White matter apoptosis is increased by delayed hypothermia and rewarming in a neonatal piglet model of hypoxic ischemic encephalopathy.

Therapeutic hypothermia is widely used to treat neonatal hypoxic ischemic (HI) brain injuries. However, potentially deleterious effects of delaying the induction of hypothermia and of rewarming on white matter injury remain unclear. We used a piglet model of HI to assess the effects of delayed hypothermia and rewarming on white matter apoptosis. Piglets underwent HI injury or sham surgery followed by normothermic or hypothermic recovery at 2h. Hypothermic groups were divided into those with no rewarming, slow rewarming at 0.5°C/h, or rapid rewarming at 4°C/h. Apoptotic cells in the subcortical white matter of the motor gyrus, corpus callosum, lateral olfactory tract, and internal capsule at 29h were identified morphologically and counted by hematoxylin & eosin staining. Cell death was verified by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay. White matter neurons were also counted, and apoptotic cells were immunophenotyped with the oligodendrocyte marker 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase). Hypothermia, slow rewarming, and rapid rewarming increased apoptosis in the subcortical white matter relative to normothermia (p<0.05). The number of white matter neurons was not lower in groups with more apoptosis after hypothermia or rapid rewarming, indicating that the apoptosis occurred among glial cells. Hypothermic piglets had more apoptosis in the lateral olfactory tract than those that were rewarmed (p<0.05). The promotion of apoptosis by hypothermia and rewarming in these regions was independent of HI. In the corpus callosum, HI piglets had more apoptosis than shams after normothermia, slow rewarming, and rapid rewarming (p<0.05). Many apoptotic cells were myelinating oligodendrocytes identified by CNPase positivity. Our results indicate that delaying the induction of hypothermia and rewarming are associated with white matter apoptosis in a piglet model of HI; in some regions these temperature effects are independent of HI. Vulnerable cells include myelinating oligodendrocytes. This study identifies a deleterious effect of therapeutic hypothermia in the developing brain.

Apparent diffusion coefficient scalars correlate with near-infrared spectroscopy markers of cerebrovascular autoregulation in neonates cooled for perinatal hypoxic-ischemic injury.

BACKGROUND AND PURPOSE: Neurologic morbidity remains high in neonates with perinatal hypoxic-ischemic injury despite therapeutic hypothermia. DTI provides qualitative and quantitative information about the microstructure of the brain, and a near-infrared spectroscopy index can assess cerebrovascular autoregulation. We hypothesized that lower ADC values would correlate with worse autoregulatory function. MATERIALS AND METHODS: Thirty-one neonates with hypoxic-ischemic injury were enrolled. ADC scalars were measured in 27 neonates (age range, 4-15 days) in the anterior and posterior centrum semiovale, basal ganglia, thalamus, posterior limb of the internal capsule, pons, and middle cerebellar peduncle on MRI obtained after completion of therapeutic hypothermia. The blood pressure range of each neonate with the most robust autoregulation was identified by using a near-infrared spectroscopy index. Autoregulatory function was measured by blood pressure deviation below the range with optimal autoregulation. RESULTS: In neonates who had MRI on day of life ≥10, lower ADC scalars in the posterior centrum semiovale (r = -0.87, P = .003, n = 9) and the posterior limb of the internal capsule (r = -0.68, P = .04, n = 9) correlated with blood pressure deviation below the range with optimal autoregulation during hypothermia. Lower ADC scalars in the basal ganglia correlated with worse autoregulation during rewarming (r = -0.71, P = .05, n = 8). CONCLUSIONS: Blood pressure deviation from the optimal autoregulatory range may be an early biomarker of injury in the posterior centrum semiovale, posterior limb of the internal capsule, and basal ganglia. Optimizing blood pressure to support autoregulation may decrease the risk of brain injury in cooled neonates with hypoxic-ischemic injury.

Prostacyclin receptor deletion aggravates hippocampal neuronal loss after bilateral common carotid artery occlusion in mouse.

