Transient Cerebral Hypoxia-ischemia Research Articles

Therapeutic hypothermia modulates the neurogenic response of the newborn piglet subventricular zone after hypoxia-ischemia

BackgroundNeuroprotection combined with neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy. To investigate the neurogenic response to hypoxia-ischemia (HI) followed by normothermia (38.5 °C) or three different hypothermic temperatures (35, 33.5, or 30 °C) in the subventricular zone (SVZ) of the neonatal piglet.MethodsFollowing transient cerebral HI and resuscitation, 28 newborn piglets were randomized to: normothermia or whole-body cooling to 35 °C, 33.5 °C, or 30 °C during 2–26 h (all n = 7). At 48 h, piglets were euthanized and SVZ obtained to evaluate its cellularity, pattern of cell death, radial glia length, doublecortin (DCX, neuroblasts) expression, and Ki67 (cell proliferation) and Ki67/Sox2 (neural stem/progenitor dividing) cell counts.ResultsNormothermic piglets showed lower total (Ki67+) and neural stem/progenitor dividing (Ki67+Sox2+) cell counts when compared to hypothermic groups. Cooling to 33.5 °C obtained the highest values of SVZ cellularity, radial glia length processes, neuroblast chains area and DCX immunohistochemistry. Cooling to 30 °C, however, revealed decreased cellularity in the lateral SVZ and shorter radial glia processes when compared with 33.5 °C.ConclusionsIn a neonatal piglet model, hypothermia to 33.5 °C modulates the neurogenic response of the SVZ after HI, highlighting the potential beneficial effect of hypothermia to 33.5 °C on endogenous neurogenesis and the detrimental effect of overcooling beyond this threshold.ImpactNeuroprotection combined with neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy.Hypothermia may modulate neurogenesis in the subventricular zone (SVZ) of the neonatal hypoxic-ischemic piglet.Cooling to 33.5 °C obtained the highest values of SVZ cellularity, radial glia length processes, neuroblast chains area and doublecortin immunohistochemistry; cooling to 30 °C, however, revealed decreased cellularity and shorter radial glia processes.In a neonatal piglet model, therapeutic hypothermia (33.5 °C) modulates the neurogenic response of the SVZ after hypoxia-ischemia, highlighting also the detrimental effect of overcooling beyond this threshold.

Stroke propensity in the Th3+/ mouse model of β-thalassemia intermedia

β-thalassemia is associated with multiple hematological and cerebrovascular symptoms linked to a hypercoagulable state that has not been fully replicated in animal models for the development of stroke treatments. Herein we compared the physiological properties and responses to transient cerebral hypoxia-ischemia (tHI) between six-month-old wildtype and heterozygous Th3/+ mice, a model of non-transfusion-dependent β-thalassemia intermedia (β-TI). We found that Th3/+ mice developed microcytic anemia, splenomegaly, higher platelet counts, and increased platelet-erythrocyte plus erythrocyte-leukocyte aggregates. Furthermore, Th3/+ mice showed diminished cerebrovascular reactivity (CVR) and cortical oxygen saturation under repetitive hypercapnic challenges. When subjected to a sub-threshold tHI insult, platelets and leukocytes in Th3/+ mice adhered to the cerebrovascular wall or formed aggregates, while their counterparts flew through smoothly in wildtype mice. Subsequently, Th3/+ mice showed increased fibrin deposition around cerebral blood vessels and larger infarction than wildtype mice, especially in female Th3/+ mice. Collectively these results showed that Th3/+ mice mimic key clinical features and a propensity to thromboembolism in β-TI patients. The hypercoagulable state in Th3/+ mice is likely caused by multiple hematological and CVR anomalies that are similar, but are not identical to those in the mouse model of sickle cell anemia. As such, we suggest that Th3/+ mice are a useful model to study the pathological mechanisms and prophylactic stroke treatments in thalassemia patients.

Neurogenesis Is Reduced at 48 h in the Subventricular Zone Independent of Cell Death in a Piglet Model of Perinatal Hypoxia-Ischemia.

