Expression, proteolysis and activation of caspases 6 and 7 during rat C6 glioma cell apoptosis.

Activation of cysteinyl aspartate-specific proteases (caspases) may underlie apoptotic cell death in brain. Terminal, executioner caspases 3, 6 and 7 likely contribute to such cell death in a stimulus- and cell type-specific manner. Here we investigate the processing and activation of caspases 3, 6 and 7 in rat C6 glioma cells induced to undergo apoptosis by staurosporine (STS) treatment as a model of apoptosis in glia.

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 bid may amplify caspase-8-induced neuronal death following focally evoked limbic seizures.

The mechanism by which seizures induce neuronal death is not completely understood. Caspase-8 is a key initiator of apoptosis via extrinsic, death receptor-mediated pathways; we therefore investigated its role in mediating seizure-induced neuronal death evoked by unilateral kainic acid injection into the amygdala of the rat, terminated after 40 min by diazepam. We demonstrate that cleaved (p18) caspase-8 was detectable immediately following seizure termination coincident with an increase in cleavage of the substrate Ile-Glu-Thr-Asp (IETD)-p-nitroanilide and the appearance of cleaved (p15) Bid.

Increased Bcl-w expression following focally evoked limbic seizures in the rat.

Control of seizure-induced neuronal death may involve members of the Bcl-2 family of cell death regulating proteins. Bcl-w is a newly described anti-apoptotic member of this family that may confer neuroprotective effects. We therefore investigated Bcl-w expression in rat brain following focally evoked limbic seizures.

Caspase-2 activation is redundant during seizure-induced neuronal death.

Seizure-induced neuronal death may be under the control of the caspase family of cell death proteases. We examined the role of caspase-2 in a model of focally evoked limbic seizures with continuous EEG recording. Seizures were elicited by microinjection of kainic acid into the amygdala of the rat and terminated after 40 min by diazepam.

Induction of oxidative DNA damage in the peri-infarct region after permanent focal cerebral ischemia.

To address the role of oxidative DNA damage in focal cerebral ischemia lacking reperfusion, we investigated DNA base and strand damage in a rat model of permanent middle cerebral artery occlusion (MCAO). Contents of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and apurinic/apyrimidinic abasic sites (AP sites), hallmarks of oxidative DNA damage, were quantitatively measured in nuclear DNA extracts from brains obtained 4-72 h after MCAO. DNA single- and double-strand breaks were detected on coronal brain sections using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), respectively.

Alterations in bcl-2 and caspase gene family protein expression in human temporal lobe epilepsy.

Objective: To address the role of cell death regulatory genes of the bcl-2 and caspase families in the neuropathology of human epilepsy using tissue extracted from patients undergoing temporal lobectomy for intractable seizures.

Methods: Using Western blotting and immunohistochemistry, the authors investigated the expression of bcl-2, bcl-xL, bax, caspase-1,and caspase-3 in temporal cortex samples from patients who had undergone temporal lobectomy surgery for intractable epilepsy (n = 19). Nonepileptic postmortem tissue from a brain bank served as control (n = 6).

Activation of poly(ADP-ribose) polymerase in the rat hippocampus may contribute to cellular recovery following sublethal transient global ischemia.

We have investigated the role of poly(ADP-ribose) polymerase (PARP) activation in rat brain in a model of sublethal transient global ischemia. Adult male rats were subjected to 15 min of ischemia with brain temperature reduced to 34 degrees C, followed by 1, 2, 4, 8, 16, 24, and 72 h of reperfusion. PARP mRNA expression was examined in the hippocampus using quantitative RT-PCR, northern blot analysis, and in situ hybridization.

Spatio-temporal profile of DNA fragmentation and its relationship to patterns of epileptiform activity following focally evoked limbic seizures.

The specific electrographic activity responsible for seizure-induced DNA damage remains little explored. We therefore examined the regional and temporal appearance of DNA fragmentation and cell death and its relationship to specific electrographic seizure patterns in a rat model of focally evoked limbic epilepsy. Animals received intra-amygdaloid injection of kainic acid (KA) to induce seizures for 45 min during continuous electroencephalographic (EEG) monitoring, after which diazepam (30 mg/kg) was administered.

Involvement of caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures.

The cysteine protease caspase-3 may be involved in the mechanism of cell death following seizures. Using a rat model of focally evoked limbic epilepsy with continuous electroencephalography monitoring, we investigated seizure-induced changes in caspase-3 protein expression and processing, enzyme activity, and the in vivo effect of caspase-3 inhibition. Seizures were induced by intraamygdaloid injection of kainic acid (0.

