We examined the expression, activation, and cellular localization of caspase-3 (CPP32) using immunohistochemistry, immunoblots, and cleavage of the fluorogenic substrate N-benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (zDEVD-afc) in adult mouse brain after temporary (2 hr) middle cerebral artery occlusion produced by filament insertion into the carotid artery. Immunoreactive caspase-3p32 but not its cleavage product caspase-3p20 was constitutively expressed in neurons throughout brain and was most prominent in neuronal perikarya within piriform cortex. Caspase-like enzyme activity was elevated in brain homogenate 0-3 hr after reperfusion and reached a peak within 30 to 60 min. Caspase-3p20 immunoreactivity became prominent in neuronal perikarya within the middle cerebral artery territory at the time of reperfusion and on immunoblots 1-12 hr later. DNA laddering (agarose gels) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-stained cells were detected 6-24 hr after reperfusion. At 12-24 hr, immunoreactive p20 was visualized in TUNEL-positive cells, a finding also observed in apoptotic mouse cerebellar granule cells on postnatal day 5. Together, these observations suggest the existence of a time-dependent evolution of ischemic injury characterized by the close correspondence between caspase-like enzyme activation and an associated increase in immunoreactive product (caspase-3p20) beginning at or before reperfusion and followed several hours later by morphological and biochemical features of apoptosis.

Poly(ADP-ribose)polymerase (PARP, EC 2.4.2.30), an abundant nuclear protein activated by DNA nicks, mediates cell death in vitro by nicotinamide adenine dinucleotide (NAD) depletion after exposure to nitric oxide. The authors examined whether genetic deletion of PARP (PARP null mice) or its pharmacologic inhibition by 3-aminobenzamide (3-AB) attenuates tissue injury after transient cerebral ischemia. Twenty-two hours after reperfusion following 2 hours of filamentous middle cerebral artery occlusion, ischemic injury was decreased in PARP-/- and PARP+/- mice compared with PARP+/+ litter mates, and also was attenuated in 129/SV wild-type mice after 3-AB treatment compared with controls. Infarct sparing was accompanied by functional recovery in PARP-/- and 3-AB-treated mice. Increased poly(ADP-ribose) immunostaining observed in ischemic cell nuclei 5 minutes after reperfusion was reduced by 3-AB treatment. Levels of NAD--the substrate of PARP--were reduced 2 hours after reperfusion and were 35% of contralateral levels at 24 hours. The decreases were attenuated in PARP-/- mice and in 3-AB-treated animals. Poly(ADP-ribose)polymerase cleavage by caspase-3 (CPP-32) has been proposed as an important step in apoptotic cell death. Markers of apoptosis, such as oligonucleosomal DNA damage, total DNA fragmentation, and the density of terminal deoxynucleotidyl transferase dUTP nick-end-labelled (TUNEL +) cells, however, did not differ in ischemic brain tissue of PARP-/- mice or in 3-AB-treated animals versus controls, although there were differences in the number of TUNEL-stained cells reflecting the decrease in infarct size. Thus, ischemic brain injury activates PARP and contributes to cell death most likely by NAD depletion and energy failure, although the authors have not excluded a role for PARP in apoptotic cell death at earlier or later stages in ischemic cell death. Inhibitors of PARP activation could provide a potential therapy in acute stroke.