Cellular and tissue damage triggered after hypoxia-ischemia (HI) can be generalized and affect the neurogenic niches present in the central nervous system. As neuroregeneration may be critical for optimizing functional recovery in neonatal encephalopathy, the goal of the present work was to investigate the neurogenic response to HI in the neurogenic niche of the subventricular zone (SVZ) in the neonatal piglet. A total of 13 large white male piglets aged <24 h were randomized into two groups: i) HI group (n = 7), animals submitted to transient cerebral HI and resuscitation; and ii) Control group (n = 6), non-HI animals. At 48 h, piglets were euthanized, and the SVZ and its surrounding regions, such as caudate and periventricular white matter, were analyzed for histology using hematoxylin-eosin staining and immunohistochemistry by evaluating the presence of cleaved caspase 3 and TUNEL positive cells, together with the cell proliferation/neurogenesis markers Ki67 (cell proliferation), GFAP (neural stem cells processes), Sox2 (neural stem/progenitor cells), and doublecortin (DCX, a marker of immature migrating neuroblasts). Hypoxic-ischemic piglets showed a decrease in cellularity in the SVZ independent of cell death, together with decreased length of neural stem cells processes, neuroblast chains area, DCX immunoreactivity, and lower number of Ki67 + and Ki67 + Sox2 + cells. These data suggest a reduction in both cell proliferation and neurogenesis in the SVZ of the neonatal piglet, which could in turn compromise the replacement of the lost neurons and the achievement of global repair.

Human umbilical cord mesenchymal stromal cells as an adjunct therapy with therapeutic hypothermia in a piglet model of perinatal asphyxia

Background: With therapeutic hypothermia (HT) for neonatal encephalopathy, disability rates are reduced, but not all babies benefit. Pre-clinical rodent studies suggest mesenchymal stromal cells (MSCs) augment HT protection. Aims:The authors studied the efficacy of intravenous (IV) or intranasal (IN) human umbilical cord-derived MSCs (huMSCs) as adjunct therapy to HT in a piglet model. Methods:A total of 17 newborn piglets underwent transient cerebral hypoxia-ischemia (HI) and were then randomized to (i) HT at 33.5°C 1–13 h after HI (n = 7), (ii) HT+IV huMSCs (30 × 106 cells) at 24 h and 48 h after HI (n = 5) or (iii) HT+IN huMSCs (30 × 106 cells) at 24 h and 48 h after HI (n = 5). Phosphorus-31 and hydrogen-1 magnetic resonance spectroscopy (MRS) was performed at 30 h and 72 h and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and oligodendrocytes quantified. In two further piglets, 30 × 106 IN PKH-labeled huMSCs were administered. Results:HI severity was similar between groups. Amplitude-integrated electroencephalogram (aEEG) recovery was more rapid for HT+IN huMSCs compared with HT from 25 h to 42 h and 49 h to 54 h (P ≤ 0.05). MRS phosphocreatine/inorganic phosphate was higher on day 2 in HT+IN huMSCs than HT (P = 0.035). Comparing HT+IN huMSCs with HT and HT+IV huMSCs, there were increased OLIG2 counts in hippocampus (P = 0.011 and 0.018, respectively), internal capsule (P = 0.013 and 0.037, respectively) and periventricular white matter (P = 0.15 for IN versus IV huMSCs). Reduced TUNEL-positive cells were seen in internal capsule with HT+IN huMSCs versus HT (P = 0.05). PKH-labeled huMSCs were detected in the brain 12 h after IN administration. Conclusions:After global HI, compared with HT alone, the authors saw beneficial effects of HT+IN huMSCs administered at 24 h and 48 h (30 × 106 cells/kg total dose) based on more rapid aEEG recovery, improved 31P MRS brain energy metabolism and increased oligodendrocyte survival at 72 h.

Quantification of the severity of hypoxic-ischemic brain injury in a neonatal preclinical model using measurements of cytochrome-c-oxidase from a miniature broadband-near-infrared spectroscopy system.