Transient global cerebral ischemia causes delayed neuronal death in the hippocampal CA1 region. It also induces an increase in cyclooxygenase 2 (COX-2), which generates several metabolites of arachidonic acid, known as prostanoids, including prostacyclin (PGI(2)). To determine the role of the PGI(2) receptor (IP) in post-ischemic delayed cell death, wild-type and IP knockout (IP(-/-)) C57Bl/6 mice were subjected to 12-min bilateral common carotid artery occlusion or sham surgery, followed by 7 days of reperfusion. In the sham-operated mice, no statistical difference in CA1 hippocampal neuronal density was observed between the wild-type (2836+/-18/mm(2)) and IP(-/-) (2793+/-43/mm(2)) mice. Interestingly, in animals subjected to ischemia, surviving neuronal density in wild-type mice decreased to 50.5+/-7.9% and that of IP(-/-) mice decreased to 23.0+/-4.5% of their respective sham-operated controls (P<0.05). The results establish a role for the IP receptor in protecting pyramidal hippocampal neurons after this global ischemic model and suggest that IP receptor agonists could be developed to prevent delayed pyramidal neuronal cell death.

Blood viscosity modulates tissue perfusion: sometimes and somewhere.

Each organ possesses specific properties for controlling microvascular perfusion. Such specificity provides an opportunity to design transfusion fluids that target thrombo-embolic or vasospasm-induced ischemia in a particular organ or that optimize overall perfusion from systemic shock. The role of viscosity in the design of these fluids might be underestimated, because viscosity is rarely monitored or considered in critical care decisions. Studies linking viscosity-dependent changes of microvascular perfusion to outcome-relevant data suggest that whole blood viscosity is negligible as a determinant of microvascular perfusion under physiological conditions when autoregulation is effective. Because autoregulation is driven to maintain oxygen supply constant, the organism will compensate for changes in blood viscosity to sustain oxygen delivery. In contrast, under pathological conditions in the brain and elsewhere, increases of overall viscosity should be avoided - including all the situations where vascular autoregulatory mechanisms are inoperative due to ischemia, structural damage or physiologic dysfunction. As latter conditions are not to identify with high certainty, the risks that accompany therapeutic correction of blood viscosity are outweighing the benefits. The ability to bedside monitor blood viscosity and to link changes in viscosity to outcome parameters in various clinical conditions would provide more solid foundation for evidence-based clinical management.

Influence of duration of focal cerebral ischemia and neuronal nitric oxide synthase on translocation of apoptosis-inducing factor to the nucleus.

Translocation of apoptosis-inducing factor (AIF) from the mitochondria to the nucleus can play a major role in neuronal death elicited by oxidant stress. The time course of nuclear translocation of AIF after experimental stroke may vary with the severity of injury and may be accelerated by oxidant stress associated with reperfusion and nitric oxide (NO) production. Western immunoblots of AIF on nuclear fractions of ischemic hemisphere of male mice showed no significant increase with 1 h of middle cerebral artery occlusion and no reperfusion, whereas increases were detectable after 6 and 24 h of permanent ischemia. However, as little as 20 min of reperfusion after 1 h of middle cerebral artery occlusion resulted in an increase in nuclear AIF coincident with an increase in poly(ADP-ribose) polymer (PAR) formation. Further nuclear AIF accumulation was seen at 6 and 24 h of reperfusion. In contrast, 20 min of reperfusion after 2 h of occlusion did not increase nuclear AIF. In this case, nuclear AIF became detectable at 6 and 24 h of reperfusion. With brief occlusion of 30 min duration, nuclear AIF remained undetectable at both 20 min and 6 h and became evident only after 24 h of reperfusion. Inhibition of neuronal NO synthase attenuated formation of PAR and nuclear AIF accumulation. Gene deletion of neuronal NO synthase also attenuated nuclear AIF accumulation. Therefore, reperfusion accelerates AIF translocation to the nucleus when focal ischemia is of moderate duration (1 h), but is markedly delayed after brief ischemia (30 min). Nuclear translocation of AIF eventually occurs with prolonged focal ischemia with or without reperfusion. Neuronally-derived NO is a major factor contributing to nuclear AIF accumulation after stroke.

Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca2+ -activated K+ channel current in cultured rat hippocampal astrocytes.

Brief hypoxia differentially regulates the activities of Ca(2+)-activated K(+) channels (K(Ca)) in a variety of cell types. We investigated the effects of hypoxia (<2% O(2)) on K(Ca) channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K(Ca) current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K(Ca) currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K(Ca) channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K(Ca) current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca(2+) external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K(Ca) channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K(Ca) currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K(Ca) single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K(Ca) currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.

