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Neuroimaging is widely used to aid in the diagnosis and clinical management of neonates with neonatal encephalopathy (NE). Yet, despite widespread use clinically, there are few published guidelines on neuroimaging for neonates with NE. This review outlines the primary patterns of brain injury associated with hypoxic-ischemic injury in neonates with NE and their frequency, associated neuropathological features, and risk factors. In addition, it provides an overview of neuroimaging methods, including the most widely used scoring systems used to characterize brain injury in these neonates and their utility as predictive biomarkers. Last, recommendations for neuroimaging in neonates with NE are presented., (Copyright © 2021. Published by Elsevier Ltd.)

Objective: To assess the impact of hypothermic neural rescue for perinatal asphyxia at birth on healthcare costs of survivors aged 6-7 years, and to quantify the relationship between costs and overall disability levels., Design: 6-7 years follow-up of surviving children from the Total Body Hypothermia for Neonatal Encephalopathy (TOBY) trial., Setting: Community study including a single parental questionnaire to collect information on children's healthcare resource use., Patients: 130 UK children (63 in the control group, 67 in the hypothermia group) whose parents consented and returned the questionnaire., Interventions: Intensive care with cooling of the body to 33.5°C for 72 hours or intensive care alone., Main Outcome Measures: Healthcare resource usage and costs over the preceding 6 months., Results: At 6-7 years, mean (SE) healthcare costs per child were £1543 (£361) in the hypothermia group and £2549 (£812) in the control group, giving a saving of -£1005 (95% CI -£2734 to £724). Greater levels of overall disability were associated with progressively higher costs, and more parents in the hypothermia group were employed (64% vs 47%). Results were sensitive to outlying observations., Conclusions: Cost results although not significant favoured moderate hypothermia and so complement the clinical results of the TOBY Children study. Estimates were however sensitive to the care requirements of two seriously ill children in the control group. A quantification of the relationship between costs and levels of disability experienced will be useful to healthcare professionals, policy makers and health economists contemplating the long-term economic consequences of perinatal asphyxia and hypothermic neural rescue., Trial Registration Number: This study reports on the follow-up of the TOBY clinical trial: ClinicalTrials. gov number NCT01092637., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

Introduction: Objective biomarkers are needed to assess neuroprotective therapies after perinatal hypoxic-ischemic encephalopathy (HIE). We tested the hypothesis that, in infants who underwent therapeutic hypothermia after perinatal HIE, neurodevelopmental performance was predicted by fractional anisotropy (FA) values in the white matter (WM) on early diffusion tensor imaging (DTI) as assessed by means of tract-based spatial statistics (TBSS)., Methods: We studied 43 term infants with HIE. Developmental assessments were carried out at a median (range) age of 24 (12-28) mo., Results: As compared with infants with favorable outcomes, those with unfavorable outcomes had significantly lower FA values (P < 0.05) in the centrum semiovale, corpus callosum (CC), anterior and posterior limbs of the internal capsule, external capsules, fornix, cingulum, cerebral peduncles, optic radiations, and inferior longitudinal fasciculus. In a second analysis in 32 assessable infants, the Griffiths Mental Development Scales (Revised) (GMDS-R) showed a significant linear correlation (P < 0.05) between FA values and developmental quotient (DQ) and all its component subscale scores., Discussion: DTI analyzed by TBSS provides a qualified biomarker that can be used to assess the efficacy of additional neuroprotective therapies after HIE.