.We describe the development of a miniaturized broadband near-infrared spectroscopy system (bNIRS), which measures changes in cerebral tissue oxyhemoglobin () and deoxyhemoglobin ([HHb]) plus tissue metabolism via changes in the oxidation state of cytochrome-c-oxidase ([oxCCO]). The system is based on a small light source and a customized mini-spectrometer. We assessed the instrument in a preclinical study in 27 newborn piglets undergoing transient cerebral hypoxia-ischemia (HI). We aimed to quantify the recovery of the HI insult and estimate the severity of the injury. The recovery in brain oxygenation (), blood volume (), and metabolism () for up to 30 min after the end of HI were quantified in percentages using the recovery fraction (RF) algorithm, which quantifies the recovery of a signal with respect to baseline. The receiver operating characteristic analysis was performed on bNIRS-RF measurements compared to proton () magnetic resonance spectroscopic (MRS)-derived thalamic lactate/N-acetylaspartate (Lac/NAA) measured at 24-h post HI insult; Lac/NAA peak area ratio is an accurate surrogate marker of neurodevelopmental outcome in babies with neonatal HI encephalopathy. The -RF cut-off threshold of 79% within 30 min of HI predicted injury severity based on Lac/NAA with high sensitivity (100%) and specificity (93%). A significant difference in thalamic Lac/NAA was noticed () between the two groups based on this cut-off threshold of 79% -RF. The severe injury group () had smaller recovery in -RF () and no significant difference was observed in -RF between groups. At 48 h post HI, significantly higher -MRS-measured inorganic phosphate/exchangeable phosphate pool (epp) () and reduced phosphocreatine/epp () were observed in the severe injury group indicating persistent cerebral energy depletion. Based on these results, the bNIRS measurement of the oxCCO recovery fraction offers a noninvasive real-time biomarker of brain injury severity within 30 min following HI insult.

Combined damage produced by multiple mild cerebral insults assessed using MRI in neonatal rats

PurposeTo determine whether damage to neonatal brain is exacerbated with multiple mild cerebral insults as detected with MRI and corroborated using histology. Materials and methodsThe combined brain injury produced by multiple procedures was compared in neonatal rats having: Sham surgery at P5, Sham surgery at P5 plus a diffuse mild transient unilateral cerebral hypoxia ischemia (HI) at P7, HI alone, and a minor photothrombotic (PT) stroke at P5 followed by HI. MRI after the ischemic insults was followed by final histology. ResultsPT produced lesions with increased T2 and decreased apparent diffusion coefficient for water (ADC) but no significant effects of a second HI. However, near the PT lesion/parietal cortex there were patchy areas of enhanced T2 and decreased ADC in 6/9, 3/8 and 0/8 animals in the PT+HI, Sham+HI and HI groups, respectively (P<0.05). Patches corresponded histologically to increased vacuolation and cell death and were more pronounced in the PT+HI and Sham+PT groups than the HI group. ConclusionThe extent of damage produced by a minor neonatal stroke followed by a diffuse HI two days later results in heterogeneous enhancement of T2, ADC and histological injury near the lesion. Surgical procedures including mechanical head manipulation followed by HI also produced some enhanced heterogeneity of hypoxic-ischemic injury affirming the need for sham controls.

Experimental Cannabinoid 2 Receptor Inhibition in CNS Injury-Induced Immunodeficiency Syndrome.

Severe CNS injury, such as stroke, traumatic brain injury, or spinal cord injury, is known to increase susceptibility to infections. The increased susceptibility to infection is due to an impaired immune response and is referred to as CIDS. The CB2 receptor on immune cells presents a potential therapeutic target in CIDS as activation of this receptor has been shown to be involved in immunosuppression. The main purpose of this study was to determine the impact of CB2 receptor inhibition on leukocyte activation within the microcirculation following endotoxin challenge in an experimental stroke model. Five experimental groups (male C57BL/6 mice, age: 6-8 weeks) were subjected to the following treatments: control; endotoxemia (LPS 5 mg/kg, i.v.); transient cerebral hypoxia-ischemia (HI) + endotoxemia; HI + endotoxemia + CB2 receptor antagonist (AM630 2.5 mg/kg, i.v.). HI was induced by unilateral carotid artery occlusion, followed by 50 minute exposure to a low oxygen atmosphere (8% O2 ). The CB2 receptor antagonist was given 15 min prior to LPS administration. Intravital microscopy (IVM) was carried out 2h after LPS administration. Brains were extracted and stained with tetrazolium chloride (TTC) to measure infarct volume. Compared to endotoxemic animals without CNS injury, mice subjected to HI displayed reduced leukocyte activation in intestinal submucosal venules indicative of CIDS. Administration of the CB2 receptor antagonist in animals with CIDS challenged with endotoxin restored peripheral leukocyte recruitment without a detrimental impact on infarct size. We conclude that the ECS is involved in the impaired immune response following CNS injury. Future studies should further explore the CB2 receptor pathway to develop novel therapies for CIDS.

Prophylactic Edaravone Prevents Transient Hypoxic-Ischemic Brain Injury: Implications for Perioperative Neuroprotection.