Bcl-w expression is increased in brain regions affected by focal cerebral ischemia in the rat.

Proteins of the bcl-2 family are important regulators of apoptosis in many tissues of the embryo and adult and may play a role in cell death following stroke. The recently isolated bcl-w gene encodes a pro-survival member of the bcl-2 family, which is widely expressed. However, it is not known whether bcl-w plays a role in determining cell survival after cerebral ischemia.

Formation of the base modification 8-hydroxyl-2'-deoxyguanosine and DNA fragmentation following seizures induced by systemic kainic acid in the rat.

The formation of oxidative DNA damage as a consequence of seizures remains little explored. We therefore investigated the regional and temporal profile of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) formation, a hallmark of oxidative DNA damage and DNA fragmentation in rat brain following seizures induced by systemic kainic acid (KA). Formation of 8-OHdG was determined via HPLC with electrochemical detection, and single- and double-stranded DNA breaks were detected using in situ DNA polymerase I-mediated biotin-dATP nick-translation (PANT) and terminal deoxynucleotidyl-transferase-mediated nick end-labeling (TUNEL), respectively.

A rat model of endothelin-3-induced middle cerebral artery occlusion with controlled reperfusion.

Surge hyperemia and mechanical damage to the cerebrovascular endothelium may serve to exacerbate the neuropathological outcome in animal models of focal cerebral ischemia. We have modified an existing model of endothelin-1-induced middle cerebral artery (MCA) occlusion to enable controlled reperfusion without damage to the cerebral vasculature. Endothelin-1 (ET-1) and endothelin-3 (ET-3) were injected via a double-injection cannula into brain parenchyma adjacent to the MCA of anesthetized rats to produce focal cerebral ischemia.

Relationship between seizure-induced transcription of the DNA damage-inducible gene GADD45, DNA fragmentation, and neuronal death in focally evoked limbic epilepsy.

We investigated the temporal and spatial profile of mRNA transcription for the growth arrest and DNA damage-inducible gene GADD45, DNA fragmentation, and neuronal death in rat brain following focally evoked limbic seizures. GADD45 mRNA was detected by in situ hybridization, whereas fragmented DNA was detected using in situ nick end-labeling by the large (Klenow) fragment of DNA polymerase I. Kainic acid (0.

Parallel dose-response studies of the voltage-dependent Na+ channel antagonist BW619C89, and the voltage-dependent Ca2+ channel antagonist nimodipine, in rat transient focal cerebral ischaemia.

We have compared two classes of putative neuroprotectants, the voltage-dependent Na+ channel antagonist BW619C87 [4-amino-2-(4-methyl-1-piperazinyl)-5-(2,3,5-trichlorophenyl) pyrimidine], and the voltage-dependent Ca2+ channel antagonist nimodipine, in a rat model of transient focal cerebral ischaemia. BW619C87 (10-50 mg/kg) or nimodipine (10-100 microg/kg) were injected intravenously 5 min before induction of 2 h transient focal cerebral ischaemia via intraluminal thread occlusion of the middle cerebral artery. BW619C87 was a potent neuroprotectant over the range tested, maximally reducing the volume of hemispheric ischaemic damage by 51% at the 50 mg/kg dose.

Bilateral intranigral NMDA infusion suppresses neuronal injury without affecting the duration of kainic acid-induced seizures in rats.

The substantia nigra pars reticulata (SNpr) is recognized as an important modulator of seizures within the limbic system. We have investigated the effects of N-methyl-D-aspartate (NMDA) infusion into SNpr upon seizure-related neuronal injury (assessed by expression of the 72-kDa heat shock protein - HSP 72) induced by systemic kainic acid (KA) in rats. Three to four days following implantation of guide cannulae for drug administration into SNpr, KA (7 mg/kg) was injected intravenously to induce seizures.

Neuroprotective actions of FK506 in experimental stroke: in vivo evidence against an antiexcitotoxic mechanism.

The cellular mechanisms underlying the neuroprotective action of the immunosuppressant FK506 in experimental stroke remain uncertain, although in vitro studies have implicated an antiexcitotoxic action involving nitric oxide and calcineurin. The present in vivo study demonstrates that intraperitoneal pretreatment with 1 and 10 mg/kg FK506, doses that reduced the volume of ischemic cortical damage by 56-58%, did not decrease excitotoxic damage induced by quinolinate, NMDA, and AMPA. Similarly, intravenous FK506 did not reduce the volume of striatal quinolinate lesions at a dose (1 mg/kg) that decreased ischemic cortical damage by 63%.