Analysis of the NMDA in Focal Cerebral Ischemia in Rats

NMDAR (N-methyl-D-aspartate receptor) is one subtype of ionotrophic glutamate receptor which is extensively distributed in the central nervous system (CNS). In the mammalian CNS, NMDAR serves prominent roles in the pathophysiologic process of cerebral ischemia. This study aimed to investigate the pattern of expression of protein and gene of the excitatory neurotransmitter NMDAR in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. 120 rats were randomly divided into 6 groups (20 animals each): control - no surgery; sham - simulation of surgery; ischemic - focal ischemia for 1 hour, without reperfusion; ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. Ten animals from each experimental group were used to establish the volume of infarct. Transient focal cerebral ischemia was obtained in rats by occlusion of the middle cerebral artery with an intraluminal suture. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the protein and gene NMDA were evaluated by immunohistochemistry and quantitative real-time PCR, respectively. Increases in the protein and gene NMDA receptor in the ischemics areas were observed and these increases were reduced by hypothermia and ketoprofen. The increase in the NMDA receptor protein and gene expression observed in the ischemic animals was reduced by neuroprotection (hypothermia and ketoprofen). The NMDA receptor increases in the ischemic area suggests that the NMDA mediated neuroexcitotoxicity plays an important role in cell death and that the neuroprotective effect of both, hypothermia and ketoprofen is directly involved with the NMDA.

Hypothermia And Early Neonatal Mortality In Preterm Infants.

To evaluate intervention practices associated with hypothermia at both 5 minutes after birth and at neonatal intensive care unit (NICU) admission and to determine whether hypothermia at NICU admission is associated with early neonatal death in preterm infants. This prospective cohort included 1764 inborn neonates of 22-33 weeks without malformations admitted to 9 university NICUs from August 2010 through April 2012. All centers followed neonatal International Liaison Committee on Resuscitation recommendations for the stabilization and resuscitation in the delivery room (DR). Variables associated with hypothermia (axillary temperature <36.0 °C) 5 minutes after birth and at NICU admission, as well as those associated with early death, were analyzed by logistic regression. Hypothermia 5 minutes after birth and at NICU admission was noted in 44% and 51%, respectively, with 6% of early neonatal deaths. Adjusted for confounding variables, practices associated with hypothermia at 5 minutes after birth were DR temperature <25 °C (OR 2.13, 95% CI 1.67-2.28), maternal temperature at delivery <36.0 °C (OR 1.93, 95% CI 1.49-2.51), and use of plastic bag/wrap (OR 0.53, 95% CI 0.40-0.70). The variables associated with hypothermia at NICU admission were DR temperature <25 °C (OR 1.44, 95% CI 1.10-1.88), respiratory support with cold air in the DR (OR 1.40, 95% CI 1.03-1.88) and during transport to NICU (OR 1.51, 95% CI 1.08-2.13), and cap use (OR 0.55, 95% CI 0.39-0.78). Hypothermia at NICU admission increased the chance of early neonatal death by 1.64-fold (95% CI 1.03-2.61). Simple interventions, such as maintaining DR temperature >25 °C, reducing maternal hypothermia prior to delivery, providing plastic bags/wraps and caps for the newly born infants, and using warm resuscitation gases, may decrease hypothermia at NICU admission and improve early neonatal survival.