MEK1 is a dual-specificity kinase that phosphorylates extracellular-signal-responsive kinase (ERK)1͞2 on threonine and tyrosine residues and activates these mitogen-activated protein (MAP) kinases (1). The ERK MAP kinases are phosphorylated in the hippocampus in response to global brain ischemia (2, 3). The use of general tyrosine kinase inhibitors, such as genistein, decreases ERK2 phosphorylation in this model and is associated with protection against neuronal cell damage (2). These data, however, do not establish a direct relationship between ERK2 phosphorylation and cell injury. Tyrosine kinase inhibitors, such as genistein, also inhibit protein kinaseindependent systems, such as fatty acid synthesis, mitochondrial oxidative phosphorylation, and lactate transport (4). It has been shown that inhibition of MEK1 protects hippocampal neurons in a cell-culture model of seizure (5). In this study, we show that inhibition of the MEK1͞ERK pathway leads to neuroprotection from brain injury resulting from occlusion of the middle cerebral artery (MCA). MethodsIschemia Model. Adult male SV-129 mice (18-22 g, Taconic Farms, Germantown, NY) were housed under diurnal lighting conditions and allowed food and water ad libitum. Animals were anesthetized with 1.5% halothane and maintained in 1.0% halothane in 70% N 2 O and 30% O 2 by using a Fluotec 3 vaporizer (Colonial Medical, Amherst, NH). Ischemia was induced with a 8.0 nylon monofilament coated with silicone resin͞hardener mixture (Xantopren and Elastomer Activator, Bayer Dental, Osaka, Japan) as described previously (6).Two microliters of PD98059, SB203580, or DMSO was injected intracerebroventricularly (bregma Ϫ0.9 mm lateral, Ϫ0.1 mm posterior, Ϫ3.1 mm deep) 30 min before ischemia by using a Hamilton injection syringe. The concentrations used are stated in Results.Immunohistochemistry. Mice were deeply anesthetized with an overdose of sodium pentobarbital (100 mg͞kg i.p.) and then transcardially perfused with 0.9% saline solution, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.4. The brains were quickly removed and stored in the same fresh buffer containing 20% sucrose. Brains were cut with a freezing microtome into coronal sections 40 m thick. The sections were processed by the free-floating method as described previously (7).Western Blot Analysis. Postischemic and nonischemic brains were Dounce homogenized in 1 ml of potassium phosphate buffer [10 mM potassium phosphate, pH 7.05͞1 mM EDTA͞5 mM EGTA͞10 mM magnesium chloride͞50 mM ␤-glycerophosphate͞1 mM sodium vanadate͞1 mM dithiothreitol (DTT)͞ 0.5% Nonidet P-40͞0.1% Brij-35]. Lysates were clarified by centrifugation at 14,000 ϫ g for 10 min. The protein concentration in the supernatant was determined by the Bradford assay (Bio-Rad). To test for phosphorylation of ERK1͞2, 40 g of total cell lysate was subjected to electrophoresis on an SDS͞10% polyacrylamide gel and transferred onto Immobilon-P membrane (Millipore). Western blotting was performed with phospho-specific p44͞42 MAP kinas...

Inhibitors of apoptosis and of excitotoxic cell death reduce brain damage after transient and permanent middle cerebral artery occlusion. We compared the neuroprotective effects of two caspase family inhibitors with the N-methyl-D-aspartate receptor antagonist (+)-MK-801 hydrogen maleate (MK-801) in a newly characterized cycloheximide-sensitive murine model of transient middle cerebral artery occlusion (30 minutes) in which apoptotic cell death is prominent. Ischemic infarction, undetected by 2,3,5-triphenyltetrazolium chloride staining at 24-hour reperfusion, featured prominently in the striatum at 72 hours and 7 days on hematoxylin-eosin-stained sections. Markers of apoptosis, such as oligonucleosomal DNA damage (laddering) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive cells first appeared at 24 hours and increased significantly at 72 hours and 7 days after reperfusion. The TUNEL-labeled cells were mostly neurons and stained negative for glial (GFAP, glial fibrillary acid protein) and leukocyte specific markers (CD-45). The caspase inhibitors, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.FMK; 120 ng intracerebroventricularly) or N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD.FMK; 480 ng intracerebroventricularly) decreased infarct size and neurologic deficits when administered 6 hours after reperfusion. The extent of protection was greater than in models of more prolonged ischemia or after permanent occlusion, and the therapeutic window was extended from 0 to 1 hours after 2-hour middle cerebral artery occlusion to at least 6 hours after brief ischemia. Also, z-VAD.FMK and z-DEVD.FMK treatment decreased oligonucleosomal DNA damage (DNA laddering) as assessed by quantitative autoradiography after gel electrophoresis. By contrast, MK-801 protected brain tissue only when given before ischemia (3 mg/kg intraperitoneally), but not at 3 or 6 hours after reperfusion. Despite a decrease in infarct size after MK-801 pretreatment, the amount of DNA laddering did not decrease 72 hours after reperfusion, thereby suggesting a mechanism distinct from inhibition of apoptosis. Hence, 30 minutes of reversible ischemia augments apoptotic cell death, which can be attenuated by delayed z-VAD.FMK and z-DEVD.FMK administration with preservation of neurologic function. By contrast, the therapeutic window for MK-801 does not extend beyond the time of occlusion, probably because its primary mechanism of action does not block the development of apoptotic cell death.

