Zeng Jin Yang

Sigma receptor ligand 4-phenyl-1-(4-phenylbutyl)-piperidine modulates neuronal nitric oxide synthase/postsynaptic density-95 coupling mechanisms and protects against neonatal ischemic degeneration of striatal neurons.

In adult stroke models, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), a sigma receptor agonist, attenuates activity of neuronal nitric oxide synthase (nNOS), blunts ischemia-induced nitric oxide production, and provides neuroprotection. Here, we tested the hypothesis that PPBP attenuates neuronal damage in a model of global hypoxia-ischemia (H-I) in newborn piglets. Piglets subjected to hypoxia followed by asphyxic cardiac arrest were treated with saline or two dosing regimens of PPBP after resuscitation. Sigma-1 receptors were found in striatal neurons. PPBP dose-dependently protected neurons in putamen at 4 days of recovery from H-I. Immunoblots of putamen extracts at 3 h of recovery showed that PPBP decreased H-I-induced recruitment of nNOS in the membrane fraction and reduced the association of nNOS with NMDA receptor NR2 subunit. The latter effect was associated with changes in the coupling of nNOS to postsynaptic density-95 (PSD-95), but not NR2-PSD-95 interactions. Moreover, PPBP suppressed NOS activity in the membrane fraction and reduced H-I-induced nitrative and oxidative damage to proteins and nucleic acids. These findings indicate that PPBP protects striatal neurons in a large animal model of neonatal H-I and that the protection is associated with decreased coupling of nNOS to PSD-95.

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.

Rewarming from therapeutic hypothermia induces cortical neuron apoptosis in a swine model of neonatal hypoxic-ischemic encephalopathy.

The consequences of therapeutic hypothermia for neonatal hypoxic–ischemic encephalopathy are poorly understood. Adverse effects from suboptimal rewarming could diminish neuroprotection from hypothermia. Therefore, we tested whether rewarming is associated with apoptosis. Piglets underwent hypoxia–asphyxia followed by normothermic or hypothermic recovery at 2 hours. Hypothermic groups were divided into those with no rewarming, rewarming at 0.5 °C/hour, or rewarming at 4 °C/hour. Neurodegeneration at 29 hours was assessed by hematoxylin and eosin staining, TUNEL assay, and immunoblotting for cleaved caspase-3. Rewarmed piglets had more apoptosis in motor cortex than did those that remained hypothermic after hypoxia–asphyxia. Apoptosis in piriform cortex was greater in hypoxic–asphyxic, rewarmed piglets than in naive/sham piglets. Caspase-3 inhibitor suppressed apoptosis with rewarming. Rapidly rewarmed piglets had more caspase-3 cleavage in cerebral cortex than did piglets that remained hypothermic or piglets that were rewarmed slowly. We conclude that rewarming from therapeutic hypothermia can adversely affect the newborn brain by inducing apoptosis through caspase mechanisms.

Noninvasive Autoregulation Monitoring in a Swine model of Pediatric Cardiac Arrest