Introduction: We sought to determine whether 18- to 22-mo neurodevelopmental outcomes predicted functional outcomes at 7-8 y for survivors of the CoolCap study of therapeutic hypothermia for neonates with hypoxic-ischemic encephalopathy., Results: WeeFIM ratings were completed at 7-8 y of age on 62 (32 cooled; 30 standard care) of 135 surviving children who had had neurodevelopmental assessment at 18 mo. There was 1 refusal, 58 lost to follow-up, and 14 children whose centers declined to participate. Disability status at 18 mo was strongly associated with WeeFIM ratings (P < 0.001); there was no significant effect of treatment (P = 0.83)., Discussion: Functional outcome at 7-8 y of survivors of neonatal encephalopathy is associated with 18-mo neurodevelopmental assessment, supporting the long-term predictive value of a favorable outcome at 18 mo assessed by published trials of therapeutic hypothermia., Methods: All surviving children who participated in the CoolCap study and were assessed at 18 mo were eligible for reassessment using the WeeFIM instrument that qualitatively measures self-care, mobility, and cognitive function. Center investigators obtained consent from the families for a certified researcher to administer the WeeFIM instrument by phone.

Evaluation of infants with hypoxic ischemic encephalopathy by magnetic resonance spectroscopy and imaging is useful to direct clinical care, and may assist the evaluation of candidate neuroprotective therapies. Cerebral metabolites measured by magnetic resonance spectroscopy, and visual analysis of magnetic resonance images during the first 30 days after birth accurately predict later neurological outcome and are valid biomarkers of the key physiological processes underlying brain injury in neonatal hypoxic ischemic encephalopathy. Visual assessment of magnetic resonance images may also be a suitable surrogate outcome in studies of neuroprotective therapies but current magnetic resonance methods are relatively inefficient for use in early phase, first in human infant studies of novel neuroprotective therapies. However, diffusion tensor imaging and analysis of fractional anisotropy with tract-based spatial statistics promises to be a highly efficient biomarker and surrogate outcome for rapid preliminary evaluation of promising therapies for neonatal hypoxic ischemic injury. Standardisation of scanning protocols and data analysis between different scanners is essential., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: To estimate the cost-effectiveness (CE) of total body hypothermia plus intensive care versus intensive care alone to treat neonatal encephalopathy., Methods: Decision analytic modeling was used to synthesize mortality and morbidity data from three randomized controlled trials, the Total Body Hypothermia for Neonatal Encephalopathy Trial (TOBY), National Institute of Child Health and Human Development (NICHD), and CoolCap trials. Cost data inputs were informed by TOBY, the sole source of prospectively collected resource utilization data for encephalopathic infants. CE was expressed in terms of incremental cost per disability-free life year (DFLY) gained. Probabilistic sensitivity analysis was performed to generate CE acceptability curves (CEACs)., Results: Cooling led to a cost increase of £3787 (95% confidence interval [CI]: -2516, 12,360) (€5115; 95% CI: -3398-16,694; US$5344; 95% CI: -3598, 26,356; using 2006 Organisation for Economic Co-operation and Development (OECD) purchasing power parities) and a DFLY gain of 0.19 (95%CI: 0.07-0.31) over the first 18 months after birth. The incremental cost per DFLY gained was £19,931 (€26,920; US$28,124). The baseline CEAC showed that if decision-makers are willing to pay £30,000 for an additional DFLY, there is a 69% probability that cooling is cost-effective. The probability of CE exceeded 99% at this threshold when the throughput of infants was increased to reflect the national incidence of neonatal encephalopathy or when the time horizon of the economic evaluation was extended to 18 years after birth., Conclusions: The probability that cooling is a cost-effective treatment for neonatal encephalopathy is finely balanced over the first 18 months after birth but increases substantially when national incidence data or an extended time horizon are considered., (© 2010, International Society for Pharmacoeconomics and Outcomes Research (ISPOR).)

Perinatal hypoxic-ischemic encephalopathy can be a devastating complication of childbirth. Herein, the authors review the pathophysiology of hypoxic-ischemic encephalopathy and the current status of neuroprotective strategies to ameliorate the injury centering on four themes: (1) monitoring in the perinatal period, (2) rapid identification of affected neonates to allow timely institution of therapy, (3) preconditioning therapy (a therapeutic that reduces the brain vulnerability) before hypoxic-ischemic encephalopathy, and (4) prompt institution of postinsult therapies to ameliorate the evolving injury. Recent clinical trials have demonstrated the significant benefit for hypothermic therapy in the postnatal period; furthermore, there is accumulating preclinical evidence that adjunctive therapies can enhance hypothermic neuroprotection. Advances in the understanding of preconditioning may lead to the administration of neuroprotective agents earlier during childbirth. Although most of these neuroprotective strategies have not yet entered clinical practice, there is a significant hope that further developments will enhance hypothermic neuroprotection.