Effect of glutamine synthetase inhibition on astrocyte swelling and altered astroglial protein expression during hyperammonemia in rats.

Inhibition of glutamine synthesis reduces astrocyte swelling and associated physiological abnormalities during acute ammonium acetate infusion in anesthetized rats. We tested the hypothesis that inhibition of glutamine accumulation during more prolonged ammonium acetate infusion in unanesthetized rats reduces cortical astrocyte swelling and immunohistochemical changes in astrocytic proteins. Rats received a continuous i.v. infusion of either sodium acetate or ammonium acetate for 24 h to increase plasma ammonia from about 30-400 mumol/l. Cohorts were pretreated with vehicle or l-methionine-S-sulfoximine (MSO; 0.83 mmol/kg). MSO reduced glutamine synthetase activity by 57% and glutamine synthetase immunopositive cell number by 69%, and attenuated cortical glutamine accumulation by 71%. Hyperammonemia increased the number of swollen astrocytes in cortex and MSO reduced this increase to control values. The number of glial fibrillary acidic protein immunopositive cells in cortex was greater in hyperammonemic rats and the increase in superficial cortical layers was attenuated by MSO. Immunoreactivity for the gap junction protein connexin-43 in the neuropil, assessed by optical density, was greater in the hyperammonemic group compared with controls, but this increase was not attenuated by MSO. No changes in the optical density of GLT1 glutamate transporter immunoreactivity in cortex were detected in any group. We conclude that glutamine synthetase inhibition reduces astrocyte swelling and ameliorates some of the reactive astroglial cytoskeletal alterations seen at 24 h of hyperammonemia, but that gap junction changes in astrocytes occur independently of glutamine accumulation and swelling.

Fetal cerebral and peripheral circulatory responses to hypoxia after nitric oxide synthase inhibition.

The increase in cerebral blood flow (CBF) during hypoxia in fetal sheep at 0.6 gestation is less than the increase at 0.9 gestation when normalized for differences in baseline CBF and oxygen consumption. Nitric oxide (NO) synthase (NOS) catalytic activity increases threefold during this period of development. We tested the hypothesis that administration of the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) decreases the CBF response to systemic hypoxia selectively at 0.9 gestation. We also tested whether any peripheral vasoconstriction during hypoxia is potentiated by L-NAME at 0.9 gestation. Administration of L-NAME increased arterial blood pressure and decreased microsphere-determined CBF during normoxia in fetal sheep at both 0.6 and 0.9 gestation. With subsequent reduction of arterial oxygen content by approximately 50%, the percent increase in forebrain CBF in a control group (57 +/- 11%; +/- SE) and L-NAME-treated group (51 +/- 6%) was similar at 0.6 gestation. Likewise, at 0.9 gestation, the increase in CBF was similar in control (90 +/- 25%) and L-NAME (80 +/- 28%) groups. At 0.9 gestation, L-NAME treatment attenuated the increase in coronary blood flow and increased gastrointestinal vascular resistance during hypoxia. We conclude that NO exerts a basal vasodilatory influence in brain as early as 0.6 gestation in fetal sheep but is not an important mechanism for hypoxic vasodilation in brain at either 0.6 or 0.9 gestation. Thus the developmental increase in NOS catalytic capacity does not appear to be responsible for developmental increases in the CBF response to hypoxia during this period. In contrast, NO modulates the vascular response to hypoxia in heart and gastrointestinal tract.

Blood-brain barrier permeability during dopamine-induced hypertension in fetal sheep.

Dopamine is often used as a pressor agent in sick newborn infants, but an increase in arterial blood pressure could disrupt the blood-brain barrier (BBB), especially in the preterm newborn. Using time-dated pregnant sheep, we tested the hypothesis that dopamine-induced hypertension increases fetal BBB permeability and cerebral water content. Barrier permeability was assessed in nine brain regions, including cerebral cortex, caudate, thalamus, brain stem, cerebellum, and spinal cord, by intravenous injection of the small tracer molecule [(14)C]aminoisobutyric acid at 10 min after the start of dopamine or saline infusion. We studied 23 chronically catheterized fetal sheep at 0.6 (93 days, n = 10) and 0.9 (132 days, n = 13) gestation. Intravenous infusion of dopamine increased mean arterial pressure from 38 +/- 3 to 53 +/- 5 mmHg in 93-day fetuses and from 55 +/- 5 to 77 +/- 8 mmHg in 132-day fetuses without a decrease in arterial O(2) content. These 40% increases in arterial pressure are close to the maximum hypertension reported for physiological stresses at these ages in fetal sheep. No significant increases in the brain transfer coefficient of aminoisobutyric acid were detected in any brain region in dopamine-treated fetuses compared with saline controls at 0.6 or 0.9 gestation. There was also no significant increase in cortical water content with dopamine infusion at either age. We conclude that a 40% increase in mean arterial pressure during dopamine infusion in normoxic fetal sheep does not produce substantial BBB disruption or cerebral edema even as early as 0.6 gestation.

