMDA antagonists. These results demonstrate that drugs that inhibit glutamate release in ischemia may be nontoxic and show promise for the treatment of stroke.

Fas (CD95) may mediate delayed cell death in hippocampal CA1 sector after global cerebral ischemia.

Cell death-regulatory genes like caspases and bcl-2 family genes are involved in delayed cell death in the CA1 sector of hippocampus after global cerebral ischemia, but little is known about the mechanisms that trigger their expression. The authors found that expression of Fas and Fas-ligand messenger ribonucleic acid and protein was induced in vulnerable CA1 neurons at 24 and 72 hours after global ischemia. Fas-associating protein with a novel death domain (FADD) also was upregulated and immunoprecipitated and co-localized with Fas. Caspase-10 was activated and interacted with FADD protein to an increasing extent as the duration of ischemia increased. Moreover, caspase-10 co-localized with both FADD and caspase-3. These findings suggest that Fas-mediated death signaling may play an important role in signaling hippocampal neuronal death in CA1 after global cerebral ischemia.

Global ischemia induces expression of acid-sensing ion channel 2a in rat brain.

Acid-sensing ion channels (ASICs) are ligand-gated cation channels that respond to acidic stimuli. They are expressed throughout the mammalian nervous system. In the peripheral nervous system, ASICs act as nociceptors, responding to the tissue acidosis that accompanies ischemic and inflammatory conditions. The function of ASICs in the central nervous system is not known. In this article, the authors present evidence that transient global ischemia induces ASIC 2a protein expression in neurons that survive ischemia. Western blot analysis with an anti-ASIC 2a antibody revealed up-regulation of an 80 kD protein in ischemic rat brain. Immunohistochemical analysis showed that ASIC 2a protein expression increased in neurons of the hippocampus and cortex. Klenow fragment-mediated labeling of DNA strand breaks determined that ASIC 2a induction did not occur in cells with detectable DNA damage. The current results suggest a possible role for ASICs in mediating a cellular response to ischemia.

Brain phenobarbital uptake during prolonged status epilepticus.

The brain uptake of phenobarbital during prolonged status epilepticus (3 h) was studied in paralyzed, ventilated sheep. The first 30 min of status epilepticus was characterized by systemic hypertension, increased CBF, increased peripheral vascular resistance, a fall in brain pH, and an elevation in brain lactate concentrations. Subsequently, hemodynamic factors normalized and brain acidosis persisted. Phenobarbital administered during the early phase of status epilepticus produced higher levels of brain phenobarbital concentration, which was greatest at the earliest sample time (5 min following infusion), compared to nonseizure controls. This elevation persisted for the first 3 h following the infusion. Phenobarbital administration during the established phase of status epilepticus, when systemic blood pressure, peripheral vascular resistance, and CBF had returned to preseizure values, resulted in attenuated brain phenobarbital uptake not different from controls for the first 30 min. These results are explained by disruption of the blood-brain barrier to phenobarbital during the early (hypertensive) phase of status epilepticus.

Overexpression of the cell death suppressor Bcl-w in ischemic brain: implications for a neuroprotective role via the mitochondrial pathway.

Bcl-w is a newly described cell death suppressor member of the Bcl-2 gene family. As these genes may have a role in the outcome of ischemic brain injury, the regional expression of Bcl-w protein in rat brain was examined at 6 to 72 hours after 90 minutes of transient middle cerebral artery occlusion. Bcl-w protein, although constitutively expressed at low levels in nonischemic brain, was found to be overexpressed in ischemic brain at all time points studied. Up-regulation of Bcl-w protein was particularly abundant in the penumbral region of the cortex and mainly in cells lacking DNA fragmentation. In the cortical penumbra, Bcl-w protein was detected predominantly in neurons and showed mitochondrial localization, as determined using double-label immunohistochemistry. Bcl-w expression was also detectable, to a lesser extent, in reactive astrocytes in the infarct border zone and in microvessel walls in the infarct regions. At the mechanistic level, incubation of isolated brain mitochondria with the addition of recombinant Bax or high concentration of calcium resulted in release of cytochrome c from the mitochondria. In the presence of recombinant Bcl-w protein, however, the release of cytochrome c induced by Bax or calcium was largely inhibited. Further, recombinant Bcl-w protein inhibited calcium-induced loss of mitochondrial transmembrane potential, indicative of permeability transition, in a dose-dependent manner. These results suggest that Bcl-w may be an endogenous neuroprotectant against ischemic neuronal death and that, like its analogues such as Bcl-2 and Bcl-x-long, Bcl-w may achieve this protection via the mitochondrial death-regulatory pathway.