Objective: To determine whether moderate hypothermia after hypoxic-ischaemic encephalopathy in neonates improves survival and neurological outcome at 18 months of age., Design: A meta-analysis was performed using a fixed effect model. Risk ratios, risk difference, and number needed to treat, plus 95% confidence intervals, were measured., Data Sources: Studies were identified from the Cochrane central register of controlled trials, the Oxford database of perinatal trials, PubMed, previous reviews, and abstracts. Review methods Reports that compared whole body cooling or selective head cooling with normal care in neonates with hypoxic-ischaemic encephalopathy and that included data on death or disability and on specific neurological outcomes of interest to patients and clinicians were selected. Results We found three trials, encompassing 767 infants, that included information on death and major neurodevelopmental disability after at least 18 months' follow-up. We also identified seven other trials with mortality information but no appropriate neurodevelopmental data. Therapeutic hypothermia significantly reduced the combined rate of death and severe disability in the three trials with 18 month outcomes (risk ratio 0.81, 95% confidence interval 0.71 to 0.93, P=0.002; risk difference -0.11, 95% CI -0.18 to -0.04), with a number needed to treat of nine (95% CI 5 to 25). Hypothermia increased survival with normal neurological function (risk ratio 1.53, 95% CI 1.22 to 1.93, P<0.001; risk difference 0.12, 95% CI 0.06 to 0.18), with a number needed to treat of eight (95% CI 5 to 17), and in survivors reduced the rates of severe disability (P=0.006), cerebral palsy (P=0.004), and mental and the psychomotor developmental index of less than 70 (P=0.01 and P=0.02, respectively). No significant interaction between severity of encephalopathy and treatment effect was detected. Mortality was significantly reduced when we assessed all 10 trials (1320 infants; relative risk 0.78, 95% CI 0.66 to 0.93, P=0.005; risk difference -0.07, 95% CI -0.12 to -0.02), with a number needed to treat of 14 (95% CI 8 to 47)., Conclusions: In infants with hypoxic-ischaemic encephalopathy, moderate hypothermia is associated with a consistent reduction in death and neurological impairment at 18 months.

Background: Moderate hypothermia in neonates with hypoxic-ischaemic encephalopathy might improve survival and neurological outcomes at up to 18 months of age, although complete neurological assessment at this age is difficult. To ascertain more precisely the effect of therapeutic hypothermia on neonatal cerebral injury, we assessed cerebral lesions on MRI scans of infants who participated in the Total Body Hypothermia for Neonatal Encephalopathy (TOBY) trial., Methods: In the TOBY trial hypoxic-ischaemic encephalopathy was graded clinically according to the changes seen on amplitude integrated EEG, and infants were randomly assigned to intensive care with or without cooling by central telephone randomisation. The relation between allocation to hypothermia or normothermia and cerebral lesions was assessed by logistic regression with perinatal factors as covariates, and adjusted odds ratios (ORs) were calculated. The TOBY trial is registered, number ISRCTN 89547571., Findings: 325 infants were recruited in the TOBY trial between 2002 and 2006. Images were available for analysis from 131 infants. Therapeutic hypothermia was associated with a reduction in lesions in the basal ganglia or thalamus (OR 0.36, 95% CI 0.15-0.84; p=0.02), white matter (0.30, 0.12-0.77; p=0.01), and abnormal posterior limb of the internal capsule (0.38, 0.17-0.85; p=0.02). Compared with non-cooled infants, cooled infants had fewer scans that were predictive of later neuromotor abnormalities (0.41, 0.18-0.91; p=0.03) and were more likely to have normal scans (2.81, 1.13-6.93; p=0.03). The accuracy of prediction by MRI of death or disability to 18 months of age was 0.84 (0.74-0.94) in the cooled group and 0.81 (0.71-0.91) in the non-cooled group., Interpretation: Therapeutic hypothermia decreases brain tissue injury in infants with hypoxic-ischaemic encephalopathy. The predictive value of MRI for subsequent neurological impairment is not affected by therapeutic hypothermia., Funding: UK Medical Research Council; UK Department of Health., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The development of the concepts of delayed post-ischaemic neuronal death and neural rescue brought about a search for clinical treatments to reduce brain damage after birth asphyxia. Cooling had long been an unproven empyrical therapy, and a 20 year programme of careful laboratory and clinical research has proved that hypothermia reduces neurological damage in infants suffering perinatal asphyxial encephalopathy.

