Judith A. Klaus

Poly(ADP-ribose) (PAR) polymer is a death signal

Excessive activation of the nuclear enzyme, poly(ADP-ribose) polymerase-1 (PARP-1) plays a prominent role in various of models of cellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer, a product of PARP-1 activity, as a previously uncharacterized cell death signal. PAR polymer is directly toxic to neurons, and degradation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG) or phosphodiesterase 1 prevents PAR polymer-induced cell death. PARP-1-dependent, NMDA excitotoxicity of cortical neurons is reduced by neutralizing antibodies to PAR and by overexpression of PARG. Neuronal cultures with reduced levels of PARG are more sensitive to NMDA excitotoxicity than WT cultures. Transgenic mice overexpressing PARG have significantly reduced infarct volumes after focal ischemia. Conversely, mice with reduced levels of PARG have significantly increased infarct volumes after focal ischemia compared with WT littermate controls. These results reveal PAR polymer as a signaling molecule that induces cell death and suggests that interference with PAR polymer signaling may offer innovative therapeutic approaches for the treatment of cellular injury.

Quantitative description of proton exchange processes between water and endogenous and exogenous agents for WEX, CEST, and APT experiments.

The proton exchange processes between water and solutes containing exchangeable protons have recently become of interest for monitoring pH effects, detecting cellular mobile proteins and peptides, and enhancing the detection sensitivity of various low‐concentration endogenous and exogenous species. In this work, the analytic expressions for water exchange (WEX) filter spectroscopy, chemical exchange‐dependent saturation transfer (CEST), and amide proton transfer (APT) experiments are derived by the use of Bloch equations with exchange terms. The effects of the initial states for the system, the difference between a steady state and a saturation state, and the relative contributions of the forward and backward exchange processes are discussed. The theory, in combination with numerical calculations, provides a useful tool for designing experimental schemes and assessing magnetization transfer (MT) processes between water protons and solvent‐exchangeable protons. As an example, the case of endogenous amide proton exchange in the rat brain at 4.7 T is analyzed in detail. Magn Reson Med 51:945–952, 2004.

Neuroprotection and P450 2C11 Upregulation After Experimental Transient Ischemic Attack

Background and Purpose— Transient ischemic attack (TIA) is a risk factor for stroke. However, TIA may also serve as a preconditioning stimulus, reducing damage from subsequent stroke. We tested the hypothesis that experimental TIA induces expression of P450 2C11, an arachidonic acid epoxygenase that produces vasodilator epoxyeicosatrienoic acids, leading to increased tissue perfusion and reduced stroke damage. Methods— Wistar rats underwent three 10-minute middle cerebral artery occlusions (TIA) or sham surgery. Three days later, animals were subjected to 2-hour middle cerebral artery occlusion and 24 hours of reperfusion. Brains were stained with 2,3,5-triphenyltetrazolium chloride for infarct size measurement or processed for quantification of P450 2C11 mRNA and protein with the use of RNase protection assay and Western blotting. Regional cerebral blood flow (CBF) at the end of 2-hour ischemia was measured in separate groups of rats with iodoantipyrine autoradiography. Results— Cerebral infarct was reduced by >50% in TIA- versus sham-preconditioned animals. 2C11 mRNA and protein were increased in ipsilateral hemisphere by 3 days after TIA but not sham surgery. Induction of 2C11 by TIA was also evident in ipsilateral hemisphere at 24 hours after 2-hour middle cerebral artery occlusion and 24 hours of reperfusion. End-ischemic regional CBF was not different between TIA- and sham-pretreated groups. Conclusions— We conclude that experimental TIA induces ischemic tolerance by a mechanism temporally linked to upregulation of P450 2C11. Enzyme induction does not attenuate ischemic severity by amplifying end-ischemic CBF.

Role of cocaine- and amphetamine-regulated transcript in estradiol-mediated neuroprotection.