P-450 epoxygenase and NO synthase inhibitors reduce cerebral blood flow response to N-methyl-D-aspartate.

Epoxyeicosatrienoic acids are cerebral vasodilators produced in astrocytes by cytochrome P-450 epoxygenase activity. The P-450 inhibitor miconazole attenuates the increase in cerebral blood flow (CBF) elicited by glutamate. We evaluated whether epoxygenase activity is involved in the CBF response to activation of the N-methyl-D-aspartate (NMDA) receptor subtype by using two structurally distinct inhibitors, miconazole and N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH), a selective epoxygenase substrate inhibitor. Drugs were delivered locally through microdialysis probes in striata of anesthetized rats. Local CBF was measured by hydrogen clearance and compared with CBF in contralateral striatum receiving vehicle. Microdialysis perfusion of NMDA doubled CBF and increased nitric oxide (NO) production estimated by recovery of labeled citrulline in the dialysate during labeled arginine infusion. Perfusion of miconazole or MS-PPOH blocked the increase in CBF without decreasing citrulline recovery. Perfusion of N(omega)-nitro-L-arginine decreased baseline CBF and inhibited the CBF response to NMDA. Perfusion of MS-PPOH did not inhibit the CBF response to sodium nitroprusside. We conclude that both the P-450 epoxygenase and NO synthase pathways are involved in the local CBF response to NMDA receptor activation, and that the signaling pathway may be more complex than simply NO diffusion from neurons to vascular smooth muscle.

Heme oxygenase-2 acts to prevent neuronal death in brain cultures and following transient cerebral ischemia.

Heme oxygenase (HO) cleaves the heme ring to form biliverdin, which is rapidly reduced to bilirubin, carbon monoxide, and iron. HO1, the first form of the enzyme discovered, is an inducible protein, concentrated in tissues that are exposed to degrading red blood cells and stimulated by hemolysis and numerous other toxic perturbations to eliminate potentially toxic heme. By contrast, HO2 is constitutive and most highly concentrated in neural tissues. Carbon monoxide, formed from HO2, is a putative neurotransmitter in the brain and peripheral autonomic nervous system. HO1 regulates the efflux of potentially toxic iron from cells, as iron efflux is deficient in mice with targeted deletion of HO1 (HO1(-/-)), and transfection of HO1 facilitates iron efflux. Bilirubin appears to be a physiologic neuroprotectant. Activation of HO2 by phorbol esters, that stimulate protein kinase C to phosphorylate HO2, augments production of bilirubin which protects brain cultures from oxidative stress. Bilirubin itself in nanomolar concentrations is neuroprotective, while HO2 deletion (HO2(-/-)) leads to increased neurotoxicity in brain cultures and increased neural damage following transient cerebral ischemia in intact mice. Mechanisms whereby HO2 provides neuroprotection have not been clarified including whether protection is primarily associated with apoptotic or necrotic cell death. Moreover, the generality of neurotoxic stimuli influenced by HO2 has been unclear. We now demonstrate increased neuronal death in cerebellar granule cultures of HO2(-/-) mice with a selective augmentation of apoptotic death. We also demonstrate that HO2 transfection rescues apoptotic death. In intact mice, we show an increased incidence of apoptotic morphology in the penumbra area surrounding the infarct core in HO2(-/-) mice undergoing transient focal ischemia.

Hypertonic saline worsens infarct volume after transient focal ischemia in rats.