Hypoperfusion-induced thrombosis is an important mechanism for postsurgery stroke and cognitive decline, but there are no perioperative neuroprotectants to date. This study investigated whether prophylactic application of Edaravone, a free radical scavenger already used in treating ischemic stroke in Japan, can prevent infarct and cognitive deficits in a murine model of transient cerebral hypoxia-ischemia. Adult male C57BL/6 mice were subjected to transient hypoxic-ischemic (tHI) insult that consists of 30-minute occlusion of the unilateral common carotid artery and exposure to 7.5% oxygen. Edaravone or saline was prophylactically applied to compare their effects on cortical oxygen saturation, blood flow, coagulation, oxidative stress, metabolites, and learning-memory using methods that include photoacoustic imaging, laser speckle contrast imaging, solid-state NMR, and Morris water maze. The effects on infarct size by Edaravone application at different time points after tHI were also compared. Prophylactic administration of Edaravone (4.5 mg/kg×2, IP, 1 hour before and 1 hour after tHI) improved vascular reperfusion, oxygen saturation, and the maintenance of brain metabolites, reducing oxidative stress, thrombosis, white-matter injury, and learning impairment after tHI insult. Delayed Edaravone treatment after 3 h post-tHI became unable to reduce infarct size. Acute application of Edaravone may be a useful strategy to prevent postsurgery stroke and cognitive impairment, especially in patients with severe carotid stenosis.

Prophylactic Subacute Administration of Zinc Increases CCL2, CCR2, FGF2, and IGF-1 Expression and Prevents the Long-Term Memory Loss in a Rat Model of Cerebral Hypoxia-Ischemia.

Prophylactic subacute administration of zinc decreases lipoperoxidation and cell death following a transient cerebral hypoxia-ischemia, thus suggesting neuroprotective and preconditioning effects. Chemokines and growth factors are also involved in the neuroprotective effect in hypoxia-ischemia. We explored whether zinc prevents the cerebral cortex-hippocampus injury through regulation of CCL2, CCR2, FGF2, and IGF-1 expression following a 10 min of common carotid artery occlusion (CCAO). Male rats were grouped as follows: (1) Zn96h, rats injected with ZnCl2 (one dose every 24 h during four days); (2) Zn96h + CCAO, rats treated with ZnCl2 before CCAO; (3) CCAO, rats with CCAO only; (4) Sham group, rats with mock CCAO; and (5) untreated rats. The cerebral cortex-hippocampus was dissected at different times before and after CCAO. CCL2/CCR2, FGF2, and IGF-1 expression was assessed by RT-PCR and ELISA. Learning in Morris Water Maze was achieved by daily training during 5 days. Long-term memory was evaluated on day 7 after learning. Subacute administration of zinc increased expression of CCL2, CCR2, FGF2, and IGF-1 in the early and late phases of postreperfusion and prevented the CCAO-induced memory loss in the rat. These results might be explained by the induction of neural plasticity because of the expression of CCL2 and growth factors.

Prenatal cerebral ischemia triggers dysmaturation of caudate projection neurons

Recently, we reported that the neocortex displays impaired growth after transient cerebral hypoxia-ischemia (HI) at preterm gestation that is unrelated to neuronal death but is associated with decreased dendritic arbor complexity of cortical projection neurons. We hypothesized that these morphological changes constituted part of a more widespread neuronal dysmaturation response to HI in the caudate nucleus (CN), which contributes to motor and cognitive disability in preterm survivors. Ex vivo magnetic resonance imaging (MRI), immunohistochemistry, and Golgi staining defined CN growth, cell death, proliferation, and dendritic maturation in preterm fetal sheep 4 weeks after HI. Patch-clamp recording was used to analyze glutamatergic synaptic currents in CN neurons. MRI-defined growth of the CN was reduced after ischemia compared to controls. However, no significant acute or delayed neuronal death was seen in the CN or white matter. Nor was there significant loss of calbindin-positive medium spiny projection neurons (MSNs) or CN interneurons expressing somatostatin, calretinin, parvalbumin, or tyrosine hydroxylase. Morphologically, ischemic MSNs showed a markedly immature dendritic arbor, with fewer dendritic branches, nodes, endings, and spines. The magnitude and kinetics of synaptic currents, and the relative contribution of glutamate receptor subtypes in the CN were significantly altered. The marked MSN dendritic and functional abnormalities after preterm cerebral HI, despite the marked resistance of immature CN neurons to cell death, are consistent with widespread susceptibility of projection neurons to HI-induced dysmaturation. These global disturbances in dendritic maturation and glutamatergic synaptic transmission suggest a new mechanism for long-term motor and behavioral disabilities in preterm survivors via widespread disruption of neuronal connectivity.