Renal hypothermia: Experience in pigs and clinical trial

Purpose: This study was designed to compare the effectiveness of two methods of inducing renal hypothermia through laparoscopy in pigs and humans. Materials and Methods: Twelve pigs were divided into four groups of three animals each. Both kidneys of the animals in Groups A, B, and C were submitted to pelvic irrigation with cold saline (4 degrees C) for 20 minutes, with flow rates of 5 mL/min, 10 mL/min, and 15 mL/min, respectively. In Group D renal hypothermia was induced by intracorporeal ice slush applied to the surface for 20 minutes. All maneuvers were performed laparoscopically and renal cortex temperature was measured by a thermocouple needle. Five human patients also underwent laparoscopic partial nephrectomy due to renal cell carcinoma. In one case renoprotection was induced by retrograde endoscopic cold saline perfusion at a flow rate of 10 mL/min. In the remaining four patients we induced renal hypothermia via laparoscopic application of ice slush. The renal temperature of the human patients was also monitored using a thermocouple needle. Results: In the pigs, at 20 minutes of renal pelvis perfusion the mean renal temperature, the temperature drop, and saline flow per gram of kidney were: Group A, -29.5 degrees C +/- 1.1 (-6.3 degrees C; 0.10 mL); Group B, -22.8 degrees C +/- 1.1 (-13.1 degrees C; 0.22 mL); and Group C, -21.1 degrees C +/- 0.9 (-14.9 degrees C; 0.31 mL). In Group D the mean renal cortex temperature at 20 minutes was 13.6 degrees C +/- 1.2, a drop of -22.5 degrees C. There were striking differences among the groups (P < 0.0001). The laparoscopic partial nephrectomy was uneventful in all five human patients. The lowest renal cortex temperature was 32.5 degrees C, seen in the patient who submitted to pelvic irrigation with cold saline, and the mean temperature drop was 19.1 degrees C +/- 2.5 degrees C in the patients who submitted to ice slush-induced renal hypothermia. Conclusions: Induction of renal hypothermia using intracorporeal ice slush confers lower kidney temperatures than endoscopically-induced cold saline perfusion.

Hypothermia during endotoxemic shock in female mice lacking inducible nitric oxide synthase

The present study was undertaken to evaluate: (1) whether lipopolysaccharide LPS-incluced hypothermic responses may be altered during two estrous cycle phases, proestrus and diestrus, and after ovariectomy, followed by hormonal supplementation and (2) whether nitric oxide (NO) plays a role on LPS-induced hypothermia responses in female mice. Experiments were performed on adult female wild-type (WT) C57BL and inducible NO synthase knockout (KO) mice weighing 18 to 30 g. Endotoxemia was induced by intraperitoneal LIPS administration from Escherichia coli at a nonlethal dose of 10 mg/kg, and body temperature was measured by biotelemetry. Hormonal replacement was performed in ovariectomized mice through 17 beta-estradiol Silastic capsules (100 mu g) and s.c. injection of progesterone (0.5 mg per animal). We observed that during the diestrus phase, mice presented more intensive hypothermia than during proestrus phase, and hormonal supplementation with 17 beta-estradiol and progesterone attenuated hypothermia in ovariectomized mice. During diestrus and ovariectomy, KO mice had higher hypothermic response when compared with the WT group. During proestrus, the lack of statistical difference between KO and WT mice could be consequent of lower ovarian hormones plasma levels. After hormonal replacement, hypothermia was reverted in KO groups probably because of higher ovarian hormonal levels. In summary, the results demonstrated that NO release by inducible NO synthase has an important thermoregulatory role in LPS-incluced hypothermia in female mice. Besides, this involvement is directly dependent on the presence of ovarian hormones and their respective levels.

Predicting Outcome after Cardiopulmonary Arrest in Therapeutic Hypothermia Patients: Clinical, Electrophysiological and Imaging Prognosticators

INTRODUCTION: Predicting outcome in comatose survivors of cardiac arrest is based on data validated by guidelines that were established before the era of therapeutic hypothermia. We sought to evaluate the predictive value of clinical, electrophysiological and imaging data on patients submitted to therapeutic hypothermia. MATERIALS AND METHODS: A retrospective analysis of consecutive patients receiving therapeutic hypothermia during years 2010 and 2011 was made. Neurological examination, somatosensory evoked potentials, auditory evoked potentials, electroencephalography and brain magnetic resonance imaging were obtained during the first 72 hours. Glasgow Outcome Scale at 6 months, dichotomized into bad outcome (grades 1 and 2) and good outcome (grades 3, 4 and 5), was defined as the primary outcome. RESULTS: A total of 26 patients were studied. Absent pupillary light reflex, absent corneal and oculocephalic reflexes, absent N20 responses on evoked potentials and myoclonic status epilepticus showed no false-positives in predicting bad outcome. A malignant electroencephalographic pattern was also associated with a bad outcome (p = 0.05), with no false-positives. Two patients with a good outcome showed motor responses no better than extension (false-positive rate of 25%, p = 0.008) within 72 hours, both of them requiring prolonged sedation. Imaging findings of brain ischemia did not correlate with outcome. DISCUSSION: Absent pupillary, corneal and oculocephalic reflexes, absent N20 responses and a malignant electroencephalographic pattern all remain accurate predictors of poor outcome in cardiac arrest patients submitted to therapeutic hypothermia. CONCLUSION: Prolonged sedation beyond the hypothermia period may confound prediction strength of motor responses.