Estrogen reduces brain injury after experimental cerebral ischemia in part through a genomic mechanism of action. Using DNA microarrays, we analyzed the genomic response of the brain to estradiol, and we identified a transcript, cocaine- and amphetamine-regulated transcript (CART), that is highly induced in the cerebral cortex by estradiol under ischemic conditions. Using in vitro and in vivo models of neural injury, we confirmed and characterized CART mRNA and protein up-regulation by estradiol in surviving neurons, and we demonstrated that i.v. administration of a rat CART peptide is protective against ischemic brain injury in vivo. We further demonstrated binding of cAMP response element (CRE)-binding protein to a CART promoter CRE site in ischemic brain and rapid activation by CART of ERK in primary cultured cortical neurons. The findings suggest that CART is an important player in estrogen-mediated neuroprotection and a potential therapeutic agent for stroke and other neurodegenerative diseases.

Contributions of poly(ADP-ribose) polymerase-1 and -2 to nuclear translocation of apoptosis-inducing factor and injury from focal cerebral ischemia

J. Neurochem. (2010) 113, 1012–1022.

Cytosolic phospholipase A2 alpha amplifies early cyclooxygenase-2 expression, oxidative stress and MAP kinase phosphorylation after cerebral ischemia in mice.

BackgroundThe enzyme cytosolic phospholipase A2 alpha (cPLA2α) has been implicated in the progression of cerebral injury following ischemia and reperfusion. Previous studies in rodents suggest that cPLA2α enhances delayed injury extension and disruption of the blood brain barrier many hours after reperfusion. In this study we investigated the role of cPLA2α in early ischemic cerebral injury.MethodsMiddle cerebral artery occlusion (MCAO) was performed on cPLA2α+/+ and cPLA2α-/- mice for 2 hours followed by 0, 2, or 6 hours of reperfusion. The levels of cPLA2α, cyclooxygenase-2, neuronal morphology and reactive oxygen species in the ischemic and contralateral hemispheres were evaluated by light and fluorescent microscopy. PGE2 content was compared between genotypes and hemispheres after MCAO and MCAO and 6 hours reperfusion. Regional cerebral blood flow was measured during MCAO and phosphorylation of relevant MAPKs in brain protein homogenates was measured by Western analysis after 6 hours of reperfusion.ResultsNeuronal cPLA2α protein increased by 2-fold immediately after MCAO and returned to pre-MCAO levels after 2 hours reperfusion. Neuronal cyclooxygenase-2 induction and PGE2 concentration were greater in cPLA2α+/+ compared to cPLA2α-/- ischemic cortex. Neuronal swelling in ischemic regions was significantly greater in the cPLA2α+/+ than in cPLA2α-/- brains (+/+: 2.2 ± 0.3 fold vs. -/-: 1.7 ± 0.4 fold increase; P < 0.01). The increase in reactive oxygen species following 2 hours of ischemia was also significantly greater in the cPLA2α+/+ ischemic core than in cPLA2α-/- (+/+: 7.12 ± 1.2 fold vs. -/-: 3.1 ± 1.4 fold; P < 0.01). After 6 hours of reperfusion ischemic cortex of cPLA2α+/+, but not cPLA2α-/-, had disruption of neuron morphology and decreased PGE2 content. Phosphorylation of the MAPKs-p38, ERK 1/2, and MEK 1/2-was significantly greater in cPLA2a+/+ than in cPLA2α-/- ischemic cortex 6 hours after reperfusion.ConclusionsThese results indicate that cPLA2α modulates the earliest molecular and injury responses after cerebral ischemia and have implications for the potential clinical use of cPLA2α inhibitors.