Optimum therapeutic hypothermia temperature after perinatal asphyxia: a magnetic resonance spectroscopy biomarker and immunohistochemistry study in the newborn piglet

BackgroundTherapeutic hypothermia for neonatal encephalopathy improves survival with normal neurological outcome. Despite treatment, however, 50% of infants have adverse outcomes; customising cooling with more precision might provide further benefit. The specific brain temperature for optimum neuroprotection is unknown. We have previously shown in the piglet that optimum neuroprotection by delayed cooling occurs at different temperatures in the cortical (33°C) and deep grey (35°C) matter. Magnetic resonance biomarkers, including lactate:creatine ratio (Lac/Cr), predict neurodevelopmental outcome after neonatal encephalopathy. We aimed to assess the optimum temperature for regional neuroprotection in a proton magnetic resonance system (1H MRS). Methods28 male piglets aged less than 24 h were anaesthetised with isoflurane and underwent trachaeal intubation, ventilation, continuous physiological monitoring, and intensive care treatment to maintain sedation and physiological homoeostasis. Within the bore of our 9·4 T magnetic resonance system all piglets underwent a hypoxic-ischaemic insult for 12·5 min, measured with real-time phosphorous MRS, before resuscitation. After this insult piglets were randomised (seven in each group) to normothermia (38·5°C), hypothermia (35°C), hypothermia (33·5°C), and hypothermia (30°C). Serial MRS assessments were acquired before, during, and up to 48 h after transient cerebral hypoxia-ischaemia. Areas under the curve (AUC) for the 1H MRS Lac/Cr peak-area ratio in ventromedial forebrain (vmFB, predominantly grey matter) and dorsal subcortical (dsc, predominantly white matter) voxels were calculated. Programmed cell death at 48 h was measured with terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining on paraffin-embedded tissue in corresponding regions. FindingsCompared with normothermia, cooling to 35°C and 33·5°C produced consistent 50–75% reduction in density of TUNEL-positive cells with significant decreases in insular cortex, hippocampus, subcortical white matter, thalamus, and putamen (p<0·05 in ANOVA and post-hoc Tukey test). Cooling to 30°C did not further reduce TUNEL-positive cell density compared with 33·5°C and 35°C in dorsal parietal cortex (dCTX); more cell death was seen in the deep grey matter (thalamus and putamen) at 30°C. MRS biomarker analysis did not show an increase in Lac/Cr AUC in the vmFB voxel at 30°C compared with 33·5° and 35°C (p=0·07). Raised Lac/Cr and increased cell death at 30°C was not seen in the dsc voxel or in dCTX. InterpretationCooling to 30°C worsened hypoxic-ischaemic injury in the thalamus and basal ganglia compared with cooling to 33·5°C and 35°C. We showed a higher threshold optimum temperature for neuroprotection in the deep grey matter than in white matter/dCTX on the basis of immunohistochemical markers of cell death. Systemic effects of cooling to 30°C might have exacerbated these detrimental effects on deep grey matter cell death. These data support previous work in the developing brain and are relevant to clinical practice for optimum therapeutic hypothermia in newborn infants with birth asphyxia. FundingUK Medical Research Council, National Institute for Health Research.

Methyl‐isobutyl amiloride reduces brain Lac/NAA, cell death and microglial activation in a perinatal asphyxia model

Na⁺/H⁺ exchanger (NHE) blockade attenuates the detrimental consequences of ischaemia and reperfusion in myocardium and brain in adult and neonatal animal studies. Our aim was to use magnetic resonance spectroscopy (MRS) biomarkers and immunohistochemistry to investigate the cerebral effects of the NHE inhibitor, methyl isobutyl amiloride (MIA) given after severe perinatal asphyxia in the piglet. Eighteen male piglets (aged < 24 h) underwent transient global cerebral hypoxia-ischaemia and were randomized to (i) saline placebo; or (ii) 3 mg/kg intravenous MIA administered 10 min post-insult and 8 hourly thereafter. Serial phosphorus-31 (³¹P) and proton (¹H) MRS data were acquired before, during and up to 48 h after hypoxia-ischaemia and metabolite-ratio time-series Area under the Curve (AUC) calculated. At 48 h, histological and immunohistochemical assessments quantified regional tissue injury. MIA decreased thalamic lactate/N-acetylaspartate and lactate/creatine AUCs (both p < 0.05) compared with placebo. Correlating with improved cerebral energy metabolism, transferase mediated biotinylated d-UTP nick end-labelling (TUNEL) positive cell density was reduced in the MIA group in cerebral cortex, thalamus and white matter (all p < 0.05) and caspase 3 immunoreactive cells were reduced in pyriform cortex and caudate nucleus (both p < 0.05). Microglial activation was reduced in pyriform and midtemporal cortex (both p < 0.05). Treatment with MIA starting 10 min after hypoxia-ischaemia was neuroprotective in this perinatal asphyxia model.