BACKGROUND AND PURPOSE: Hypertonic saline (HS) has been advocated as a hyperosmolar agent for the treatment of cerebral edema, especially after traumatic brain injury. We tested the hypothesis that continuous intravenous HS administered during reperfusion from transient focal cerebral ischemia attenuates infarct volume. METHODS: Halothane-anesthetized male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. At the onset of reperfusion, rats received a 10-mL/kg intravenous bolus of 0.9% saline (SAL, n=8) or 7.5% SAL (chloride:acetate 50:50, n=8) followed by a continuous infusion for 22 hours. In a second series of experiments, ischemic damage was determined in cohorts treated with equivolumetric 3% saline (n=8) or 20% mannitol (n=8). In a third series, regional cerebral blood flow was measured ([(14)C]iodoantipyrine autoradiography) at 6 hours of reperfusion in 7.5%-SAL-treated (n=5) or SAL-treated (n=5) animals. RESULTS: In SAL rats, serum Na(+) was 137+/-3 and 138+/-2 mEq/L (mean+/-SEM) at baseline and 22 hours of reperfusion, respectively. In 7.5% SAL, serum Na(+) was 136+/-2 and 154+/-2 mEq/L at baseline and reperfusion, respectively. Physiological variables and reduction in laser-Doppler signal during MCAO and early reperfusion were not different between the 2 treatment groups. Cortical infarct volume was larger in 7.5%-SAL-treated rats (121+/-14 mm(3); 30+/-3% of contralateral cortex; P<0.05) than in SAL (64+/-15 mm(3); 16+/-4% of contralateral cortex). Striatal infarct volume was unchanged by HS therapy. Ipsilateral cortical tissue volume was increased relative to the contralateral side (by 26+/-5% with SAL; by 41+/-5% with 7.5% SAL). In contrast, ischemic damage was unaffected by 3%-SAL or 20%-mannitol treatment compared with SAL. Regional cerebral blood flow during reperfusion was heterogeneous in all animals, but there was no evidence of postischemic hypoperfusion or blood flow maldistribution in 7.5%-SAL-treated animals. CONCLUSIONS: These data demonstrate that hypernatremia resulting from postischemic HS infusion worsens cortical infarct volume in transient focal cerebral ischemia. The deleterious effect is not linked to exacerbation of delayed hypoperfusion during early reperfusion (6 hours); however, blood flow defects at later recovery time points remain to be excluded. These results may have implications for HS therapy in clinical ischemic stroke.

Interaction of glutamine and arginine on cerebrovascular reactivity to hypercapnia.

Glutamine is purported to inhibit recycling of citrulline to arginine and to limit nitric oxide release in vitro. However, vasoactive effects of glutamine have not been clearly demonstrated in vivo. During hyperammonemia, impaired cerebrovascular reactivity to CO(2) is related to glutamine accumulation. We tested the hypotheses that 1) glutamine infusion in the absence of hyperammonemia impairs cerebrovascular CO(2) reactivity and 2) arginine infusion preserves CO(2) reactivity during glutamine infusion and during hyperammonemia. Pentobarbital sodium-anesthetized rats were equipped with a closed cranial window for measuring pial arteriolar diameter. Intravenous infusion of 3 mmol. kg(-1). h(-1) of L-glutamine for 6 h produced threefold increases in plasma and cerebrospinal fluid concentrations. Dilation to hypercapnia was reduced by 45% compared with that of a time control group at 6 h but not at 3 h of glutamine infusion. Coinfusion of 2 mmol. kg(-1). h(-1) of L-arginine with glutamine maintained the hypercapnic vasodilation at the control value. Infusion of ammonium acetate at a rate known to produce threefold increases in cortical tissue glutamine concentration resulted in no significant hypercapnic vasodilation. Coinfusion of arginine with ammonium acetate maintained hypercapnic vasodilation at 60% of the control value. Arginine infusion did not augment hypercapnic vasodilation in a control group. We conclude that glutamine modulates cerebrovascular CO(2) reactivity in vivo. Glutamine probably acts by limiting arginine availability because the vascular inhibitory effect required >3 h to develop and because arginine infusion counteracted the vascular effect of both endogenously and exogenously produced increases in glutamine.

Heme oxygenase-2 is neuroprotective in cerebral ischemia.