Prognostication of neurologic outcome in cardiac arrest patients after mild therapeutic hypothermia: a meta-analysis of the current literature.

PURPOSE: To assess the sensitivity and false positive rate (FPR) of neurological examination and somatosensory evoked potentials (SSEPs) to predict poor outcome in adult patients treated with therapeutic hypothermia after cardiopulmonary resuscitation (CPR). METHODS: MEDLINE and EMBASE were searched for cohort studies describing the association of clinical neurological examination or SSEPs after return of spontaneous circulation with neurological outcome. Poor outcome was defined as severe disability, vegetative state and death. Sensitivity and FPR were determined. RESULTS: A total of 1,153 patients from ten studies were included. The FPR of a bilaterally absent cortical N20 response of the SSEP could be calculated from nine studies including 492 patients. The SSEP had an FPR of 0.007 (confidence interval, CI, 0.001-0.047) to predict poor outcome. The Glasgow coma score (GCS) motor response was assessed in 811 patients from nine studies. A GCS motor score of 1-2 at 72 h had a high FPR of 0.21 (CI 0.08-0.43). Corneal reflex and pupillary reactivity at 72 h after the arrest were available in 429 and 566 patients, respectively. Bilaterally absent corneal reflexes had an FPR of 0.02 (CI 0.002-0.13). Bilaterally absent pupillary reflexes had an FPR of 0.004 (CI 0.001-0.03). CONCLUSIONS: At 72 h after the arrest the motor response to painful stimuli and the corneal reflexes are not a reliable tool for the early prediction of poor outcome in patients treated with hypothermia. The reliability of the pupillary response to light and the SSEP is comparable to that in patients not treated with hypothermia.

Evolution of insulin sensitivity and its variability in out-of-hospital cardiac arrest (OHCA) patients treated with hypothermia.

INTRODUCTION: Therapeutic hypothermia (TH) is often used to treat out-of-hospital cardiac arrest (OHCA) patients who also often simultaneously receive insulin for stress-induced hyperglycaemia. However, the impact of TH on systemic metabolism and insulin resistance in critical illness is unknown. This study analyses the impact of TH on metabolism, including the evolution of insulin sensitivity (SI) and its variability, in patients with coma after OHCA. METHODS: This study uses a clinically validated, model-based measure of SI. Insulin sensitivity was identified hourly using retrospective data from 200 post-cardiac arrest patients (8,522 hours) treated with TH, shortly after admission to the intensive care unit (ICU). Blood glucose and body temperature readings were taken every one to two hours. Data were divided into three periods: 1) cool (T <35°C); 2) an idle period of two hours as normothermia was re-established; and 3) warm (T >37°C). A maximum of 24 hours each for the cool and warm periods was considered. The impact of each condition on SI is analysed per cohort and per patient for both level and hour-to-hour variability, between periods and in six-hour blocks. RESULTS: Cohort and per-patient median SI levels increase consistently by 35% to 70% and 26% to 59% (P <0.001) respectively from cool to warm. Conversely, cohort and per-patient SI variability decreased by 11.1% to 33.6% (P <0.001) for the first 12 hours of treatment. However, SI variability increases between the 18th and 30th hours over the cool to warm transition, before continuing to decrease afterward. CONCLUSIONS: OCHA patients treated with TH have significantly lower and more variable SI during the cool period, compared to the later warm period. As treatment continues, SI level rises, and variability decreases consistently except for a large, significant increase during the cool to warm transition. These results demonstrate increased resistance to insulin during mild induced hypothermia. Our study might have important implications for glycaemic control during targeted temperature management.