Anticonvulsant Lamotrigine Administered on Reperfusion Fails To Improve Experimental Stroke Outcomes

Background and Purpose— Recent results suggest that selective inhibitors of presynaptic neuronal ion channels can diminish glutamate release during cerebral ischemia and modulate excitotoxic cell death. The aim of the present study was to evaluate lamotrigine (LTG), an antiepileptic that inhibits presynaptic sodium and voltage-sensitive calcium channels, as a potential stroke resuscitation agent in the rat. Three dosages of LTG were examined for effect on infarction volume and sensorimotor behavioral recovery after middle cerebral artery (MCA) occlusion. Methods— Halothane-anesthetized male Wistar rats were subjected to 2 hours of MCA occlusion by the intraluminal occlusion technique. Physiological variables were controlled, and ipsilateral cortical perfusion was monitored by laser Doppler flowmetry throughout ischemia. At onset of reperfusion, rats received intravenous LTG 5, 10, or 20 mg/kg or PBS (n=9 to 11 per group) during 15 minutes. Behavioral assessment was completed at 3 and 7 days after stroke, and the brain was harvested for histology (triphenyltetrazolium chloride staining). Results— Values are mean±SE. Cortical infarction volumes were unchanged in LTG-treated animals: 14±6% of contralateral cortex at 5 mg/kg LTG, 17±7% at 10 mg/kg, and 30±6% at 20 mg/kg, versus saline-treated cohorts (12±3%;P =0.19; n=9). Caudate-putamen infarction injury was also unchanged (37±11% of contralateral caudate-putamen at 5 mg/kg LTG, 44±8% at 10 mg/kg, and 65±9% at 20 mg/kg versus saline (38±11%;P =0.18). Total infarction was not different among groups (P =0.15). Consistent with histology, behavioral outcomes were unimproved by treatment. Conclusions— Histological damage and behavioral recovery at 7 days after MCA occlusion was not altered by LTG treatment over the dosage range used in the present study.

Delayed Tolerance With Repetitive Transient Focal Ischemic Preconditioning in the Mouse

Background and Purpose— Transient ischemic attacks have long been regarded as a risk factor for the incidence of stroke but may reduce the severity of stroke by inducing ischemic tolerance. The present objective was to develop an ischemic preconditioning (IPC) model of delayed tolerance in the mouse based on repetitive, transient middle cerebral artery occlusion (MCAO). Methods— Mice anesthetized with halothane or isoflurane underwent IPC, which consisted of repetitive MCAO at 45-minute intervals by the intraluminal filament technique. A 90-minute test MCAO was performed 24 to 96 hours later. Results— Using an IPC of 2 5-minute MCAO episodes, the reduction in infarct volume from the test MCAO was maximal with a 72-hour delay in striatum (70%) and cerebral cortex (64%) when halothane was used for surgical anesthesia. With isoflurane anesthesia, the reduction in infarct volume was less prominent in striatum (34%) and not significant in cortex (9%) despite similar levels of arterial pressure and decreases in cortical perfusion. Neuronal cell death was rare 6 days after this IPC stimulus alone with halothane or isoflurane. Increasing the severity of IPC to 3 5-minute bouts or 1 15-minute bout of MCAO in the presence of isoflurane anesthesia augmented the reduction in infarct volume in striatum and cortex, but it also augmented selective neuronal cell death in striatum after the IPC stimulus alone. Conclusions— These data demonstrate that a repetitive focal IPC stimulus can be titrated to induce delayed tolerance in both striatum and cortex of the mouse without inducing neuronal death by itself.

Early Treatment of Transient Focal Cerebral Ischemia with Bovine PEGylated Carboxy Hemoglobin Transfusion

Abstract: The effect of transfusion of PEGylated hemoglobin (PEG-Hb) was evaluated in anesthetized rats subjected to 2 hours of focal cerebral ischemia and 1 day of reperfusion. PEG-Hb was stored in the carboxy state (PEG-COHb) to reduce autooxidation and increase the shelf life. Transfusion of 10 ml/kg of PEG-COHb at 20 minutes of ischemia did not alter arterial blood pressure or increase red cell flux in the ischemic core. Plasma hemoglobin increased to only 0.6 g/dL, yet infarct volume was markedly decreased and neurological deficits were improved. We conclude that early topload transfusion of PEG-COHb protects the brain from ischemic stroke.