Background: Whether hypothermic therapy improves neurodevelopmental outcomes in newborn infants with asphyxial encephalopathy is uncertain., Methods: We performed a randomized trial of infants who were less than 6 hours of age and had a gestational age of at least 36 weeks and perinatal asphyxial encephalopathy. We compared intensive care plus cooling of the body to 33.5 degrees C for 72 hours and intensive care alone. The primary outcome was death or severe disability at 18 months of age. Prespecified secondary outcomes included 12 neurologic outcomes and 14 other adverse outcomes., Results: Of 325 infants enrolled, 163 underwent intensive care with cooling, and 162 underwent intensive care alone. In the cooled group, 42 infants died and 32 survived but had severe neurodevelopmental disability, whereas in the noncooled group, 44 infants died and 42 had severe disability (relative risk for either outcome, 0.86; 95% confidence interval [CI], 0.68 to 1.07; P=0.17). Infants in the cooled group had an increased rate of survival without neurologic abnormality (relative risk, 1.57; 95% CI, 1.16 to 2.12; P=0.003). Among survivors, cooling resulted in reduced risks of cerebral palsy (relative risk, 0.67; 95% CI, 0.47 to 0.96; P=0.03) and improved scores on the Mental Developmental Index and Psychomotor Developmental Index of the Bayley Scales of Infant Development II (P=0.03 for each) and the Gross Motor Function Classification System (P=0.01). Improvements in other neurologic outcomes in the cooled group were not significant. Adverse events were mostly minor and not associated with cooling., Conclusions: Induction of moderate hypothermia for 72 hours in infants who had perinatal asphyxia did not significantly reduce the combined rate of death or severe disability but resulted in improved neurologic outcomes in survivors. (Current Controlled Trials number, ISRCTN89547571.), (2009 Massachusetts Medical Society)

Objective: Our goal was to evaluate whether head cooling with mild systemic hypothermia for neonatal encephalopathy is associated with greater requirement for volume or inotrope support., Patients and Methods: We studied term infants (>/=36 weeks) with moderate-to-severe neonatal encephalopathy plus abnormal amplitude integrated electroencephalography, randomly assigned to head cooling for 72 hours starting within 6 hours of birth, with the rectal temperature maintained at 34.5 degrees C +/- 0.5 degrees C (n = 112), or conventional care (n = 118)., Design: This was a multicenter randomized, controlled study (the CoolCap trial). The primary outcome was the time relationship between mean arterial blood pressure and subsequent administration of inotropes or volume administration., Results: Pooled data from 0 to 76 hours after randomization revealed no difference in mean arterial blood pressure between groups and significantly lower mean heart rate during cooling. The use of inotropes or volume was related to preceding mean arterial blood pressure and not to treatment group in the first 24 hours. In contrast, from 24 to 76 hours, there was no effect of mean arterial blood pressure, but there was an overall reduction in pressure support over time and significantly more frequent pressure support in the cooled group than in controls., Conclusions: Mild systemic hypothermia did not affect arterial blood pressure or initial treatment with inotropes or volume in infants with moderate-to-severe encephalopathy but was associated with an apparent change in physician behavior, with slower withdrawal of therapy in cooled infants.