BACKGROUND: Cerebrovascular autoregulation after resuscitation has not been well studied in an experimental model of pediatric cardiac arrest. Furthermore, developing noninvasive methods of monitoring autoregulation using near-infrared spectroscopy (NIRS) would be clinically useful in guiding neuroprotective hemodynamic management after pediatric cardiac arrest. We tested the hypotheses that the lower limit of autoregulation (LLA) would shift to a higher arterial blood pressure between 1 and 2 days of recovery after cardiac arrest and that the LLA would be detected by NIRS-derived indices of autoregulation in a swine model of pediatric cardiac arrest. We also tested the hypothesis that autoregulation with hypertension would be impaired after cardiac arrest. METHODS: Data on LLA were obtained from neonatal piglets that had undergone hypoxic–asphyxic cardiac arrest and recovery for 1 day (n = 8) or 2 days (n = 8), or that had undergone sham surgery with 2 days of recovery (n = 8). Autoregulation with hypertension was examined in a separate cohort of piglets that underwent hypoxic–asphyxic cardiac arrest (n = 5) or sham surgery (n = 5) with 2 days of recovery. After the recovery period, piglets were reanesthetized, and autoregulation was monitored by standard laser-Doppler flowmetry and autoregulation indices derived from NIRS (the cerebral oximetry [COx] and hemoglobin volume [HVx] indices). The LLA was determined by decreasing blood pressure through inflation of a balloon catheter in the inferior vena cava. Autoregulation during hypertension was evaluated by inflation of an aortic balloon catheter. RESULTS: The LLAs were similar between sham-operated piglets and piglets that recovered for 1 or 2 days after arrest. The NIRS-derived indices accurately detected the LLA determined by laser-Doppler flowmetry. The area under the curve of the receiver operator characteristic curve for cerebral oximetry index was 0.91 at 1 day and 0.92 at 2 days after arrest. The area under the curve for hemoglobin volume index was 0.92 and 0.89 at the respective time points. During induced hypertension, the static rate of autoregulation, defined as the percentage change in cerebrovascular resistance divided by the percentage change in cerebral perfusion pressure, was not different between postarrest and sham-operated piglets. At 2 days recovery from arrest, piglets exhibited neurobehavioral deficits and histologic neuronal injury. CONCLUSIONS: In a swine model of pediatric hypoxic–asphyxic cardiac arrest with confirmed brain damage, the LLA did not differ 1 and 2 days after resuscitation. The NIRS-derived indices accurately detected the LLA in comparison with laser-Doppler flow measurements at those time points. Autoregulation remained functional during hypertension.

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.

Attenuation of neonatal ischemic brain damage using a 20-HETE synthesis inhibitor

J. Neurochem. (2012) 121, 168–179.

Adenosine A2A receptor contributes to ischemic brain damage in newborn piglet

Pharmacologic inactivation or genetic deletion of adenosine A2A receptors protects ischemic neurons in adult animals, but studies in neonatal hypoxia-ischemia (H-I) are inconclusive. The present study in neonatal piglets examined the hypothesis that A2A receptor signaling after reoxygenation from global H-I contributes to injury in highly vulnerable striatal neurons where A2A receptors are enriched. A2A receptor immunoreactivity was detected in striatopallidal neurons. In nonischemic piglets, direct infusion of the selective A2A receptor agonist CGS 21680 through microdialysis probes into putamen increased phosphorylation of N-methyl-D-aspartic acid (NMDA) receptor NR1 subunit and Na+, K+-ATPase selectively at protein kinase A (PKA)-sensitive sites. In ischemic piglets, posttreatment with SCH 58261, a selective A2A receptor antagonist, improved early neurologic recovery and preferentially protected striatopallidal neurons. SCH 58261 selectively inhibited the ischemia-induced phosphorylation of NR1, Na+, K+-ATPase, and cAMP-regulated phosphoprotein 32 KDa (DARPP32) at PKA-sensitive sites at 3 hours of recovery and improved Na+, K+-ATPase activity. SCH 58261 also suppressed ischemia-induced protein nitration and oxidation. Thus, A2A receptor activation during reoxygenation contributes to the loss of a subpopulation of neonatal putamen neurons after H-I. Its toxic signaling may be related to DARPP32-dependent phosphorylation of PKA-sensitive sites on NR1 and Na+, K+-ATPase, thereby augmenting excitotoxicity-induced oxidative stress after reoxygenation.

Neuroprotective effect of acid-sensing ion channel inhibitor psalmotoxin-1 after hypoxia-ischemia in newborn piglet striatum

Na+,Ca2+-permeable acid-sensing ion channel 1a (ASIC1a) is involved in the pathophysiologic process of adult focal brain ischemia. However, little is known about its role in the pathogenesis of global cerebral ischemia or newborn hypoxia-ischemia (H-I). Here, using a newborn piglet model of asphyxia-induced cardiac arrest, we investigated the effect of ASIC1a-specific blocker psalmotoxin-1 on neuronal injury. During asphyxia and the first 30min of recovery, brain tissue pH fell below 7.0, the approximate activation pH of ASIC1a. Psalmotoxin-1 injection at 20min before hypoxia, but not at 20min of recovery, partially protected the striatonigral and striatopallidal neurons in putamen. Psalmotoxin-1 pretreatment largely attenuated the increased protein kinase A-dependent phosphorylation of DARPP-32 and N-methyl-d-aspartate (NMDA) receptor NR1 subunit and decreased nitrative and oxidative damage to proteins at 3h of recovery. Pretreatment with NMDA receptor antagonist MK-801 also provided partial neuroprotection in putamen, and combined pretreatment with psalmotoxin-1 and MK-801 yielded additive neuroprotection. These results indicate that ASIC1a activation contributes to neuronal death in newborn putamen after H-I through mechanisms that may involve protein kinase A-dependent phosphorylation of NMDA receptor and nitrative and oxidative stress.