Heme oxygenase (HO) is believed to be a potent antioxidant enzyme in the nervous system; it degrades heme from heme-containing proteins, giving rise to carbon monoxide, iron, and biliverdin, which is rapidly reduced to bilirubin. The first identified isoform of the enzyme, HO1, is an inducible heat-shock protein expressed in high levels in peripheral organs and barely detectable under normal conditions in the brain, whereas HO2 is constitutive and most highly concentrated in the brain. Interestingly, although HO2 is constitutively expressed, its activity can be modulated by phosphorylation. We demonstrated that bilirubin, formed from HO2, is neuroprotectant, as neurotoxicity is augmented in neuronal cultures from mice with targeted deletion of HO2 (HO2(-/-)) and reversed by low concentrations of bilirubin. We now show that neural damage following middle cerebral artery occlusion (MCAO) and reperfusion, a model of focal ischemia of vascular stroke, is substantially worsened in HO2(-/-) animals. By contrast, stroke damage is not significantly altered in HO1(-/-) mice, despite their greater debility. Neural damage following intracranial injections of N-methyl-d-aspartate (NMDA) is also accentuated in HO2(-/-) animals. These findings establish HO2 as an endogenous neuroprotective system in the brain whose pharmacologic manipulation may have therapeutic relevance.

Quantitative EEG during early recovery from hypoxic-ischemic injury in immature piglets: burst occurrence and duration.

This study examined the course of EEG recovery in an animal model of hypoxic-ischemic injury. The model used periods of hypoxia, room air and asphyxia to induce cardiac arrest. One-week-old piglets (n = 16) were exposed to a period of hypoxia, room air and complete asphyxia for 7 minutes. After cardiac arrest and resuscitation, two EEG features were evaluated as prognostic indicators of behavioral outcome as assessed by a neuroscore at 24 hours after insult. A prominent EEG feature was the number and duration of bursts evident during recovery. Episodes of bursting were detected through the thresholds on sustained periods of elevated power. After the animal was resuscitated, the EEG was monitored continuously for 4 hours. To assess outcome in the recovering animal, a behavioral testing scale was used to test the animal's neurological capabilities. Trends of EEG burst counts were measured through thresholds on sustained power changes. Bursts are energy transients in the EEG record. High degrees of bursting were characteristic of animals having good neurological condition whereas piglets having low burst counts had poor 24 hr neuroscores. At 100 min the average burst rate of the good neuroscore outcome group was more than 8 per min and was significantly different from the poor outcome group's level of 2.7 (p < or = 0.05). When these counts were weighted by their total duration, differences between groups increased (p < or = 0.02). This study showed that the QEEG measure of burst counts and duration together provided a strong prognostic indication of the 24 hour outcome after asphyxic injury in a neonatal animal model. The critical determinant of the bursting character was the time when bursting occurred. Bursting occurring early in recovery was a good gauge of outcome. We conclude that quantitative EEG analysis and interpretation can be an important tool for the outcome determination during recovery from cerebral injury states.

Effects of the AMPA receptor antagonist NBQX on outcome of newborn pigs after asphyxic cardiac arrest.

In neonates, asphyxia is a common cause of neuronal injury and often results in seizures. The authors evaluated whether blockade of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors during asphyxia and early recovery with 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-(F)-quinoxaline (NBQX) ameliorates neurologic deficit and histopathology in 1-week-old piglets. Anesthetized piglets were exposed to a sequence of 30 minutes of hypoxia, 5 minutes of room air ventilation, 7 minutes of airway occlusion, and cardiopulmonary resuscitation. Vehicle or NBQX was administered intravenously before asphyxia (30 mg/kg) and during the first 4 hours of recovery (15 mg/kg/h). Neuropathologic findings were evaluated at 96 hours of recovery by light microscopic and cytochrome oxidase histochemical study. Cardiac arrest occurred at 5 to 6 minutes of airway occlusion, and cardiopulmonary resuscitation restored spontaneous circulation independent of treatment modalities in about 2 to 3 minutes. Neurologic deficit over the 96-hour recovery period was not ameliorated by NBQX. Seizure activity began after 24 to 48 hours in 7 of 10 animals with vehicle and in 9 of 10 of animals with NBQX. In each group, four animals died in status epilepticus. Neuropathologic outcomes were not improved by NBQX. The density of remaining viable neurons was decreased in parietal cortex and putamen by NBQX treatment. Metabolic defects in cytochrome oxidase activity were worsened by NBQX treatment. Seizure activity during recovery was associated with reduced neuronal viability in neocortex and striatum in piglets from both groups that survived for 96 hours. This neonatal model of asphyxic cardiac arrest and resuscitation generates neurologic deficits, clinical seizure activity, and selective damage in regions of basal ganglia and sensorimotor cortex. In contrast to other studies in mature brain, AMPA receptor blockade with NBQX failed to protect against neurologic damage in the immature piglet and worsened postasphyxic histopathologic outcome in neocortex and putamen.