Calcium overload in selectively vulnerable neurons of the hippocampus during and after ischemia: an electron microscopy study in the rat.

Light and electron microscopy has been used to study the cytopathological changes in the rat hippocampus directly after a 30-min period of forebrain ischemia and after 30 or 120 min of reperfusion. The fine structural localization of calcium has been demonstrated using the oxalate/pyroantimonate procedure. Cellular changes considered typical of ischemia (swelling of astrocytic processes, distention of mitochondria, condensation of cytoplasm, "ischemic cell change") are most prominent after 30 min of reperfusion. At this time, dense calcium pyroantimonate deposits are evident in swollen mitochondria in pyramidal and hilar neurons. After 120 min of reperfusion, substantial restitution has occurred; most mitochondria appear normal and there are few calcium deposits. However, a small number of selectively vulnerable neurons (hilar and pyramidal neurons) show dense condensation (ischemic cell change) with multiple vacuoles containing calcium deposits. The role of excessive calcium entry and mitochondrial calcium overload during the reperfusion period in determining the death of selectively vulnerable neurons is discussed.

Altered expression of the neuropeptide-processing enzyme carboxypeptidase E in the rat brain after global ischemia.

Carboxypeptidase E, an exoprotease involved in the processing of bioactive peptides released by a regulated secretory pathway, was identified in a subtractive complementary DNA library derived from an ischemic rat brain by differential screening. In situ hybridization and immunocytochemical analysis showed the presence of carboxypeptidase E messenger RNA and protein in the cerebral cortex, thalamus, striatum, and hippocampus of a healthy rat brain. After 15 minutes of transient global ischemia followed by 8 hours of reperfusion, increased levels of carboxypeptidase E messenger RNA and protein were observed in the hippocampal CA1 and CA3 regions and in the cortex, as detected by Northern and Western blot analyses and in situ hybridization. After extended reperfusion (24 to 72 hours), both carboxypeptidase E messenger RNA and protein levels were decreased. The ischemia-induced changes in carboxypeptidase E expression suggest that this enzyme may play a role in modulating the brain's response to ischemia.

bcl-2 Antisense treatment prevents induction of tolerance to focal ischemia in the rat brain.

In the rat, 60 minutes of transient ischemia to the middle cerebral artery results in infarction of the caudate putamen. Ischemic preconditioning with 20 minutes of transient focal ischemia produced tolerance (attenuated infarction volume) to 60 minutes of subsequent focal ischemia administered three days, five days, or seven days later. Western blots from tolerant caudate putamen demonstrated increased bcl-2 expression, maximum at 3 days and persisting through 7 days. Immunocytochemical examination found that bcl-2 was expressed in cells with both neuronal and nonneuronal morphology in striatum after preconditioning ischemia. bcl-2 antisense oligodeoxynucleotides (ODNs), bcl-2 sense ODNs, or artificial cerebrospinal fluid (CSF, vehicle) was infused into the lateral ventricle for the 72 hours between the 20-minute ischemic preconditioning and the 60-minute period of ischemia. Antisense ODN treatment reduced expression of bcl-2 in the striatum and blocked the induction of tolerance by preconditioning ischemia. Sense and CSF treatments had no effect on either bcl-2 expression or tolerance. In this model of induced tolerance to focal ischemia, bcl-2 appears to be a major determinant.