166 Simultaneous 31P-Magnetic Resonance And Near-Infrared Spectroscopic Investigation of Brain Tissue Oxygenation, Cytochrome-C-Oxidase And Intracellular Metabolites During Perinatal Cerebral Hypoxia-Ischaemia

BackgroundHypoxic-ischaemic neonatal encephalopathy is associated with high mortality and morbidity rates worldwide.AimsTo investigate brain haemodynamic, cytochrome-c-oxidase (CCO) and energy-resource changes during transient hypoxia-ischaemia (HI) and recovery using simultaneous broadband near-infrared...

Xenon augmented hypothermia reduces early lactate/N‐acetylaspartate and cell death in perinatal asphyxia

Additional treatments for therapeutic hypothermia are required to maximize neuroprotection for perinatal asphyxial encephalopathy. We assessed neuroprotective effects of combining inhaled xenon with therapeutic hypothermia after transient cerebral hypoxia-ischemia in a piglet model of perinatal asphyxia using magnetic resonance spectroscopy (MRS) biomarkers supported by immunohistochemistry. Thirty-six newborn piglets were randomized (all groups n = 9), with intervention from 2 to 26 hours, to: (1) normothermia; (2) normothermia + 24 hours 50% inhaled xenon; (3) 24 hours hypothermia (33.5°C); or (4) 24 hours hypothermia (33.5°C) + 24 hours 50% inhaled xenon. Serial MRS was acquired before, during, and up to 48 hours after hypoxia-ischemia. Mean arterial blood pressure was lower in all treatment groups compared with normothermia (p < 0.01) (although >40mmHg); the combined therapy group required more fluid boluses (p < 0.05) and inotropes (p < 0.001). Compared with no intervention, both hypothermia and xenon-augmented hypothermia reduced the temporal regression slope magnitudes for phosphorus-MRS inorganic phosphate/exchangeable phosphate pool (EPP) and phosphocreatine/EPP (both p < 0.05); for lactate/N-acetylaspartate (NAA), only xenon-augmented hypothermia reduced the slope (p < 0.01). Xenon-augmented hypothermia also reduced transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)(+) nuclei and caspase 3 immunoreactive cells in parasagittal cortex and putamen and increased microglial ramification in midtemporal cortex compared with the no treatment group (p < 0.05). Compared with hypothermia, however, combination treatment did not reach statistical significance for any measure. Lactate/NAA showed a strong positive correlation with TUNEL; nucleotide triphosphate/EPP showed a strong negative correlation with microglial ramification (both p < 0.01). Compared with no treatment, xenon-augmented hypothermia reduced cerebral MRS abnormalities and cell death markers in some brain regions. Compared with hypothermia, xenon-augmented hypothermia did not reach statistical significance for any measure. The safety and possible improved efficacy support phase II trials.

Delayed Whole-Body Cooling to 33 or 35°C and the Development of Impaired Energy Generation Consequential to Transient Cerebral Hypoxia-Ischemia in the Newborn Piglet