Objective: To evaluate whether therapeutic hypothermia alters the prognostic value of clinical grading of neonatal encephalopathy., Study Design: This study was a secondary analysis of a multicenter study of 234 term infants with neonatal encephalopathy randomized to head cooling for 72 hours starting within 6 hours of birth, with rectal temperature maintained at 34.5 degrees C +/- 0.5 degrees C, followed by re-warming for 4 hours, or standard care at 37.0 degrees C +/- 0.5 degrees C. Severity of encephalopathy was measured pre-randomization and on day 4, after re-warming, in 177 infants; 31 infants died before day 4, and data were missing for 10 infants. The primary outcome was death or severe disability at 18 months of age., Results: Milder pre-randomization encephalopathy, greater improvement in encephalopathy from randomization to day 4, and cooling were associated with favorable outcome in multivariate binary logistic regression. Hypothermia did not affect severity of encephalopathy at day 4, however, in infants with moderate encephalopathy at day 4, those treated with hypothermia had a significantly higher rate of favorable outcome (31/45 infants, 69%, P = .006) compared with standard care (12/33, 36%)., Conclusion: Infants with moderate encephalopathy on day 4 may have a more favorable prognosis after hypothermia treatment than expected after standard care.

Experimental studies show that, following hypoxic ischaemic injury, mild induced hypothermia-a reduction of body temperature by about 3 degrees C -- preserves cerebral energy metabolism, reduces cerebral tissue injury and improves neurological function. Randomized trials in full-term and near-full-term newborns suggest that treatment with mild hypothermia is safe and improves survival without disabilities up to 18 months of age. Although the optimal time of initiation, the depth and duration, and the method of cooling are uncertain, in the absence of specific treatments many clinicians will wish to consider treating asphyxiated infants with hypothermia. Guidance now needs to be provided to promote uniform practice, to avoid inappropriate treatment and to foster continuing collaboration in future studies of neuroprotection following asphyxia. If the promising results of the current trials are confirmed by the findings from other on-going studies, with longer follow-up, the impact of such a treatment on the babies, their families and health resources in the shorter and longer terms will be considerable.

c-Jun N-terminal kinase 3 (JNK3) is a member of the stress-activated group of mitogen-activated protein kinases. c-Jun N-terminal kinase 3 is a potent mediator of apoptosis and the use of JNK inhibitors or jnk3 gene deletion each protect against brain injury in adults. However, little is known about the role of JNK3 or its mechanism of action in neonatal brain injury. The aim of the present study was to compare the vulnerability of neonatal JNK3 knockout (JNK3 KO) mice and wild-type (WT) mice to cerebral hypoxic-ischaemic injury (HII) using unilateral-carotid occlusion combined with transient hypoxia. The degree of neural tissue loss in JNK3 KO mice was substantially reduced compared with WT mice (JNK3 KO 27.8%+/-2.8% versus WT 48.3%+/-2.0%, P

Objective: The goal of this study was to evaluate the role of factors that may determine the efficacy of treatment with delayed head cooling and mild systemic hypothermia for neonatal encephalopathy., Methods: A total of 218 term infants with moderate to severe neonatal encephalopathy plus abnormal amplitude-integrated electroencephalographic recordings, assigned randomly to head cooling for 72 hours, starting within 6 hours after birth (with the rectal temperature maintained at 34.5 +/- 0.5 degrees C), or conventional care, were studied. Death or severe disability at 18 months of age was assessed in a multicenter, randomized, controlled study (the CoolCap trial)., Results: Treatment, lower encephalopathy grade, lower birth weight, greater amplitude-integrated electroencephalographic amplitude, absence of seizures, and higher Apgar score, but not gender or gestational age, were associated significantly with better outcomes. In a multivariate analysis, each of the individually predictive factors except for Apgar score remained predictive. There was a significant interaction between treatment and birth weight, categorized as > or =25th or <25th percentile for term, such that larger infants showed a lower frequency of favorable outcomes in the control group but greater improvement with cooling. For larger infants, the number needed to treat was 3.8. Pyrexia (> or =38 degrees C) in control infants was associated with adverse outcomes. Although there was a small correlation with birth weight, the adverse effect of greater birth weight in control infants remained significant after adjustment for pyrexia and severity of encephalopathy., Conclusions: Outcomes after hypothermic treatment were strongly influenced by the severity of neonatal encephalopathy. The protective effect of hypothermia was greater in larger infants.