Effect of arrest time and cerebral perfusion pressure during cardiopulmonary resuscitation on cerebral blood flow, metabolism, adenosine triphosphate recovery, and pH in dogs.

OBJECTIVES: To test the hypothesis that greater cerebral perfusion pressure (CPP) is required to restore cerebral blood flow (CBF), oxygen metabolism, adenosine triphosphate (ATP), and intracellular pH (pHi) levels after variable periods of no-flow than to maintain them when cardiopulmonary resuscitation (CPR) is started immediately. DESIGN: Prospective, randomized, comparison of three arrest times and two perfusion pressures during CPR in 24 anesthetized dogs. SETTING: University cerebral resuscitation laboratory. INTERVENTIONS: We used radiolabeled microspheres to determine CBF and magnetic resonance spectroscopy to derive ATP and pHi levels before and during CPR. Ventricular fibrillation was induced, epinephrine administered, and thoracic vest CPR adjusted to provide a CPP of 25 or 35 mm Hg after arrest times of O, 6, or 12 mins. MEASUREMENTS AND MAIN RESULTS: When CPR was started immediately after arrest with a CPP of 25 mm Hg, CBF and ATP were 57 +/- 10% and 64 +/- 14% of prearrest (at 10 mins of CPR). In contrast, CBF and ATP were minimally restored with a CPP at 25 mm Hg after a 6-min arrest time (23 +/- 5%, 16 +/- 5%, respectively). With a CPP of 35 mm Hg, extending the no-flow arrest time from 6 to 12 mins reduced reflow from 71 +/- 11% to 37 +/- 7% of pre-arrest and reduced ATP recovery from 60 +/- 11% to 2 +/- 1% of pre-arrest. After 6- or 12-min arrest times, brainstem blood flow was restored more than supratentorial blood flow, but cerebral pHi was never restored. CONCLUSIONS: A CPP of 25 mm Hg maintains supratentorial blood flow and ATP at 60% to 70% when CPR starts immediately on arrest, but not after a 6-min delay. A higher CPP of 35 mm Hg is required to restore CBF and ATP when CPR is delayed for 6 mins. After a 12-min delay, even the CPP of 35 mm Hg is unable to restore CBF and ATP. Therefore, increasing the arrest time at these perfusion pressures increases the resistance to reflow sufficient to impair restoration of cerebral ATP.

GLT1, glial glutamate transporter, is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain.

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high-affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin-like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin-positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development.

Poor outcome after hypoxia-ischemia in newborns is associated with physiological abnormalities during early recovery. Possible relevance to secondary brain injury after head trauma in infants.

"Secondary hypoxia/ischemia" (i.e. regional impairment of oxygen and substrate delivery) results in secondary deterioration after traumatic brain injury in adults as well as in children and infants. However, detailed analysis regarding critical physiological abnormalities resulting from hypoxia/ischemia in the immature brain, e.g. acid-base-status, serum glucose levels and brain temperature, and their influence on outcome, are only available from non-traumatic experimental models. In recent studies on hypoxic/asphyxic cardiac arrest in neonatal piglets, we were able to predict short-term outcome using specific physiologic abnormalities immediately after the insult. Severe acidosis, low serum glucose levels and fever after resuscitation were associated with an adverse neurologic recovery one day after the insult. The occurrence of clinically apparent seizure activity during later recovery increased mortality (epileptic state), and survivors had greater neocortical and striatal brain damage. Brain damage after transient hypoxia/ischemia and "secondary brain injury" after head trauma may have some mechanistic overlap, and these findings on physiological predictors of outcome may also apply to pathologic conditions in the post-traumatic immature brain. Evaluation of data from other models of brain injury will be important to develop candidate treatment strategies for head-injured infants and children and may help to initiate specific studies about the possible role of these physiological predictors of brain damage in the traumatically injured immature brain.