Fundamental questions remain about the precise temperature providing optimal neuroprotection after perinatal hypoxia-ischemia (HI). Furthermore, if hypothermia delays the onset of the neurotoxic cascade and the secondary impairment in cerebral energy generation, the "latent phase" may be prolonged, thus extending the period when additional treatments may be effective. The aims of this study were to investigate the effects of delayed systemic cooling at either 33 degrees C or 35 degrees C on the following: (1) latent-phase duration, and (2) cerebral metabolism during secondary energy failure itself, in the 48-hour period after transient HI. Piglets were randomly assigned to the following: (1) HI-normothermic (HI-n) rectal temperature (Trectal; n = 12), (2) HI-Trectal 35 degrees C (HI-35; n = 7), and (3) HI-Trectal 33 degrees C (HI-33; n = 10). Groups were cooled to the target Trectal between 2 and 26 hours after HI. Serial magnetic resonance spectroscopy was performed over 48 hours. The effect of cooling on secondary energy failure severity (indexed by the nucleotide triphosphate/exchangeable phosphate pool [NTP/EPP] and phosphocreatine/inorganic phosphate [PCr/Pi] ratios) was assessed. Compared with HI-n, HI-35 and HI-33 had a longer NTP/EPP latent phase and during the entire study duration had higher mean NTP/EPP and PCr/Pi. The latent phase (both PCr/Pi and NTP/EPP) and the whole-brain cerebral energetics were similar for HI-35 and HI-33. During the hypothermic period, compared with HI-n, PCr/Pi was preserved in the cooled groups, but this advantage was not maintained after rewarming. Compared with HI-n, HI-35 and HI-33 had higher NTP/EPP after rewarming. Whole-body hypothermia for 24 hours at either 35 or 33 degrees C, commenced 2 hours after resuscitation, prolonged the NTP/EPP latent phase and reduced the overall secondary falls in mean PCr/Pi and NTP/EPP during 48 hours after HI. Reducing the temperature from 35 to 33 degrees C neither increased mean PCr/Pi and NTP/EPP nor further lengthened the latent phase.

268 Effect of Delayed Whole-Body Cooling to 33°C or 35°C After Transient Cerebral Hypoxia-Ischaemia in the Newborn Piglet

Background: Although delayed cooling after transient perinatal cerebral hypoxia-ischaemia (HI) shows great therapeutic potential in clinical and experimental studies, fundamental questions remain about the optimal cooling temperature and mode of cooling. Aims: To investigate: (i) the effect of delayed cooling at 33°C or 35°C on the evolution of secondary energy failure (SEF) (ii) the relationship between cerebral energy metabolism during SEF and neuronal death. Methods: Piglets were randomised to: (i) HI-normothermic rectal temperature (T) (n=12), (ii) HI-T35°C (n=7), and (iii) HI-T33°C (n=10). Groups (ii) and (iii) were cooled to the target rectal temperature between 2–26 hours following HI. Serial global phosphorous magnetic resonance spectroscopy was used to assess SEF over 48 hrs. At 48 hrs the brain was perfusion fixed and dead neurons assessed in the cortex and deep grey matter. The effect of cooling on SEF (indexed by the nucleotide triphosphate/exchangeable phosphate pool ratio (NTP/EPP)) was assessed using a linear model, which incorporated a random effect for each subject and fixed effects for groups. Results: The cooled groups had significantly higher NTP/EPP than the normothermia group; HI-normothermic mean (95% CI) NTP/EPP was 0.131 (0.118, 0.144); HI-T35°C 0.149 (0.136, 0.162); HI-T33°C 0.149 (0.136, 0.162) (p= 0.032 for HI-T35°C; p=0.044 for HI-T33°C, both vs normothermia). There was no difference in NTP/EPP between HI-T35°C and HI-T33°C (p=0.996). Minimal NTP/EPP 36–48 hours after HI was linearly correlated with neuronal death in cortical and deep grey matter (p<0.05). Conclusions: Compared to normothermia, delayed whole body cooling (33°C and 35°C) ameliorated global SEF. No advantage in global cerebral energy metabolism was detected with cooling to 33°C compared to 35°C, however further work is required to assess whether there is regional variation in the optimal brain temperature for neuroprotection. Minimal NTP/EPP during SEF correlated with neuronal death.

Differential Effects of Hypoxia-Ischemia on Phosphorylation of the N-Methyl-D-Aspartate Receptor in One- and Three-Week-Old Rats

The effects of transient cerebral hypoxia-ischemia (HI) on phosphorylation of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor were investigated in 7 (P7)- and 21 (P21)-day-old rats. Unilateral HI was induced by ligation of the right common carotid artery and exposure to 8% O₂/92% N₂ for 120 (P7) or 90 (P21) min. Phosphorylation by protein kinase A (PKA; S897) and PKC (S896 and S890) was depressed in the ipsilateral hemisphere relative to both naïve controls and the contralateral hemisphere immediately following HI at both ages. At P7, but not P21, reperfusion resulted in an initial recovery to control phosphorylation levels at all 3 sites followed by a secondary decline. At both ages, pS896 was less than control values after 24 h of recovery, whereas pS890 had returned to control levels by this time. pS897 recovered to control levels by 24 h in P21 animals but not in P7 animals. Differential effects of HI on phosphorylation of the NMDA receptor at P7 and P21 may contribute to age-related changes in sensitivity to HI.