Objective: To observe amplitude integrated electroencephalography (aEEG) in neonates receiving ECMO and to determine whether mild hypothermia influenced the aEEG recording., Methods: Twenty-six consecutive neonates enrolled in a pilot study of mild hypothermia during ECMO were studied. The first group (N=6) was maintained at 37 degrees C throughout the study period. Subsequent groups were cooled to 36 degrees C (N=4), 35 degrees C (N=5), and finally 34 degrees C (N=6) respectively for 24 h and the final group (N=5) to 34 degrees C for 48 h before being rewarmed to 37 degrees C. The aEEG was recorded continuously during the first 5 days of ECMO. The aEEG was classified as normal, moderately or severely suppressed and examined for the occurrence of seizures. To assess the effect of temperature, the aEEG was compared over 12 h during the final 6 h of cooling and during the first 6 h once infants were rewarmed., Results: No change in aEEG amplitude was noted over the temperature range studied. Of the 26 traces obtained, 16 (62%) were normal throughout, 6 (23%) were intermittently moderately abnormal and 1 (14%) was severely abnormal. Three (11%) traces had periods of frequent seizure activity and these were not associated with clinical manifestations in two neonates. In one infant who suffered a cerebral haemorrhage, the aEEG became abnormal before cranial ultrasound abnormalities were apparent., Conclusions: Continuous cerebral monitoring with aEEG is feasible during ECMO and may add information to clinical examination. Mild hypothermia to 34 degrees C for up to 48 h does not influence the aEEG suggesting that cerebral monitoring with aEEG is possible during mild hypothermia.

Well constructed and carefully analysed trials of hypothermic neural rescue therapy for infants with neonatal encephalopathy have recently been reported. The data suggest that either selective head cooling or total body cooling reduces the combined chance of death or disability after birth asphyxia. However, as there are still unanswered questions about these treatments, many may still feel that further data are needed before health care policy can be changed to make cooling the standard of care for all babies with suspected birth asphyxia.

Hypothermia induced by whole-body cooling (WBC) and selective head cooling (SHC) both reduce brain injury after hypoxia-ischemia in newborn animals, but it is not known how these treatments affect the incidence or pattern of brain injury in human newborns. To assess this, 14 term infants with hypoxic-ischemic encephalopathy (HIE) treated with SHC, 20 infants with HIE treated with WBC, and 52 noncooled infants with HIE of similar severity were studied with magnetic resonance imaging in the neonatal period. Infants fulfilling strict criteria for HIE were recruited into the study after assessment of an amplitude-integrated electroencephalography (aEEG). Cooling was commenced within 6 hours of birth and continued for 48 to 72 hours. Hypothermia was not associated with unexpected or unusual lesions, and the prevalence of intracranial hemorrhage was similar in all 3 groups. Both modes of hypothermia were associated with a decrease in basal ganglia and thalamic lesions, which are predictive of abnormal outcome. This decrease was significant in infants with a moderate aEEG finding but not in those with a severe aEEG finding. A decrease in the incidence of severe cortical lesions was seen in the infants treated with SHC.