Striatal neuroprotection from neonatal hypoxia-ischemia in piglets by antioxidant treatment with EUK-134 or edaravone.

Striatal neurons are highly vulnerable to hypoxia-ischemia (HI) in term newborns. In a piglet model of HI, striatal neurons develop oxidative stress and organelle disruption by 3–6 h of recovery and ischemic cytopathology over 6–24 h of recovery. We tested the hypothesis that early treatment with the antioxidants EUK-134 (a manganese-salen derivative that acts as a scavenger of superoxide, hydrogen peroxide, nitric oxide or NO and peroxynitrite) or edaravone (MCI-186, a scavenger of hydroxyl radical and NO) protects striatal neurons from HI. Anesthetized newborn piglets were subjected to 40 min of hypoxia and 7 min of airway occlusion. At 30 min after resuscitation, the piglets received vehicle, EUK-134 or edaravone. Drug treatment did not affect arterial blood pressure, blood gases, blood glucose or rectal temperature. At 4 days of recovery, the density of viable neurons in the putamen of vehicle-treated piglets was 12 ± 6% (±SD) of sham-operated control density. Treatment with EUK-134 increased viability to 41 ± 17%, and treatment with edaravone increased viability to 39 ± 19%. In the caudate nucleus, neuronal viability was increased from 54 ± 11% in the vehicle group to 78 ± 15% in the EUK-134 group and to 73 ± 13% in the edaravone group. Antioxidant drug treatment accelerated recovery from neurologic deficits and decreased oxidative and nitrative damage to nucleic acids. Treatment with EUK-134 reduced the HI-induced formation of protein carbonyl groups and tyrosine nitration at 3 h of recovery. We conclude that systemic administration of antioxidant agents by 30 min after resuscitation from HI can reduce oxidative stress and salvage neurons in the highly vulnerable striatum in a large-animal model of neonatal HI. Therefore, oxidative stress is an important mechanism for this injury, and antioxidant therapy is a rational, mechanism-based approach to neuroprotection in the newborn brain.

Endonuclease G does not play an obligatory role in poly(ADP-ribose) polymerase-dependent cell death after transient focal cerebral ischemia

Activation of poly(ADP-ribose) polymerase (PARP) and subsequent translocation of apoptosis-inducing factor contribute to caspase-independent neuronal injury from N-methyl-d-aspartate, oxygen-glucose deprivation, and ischemic stroke. Some studies have implicated endonuclease G in the DNA fragmentation associated with caspase-independent cell death. Here, we compared wild-type and endonuclease G null mice to investigate whether endonuclease G plays a role in the PARP-dependent injury that results from transient focal cerebral ischemia. Latex casts did not reveal differences in the cerebral arterial distribution territory or posterior communicating arterial diameter, and the decrease in laser-Doppler flux during middle cerebral artery occlusion was similar in wild-type and endonuclease G null mice. After 90 min of occlusion and 1 day of reperfusion, similar degrees of nuclear translocation of apoptosis-inducing factor and DNA degradation were evident in male wild-type and null mice. At 3 days of reperfusion, infarct volume and neurological deficit scores were not different between male wild-type and endonuclease G null mice or between female wild-type and endonuclease G null mice. These data demonstrate that endonuclease G is not required for the pathogenesis of transient focal ischemia in either male or female mice. Treatment with a PARP inhibitor decreased infarct volume and deficit scores equivalently in male wild-type and endonuclease G null mice, indicating that the injury in endonuclease G null mice remains dependent on PARP. Thus endonuclease G is not obligatory for executing PARP-dependent injury during ischemic stroke.