Effects of alpha-phenyl-N-tert-butyl nitrone (PBN)on brain cell membrane function and energy metabolism during transient global cerebral hypoxia-ischemia and reoxygenation-reperfusion in newborn piglets.

We sought to know whether a free radical spin trap agent, α-phenyl-N-tert-butyl nitrone (PBN) influences brain cell membrane function and energy metabolism during and after transient global hypoxia-ischemia (HI) in the newborn piglets. Cerebral HI was induced by temporary complete occlusion of bilateral common carotid arteries and simultaneous breathing with 8% oxygen for 30 min, followed by release of carotid occlusion and normoxic ventilation for 1 hr (reoxygenation-reperfusion, RR). PBN (100 mg/kg) or vehicle was administered intravenously just before the induction of HI or RR. Brain cortex was harvested for the biochemical analyses at the end of HI or RR. The level of conjugated dienes significantly increased and the activity of Na+, K+-ATPase significantly decreased during HI, and they did not recover during RR. The levels of ATP and phosphocreatine (PCr) significantly decreased during HI, and recovered during RR. PBN significantly decreased the level of conjugated dienes both during HI and RR, but did not influence the activity of Na+, K+-ATPase and the levels of ATP and PCr. We demonstrated that PBN effectively reduced brain cell membrane lipid peroxidation, but did not reverse ongoing brain cell membrane dysfunction nor did restore brain cellular energy depletion, in our piglet model of global hypoxic-ischemic brain injury.

Relationship between cerebral interstitial levels of amino acids and phosphorylation potential during secondary energy failure in hypoxic-ischemic newborn piglets.

The aim of this study was to determine the validity of the hypothesis that excitatory amino acids are related to phosphorylation potential during primary and secondary cerebral energy failure observed in asphyxiated infants. We report here the results of experiments using newborn piglets subjected to severe transient cerebral hypoxia-ischemia followed by resuscitation. We examined cerebral energy metabolism by phosphorus nuclear magnetic resonance spectroscopy and changes in levels of amino acid neurotransmitters in the cortex by microdialysis before, during, and up to 24 h after the hypoxic-ischemic insult. The concentrations of aspartate, glutamate, taurine, and gamma-aminobutyric acid were significantly elevated during the hypoxic-ischemic insult compared with prebaseline values. Shortly after resuscitation, glutamate, taurine, and gamma-aminobutyric acid concentrations decreased but then began to increase again. These secondary elevations were greater than the primary elevations. A negative linear correlation was found between primary interstitial levels of glutamate and taurine and minimum values of phosphocreatine/inorganic phosphate during the secondary energy failure. The cerebral energy state depended on the time course of changes in excitatory amino acids, suggesting that amino acids play distinct roles during the early and delayed phases of injury.

Neuroprotection of Creatine Supplementation in Neonatal Rats with Transient Cerebral Hypoxia-Ischemia

We hypothesized that creatine (Cr) supplementation would preserve energy metabolism and thus ameliorate the energy failure and the extent of brain edema seen after severe but transient cerebral hypoxia-ischemia (HI) in the neonatal rat model. Six-day-old (P6) rats received subcutaneous Cr monohydrate injections for 3 consecutive days (3 g/kg body weight/day), followed by ³¹P-magnetic resonance spectroscopy (MRS) at P9. In a second group, P4 rats received the same Cr dose as above for 3 days prior to unilateral common carotid artery ligation followed 1 h later by 100 min of hypoxia (8% O₂) at P7. Rats were maintained at 37°C rectal temperature until magnetic resonance imaging was performed 24 h after HI. Cr supplementation for 3 days significantly increased the energy potential, i.e. the ratio of phosphocreatine to β-nucleotide triphosphate (PCr/βNTP) and PCr/inorganic phosphate (PCr/Pi) as measured by ³¹P-MRS. Rats with hemispheric cerebral hypoxic-ischemic insult that had received Cr showed a significant reduction (25%) of the volume of edemic brain tissue compared with controls as calculated from diffusion-weighted images (DWI). Thus, prophylactic Cr supplementation demonstrated a significant neuroprotective effect 24 h after transient cerebral HI. We hypothesize that neuroprotection is probably due to the availability of a larger metabolic substrate pool leading to a reduction of the secondary energy failure because DWI has been reported to correlate with the PCr/Pi ratio in the acute phase of injury. Additional protection by Cr may be related to prevention of calcium overload, prevention of mitochondrial permeability transition pore opening and direct antioxidant effects.