Background: Cerebral hypothermia can improve outcome of experimental perinatal hypoxia-ischaemia. We did a multicentre randomised controlled trial to find out if delayed head cooling can improve neurodevelopmental outcome in babies with neonatal encephalopathy., Methods: 234 term infants with moderate to severe neonatal encephalopathy and abnormal amplitude integrated electroencephalography (aEEG) were randomly assigned to either head cooling for 72 h, within 6 h of birth, with rectal temperature maintained at 34-35 degrees C (n=116), or conventional care (n=118). Primary outcome was death or severe disability at 18 months. Analysis was by intention to treat. We examined in two predefined subgroup analyses the effect of hypothermia in babies with the most severe aEEG changes before randomisation--ie, severe loss of background amplitude, and seizures--and those with less severe changes., Findings: In 16 babies, follow-up data were not available. Thus in 218 infants (93%), 73/110 (66%) allocated conventional care and 59/108 (55%) assigned head cooling died or had severe disability at 18 months (odds ratio 0.61; 95% CI 0.34-1.09, p=0.1). After adjustment for the severity of aEEG changes with a logistic regression model, the odds ratio for hypothermia treatment was 0.57 (0.32-1.01, p=0.05). No difference was noted in the frequency of clinically important complications. Predefined subgroup analysis suggested that head cooling had no effect in infants with the most severe aEEG changes (n=46, 1.8; 0.49-6.4, p=0.51), but was beneficial in infants with less severe aEEG changes (n=172, 0.42; 0.22-0.80, p=0.009)., Interpretation: These data suggest that although induced head cooling is not protective in a mixed population of infants with neonatal encephalopathy, it could safely improve survival without severe neurodevelopmental disability in infants with less severe aEEG changes.

Since hypothermia may be a potential treatment for perinatal cerebral hypoxic-ischemic injury, we used an established neonatal model of hypoxia-ischemia to determine the time after injury at which cooling had the best protective effect. Fourteen-day-old Wistar rats were subjected to right carotid artery ligation and hypoxia (8% O(2) for 90 min). Immediately at the end of hypoxia (defined as 0h), animals were either maintained at normal body temperature until sacrifice (normothermia) or subjected to hypothermia. In a preliminary study, the effects of a reduction in temperature and the duration of such cooling were investigated; animals were cooled (until brain temperature reached 33 degrees C or 30 degrees C) for 2, 4, or 6 h commencing immediately after hypoxia. In a second study, animals were cooled (brain temperature 30 degrees C) for 6 h commencing at either 0, 2, 4, or 6 h after the end of hypoxia. Sham-operated animals were used as controls. Twenty-four hours after hypoxia-ischemia, cerebral energy metabolism was measured by phosphorus magnetic resonance spectroscopy, and at 5 d cerebral infarction was measured by planimetry. In normothermic animals the ratio of phosphocreatine/inorganic phosphate (PCr/Pi) had fallen markedly 24 h following hypoxia-ischemia. In contrast, animals cooled between 6 and 12 h displayed high PCr/Pi ratios similar to those in control animals. Similarly, after 5 d, infarct area was significantly reduced only in animals cooled between 6 and 12 h after injury. These results indicate that cooling between 6 and 12 h after hypoxia-ischemia is more effective in reducing cerebral injury than other cooling regimes and suggest that the physiologic events during this period are critical for understanding cerebral infarction.

Fas/CD95/Apo-1 is a cell surface receptor that transduces apoptotic death signals following activation and has been implicated in triggering apoptosis in infected or damaged cells in disease states. Apoptosis is a major mechanism of neuronal loss following hypoxic-ischemic injury to the developing brain, although the role of Fas in this process has not been studied in detail. In the present study, we have investigated the expression and function of Fas in neuronal cells in vitro and in vivo. Fas was found to be expressed in the 14 day old rat brain, with strongest expression in the cortex, hippocampus and cerebellum. Cross-linking of Fas induced neuronal apoptosis both in neuronal PC12 cells in culture and following intracerebral injection in vivo, indicating that neuronal Fas was functional as a death receptor. This death was shown to be caspase dependent in primary neuronal cultures and was blocked by the selective caspase 8 inhibitor IETD. Finally, cerebral hypoxia-ischemia resulted in a strong lateralised upregulation of Fas in the hippocampus, that peaked six to twelve hours after the insult and was greater on the side of injury. These results suggest that Fas may be involved in neuronal apoptosis following hypoxic-ischemic injury to the developing brain.