Brain subjected to acute ischemic attack caused by an arterial blockage needs immediate arterial recanalization. However, restoration of cerebral blood flow can cause tissue injury, which is termed reperfusion injury. It is important to inhibit reperfusion injury to achieve greater brain protection. Because oxidative stress has been shown to activate mitogen-activated protein kinases (MAPKs), and because oxidative stress contributes to reperfusion injury, MAPK may be a potential target to inhibit reperfusion injury after brain ischemia. Here, we demonstrate that reperfusion after forebrain ischemia dramatically increases phosphorylation level of extracellular signal-regulated kinase 2 (ERK2) in the gerbil hippocampus. In addition, i.v. administration of U0126 (100 -200 mg͞kg), a specific inhibitor of MEK (MAPK͞ERK kinase), protects the hippocampus against forebrain ischemia. Moreover, treatment with U0126 at 3 h after ischemia significantly reduces infarct volume after transient (3 h) focal cerebral ischemia in mice. This protection is accompanied by reduced phosphorylation level of ERK2, substrates for MEK, in the damaged brain areas. Furthermore, U0126 protects mouse primary cultured cortical neurons against oxygen deprivation for 9 h as well as nitric oxide toxicity. These results provide further evidence for the role of MEK͞ERK activation in brain injury resulting from ischemia͞reperfusion, and indicate that MEK inhibition may increase the resistance of tissue to ischemic injury. C ardiac arrest or cerebral arterial occlusion can cause a brain attack. Quickly restoring the cerebral blood flow is needed to stop brain injury. The most exciting new development in the field of stroke research is the recent approval of i.v. injection of tissue plasminogen activator that dissolves the blood clot (1, 2). Restoration of blood flow not only brings oxygen and nutrients into the damaged brain, but also produces free radicals such as reactive oxygen and reactive nitrogen species. These free radicals have been shown to contribute to oxidative injury. The tissue damage by the restoration of blood flow is termed reperfusion injury (3, 4). Thus, inhibition of reperfusion injury may be important to achieve greater brain protection.Mitogen-activated protein kinase (MAPK) family members, including extracellular signal-regulated kinases (ERK1͞2), p38 MAPK, and c-Jun N-terminal kinase (JNK), respond to various extracellular stimuli, thereby transmitting extracellular signals into the nucleus. ERK1͞2 are activated by MAPK͞ERK kinase1͞2 (MEK1͞2) by phosphorylating these MAPKs (5). The MEK͞ERK pathway plays a crucial role in cell growth and differentiation (6, 7). ERK1͞2 are constitutively expressed in the adult brain (8); however, little is known about the function of ERK1͞2 in postmitotic, terminally differentiated neurons. The MEK͞ERK pathway is also activated by reactive oxygen and reactive nitrogen species (9-11). Several in vivo studies showed that ERK1͞2 are phosphorylated in the damaged brain after ischemia, hypoglycemia, a...

Caspase-11, a member of the murine caspase family, has been shown to be an upstream activator of caspase-1 in regulating cytokine maturation. We demonstrate here that in addition to its defect in cytokine maturation, caspase-11–deficient mice have a reduced number of apoptotic cells and a defect in caspase-3 activation after middle cerebral artery occlusion (MCAO), a mouse model of stroke. Recombinant procaspase-11 can autoprocess itself in vitro. Purified active recombinant caspase-11 cleaves and activates procaspase-3 very efficiently. Using a positional scanning combinatorial library method, we found that the optimal cleavage site of caspase-11 was (I/L/V/P)EHD, similar to that of upstream caspases such as caspase-8 and -9. Our results suggest that caspase-11 is a critical initiator caspase responsible for the activation of caspase-3, as well as caspase-1 under certain pathological conditions.

DNA methylation is important for controlling the profile of gene expression and is catalyzed by DNA methyltransferase (MTase), an enzyme that is abundant in brain. Because significant DNA damage and alterations in gene expression develop as a consequence of cerebral ischemia, we measured MTase activity in vitro and DNA methylation in vivo after mild focal brain ischemia. After 30 min middle cerebral artery occlusion (MCAo) and reperfusion, MTase catalytic activity and the 190 kDa band on immunoblot did not change over time. However, [(3)H]methyl-group incorporation into DNA increased significantly in wild-type mice after reperfusion, but not in mutant mice heterozygous for a DNA methyltransferase gene deletion (Dnmt(S/+)). Dnmt(S/+) mice were resistant to mild ischemic damage, suggesting that increased DNA methylation is associated with augmented brain injury after MCA occlusion. Consistent with this formulation, treatment with the MTase inhibitor 5-aza-2'-deoxycytidine and the deacetylation inhibitor trichostatin A conferred stroke protection in wild-type mice. In contrast to mild stroke, however, DNA methylation was not enhanced, and reduced dnmt gene expression was not protective in an ischemia model of excitotoxic/necrotic cell death. In conclusion, our results demonstrate that MTase activity contributes to poor tissue outcome after mild ischemic brain injury.

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