Prognostication after cardiac arrest and hypothermia: a prospective study.

Current American Academy of Neurology (AAN) guidelines for outcome prediction in comatose survivors of cardiac arrest (CA) have been validated before the therapeutic hypothermia era (TH). We undertook this study to verify the prognostic value of clinical and electrophysiological variables in the TH setting. A total of 111 consecutive comatose survivors of CA treated with TH were prospectively studied over a 3-year period. Neurological examination, electroencephalography (EEG), and somatosensory evoked potentials (SSEP) were performed immediately after TH, at normothermia and off sedation. Neurological recovery was assessed at 3 to 6 months, using Cerebral Performance Categories (CPC). Three clinical variables, assessed within 72 hours after CA, showed higher false-positive mortality predictions as compared with the AAN guidelines: incomplete brainstem reflexes recovery (4% vs 0%), myoclonus (7% vs 0%), and absent motor response to pain (24% vs 0%). Furthermore, unreactive EEG background was incompatible with good long-term neurological recovery (CPC 1-2) and strongly associated with in-hospital mortality (adjusted odds ratio for death, 15.4; 95% confidence interval, 3.3-71.9). The presence of at least 2 independent predictors out of 4 (incomplete brainstem reflexes, myoclonus, unreactive EEG, and absent cortical SSEP) accurately predicted poor long-term neurological recovery (positive predictive value = 1.00); EEG reactivity significantly improved the prognostication. Our data show that TH may modify outcome prediction after CA, implying that some clinical features should be interpreted with more caution in this setting as compared with the AAN guidelines. EEG background reactivity is useful in determining the prognosis after CA treated with TH.

Therapeutic hypothermia for traumatic brain injury.

Experimental evidence demonstrates that therapeutic temperature modulation with the use of mild induced hypothermia (MIH, defined as the maintenance of body temperature at 32-35 °C) exerts significant neuroprotection and attenuates secondary cerebral insults after traumatic brain injury (TBI). In adult TBI patients, MIH has been used during the acute "early" phase as prophylactic neuroprotectant and in the sub-acute "late" phase to control brain edema. When used to control brain edema, MIH is effective in reducing elevated intracranial pressure (ICP), and is a valid therapy of refractory intracranial hypertension in TBI patients. Based on the available evidence, we recommend: applying standardized algorithms for the management of induced cooling; paying attention to limit potential side effects (shivering, infections, electrolyte disorders, arrhythmias, reduced cardiac output); and using controlled, slow (0.1-0.2 °C/h) rewarming, to avoid rebound ICP. The optimal temperature target should be titrated to maintain ICP <20 mmHg and to avoid temperatures <35 °C. The duration of cooling should be individualized until the resolution of brain edema, and may be longer than 48 h. Patients with refractory elevated ICP following focal TBI (e.g. hemorrhagic contusions) may respond better to MIH than those with diffuse injury. Randomized controlled trials are underway to evaluate the impact of MIH on neurological outcome in adult TBI patients with elevated ICP. The use of MIH as prophylactic neuroprotectant in the early phase of adult TBI is not supported by clinical evidence and is not recommended.

Dietary fish oil is antihypertrophic but does not enhance postischemic myocardial function in female mice.

Clinically and experimentally, a case for omega-3 polyunsaturated fatty acid (PUFA) cardioprotection in females has not been clearly established. The goal of this study was to investigate whether dietary omega-3 PUFA supplementation could provide ischemic protection in female mice with an underlying genetic predisposition to cardiac hypertrophy. Mature female transgenic mice (TG) with cardiac-specific overexpression of angiotensinogen that develop normotensive cardiac hypertrophy and littermate wild-type (WT) mice were fed a fish oil-derived diet (FO) or PUFA-matched control diet (CTR) for 4 wk. Myocardial membrane lipids, ex vivo cardiac performance (intraventricular balloon) after global no-flow ischemia and reperfusion (15/30 min), and reperfusion arrhythmia incidence were assessed. FO diet suppressed cardiac growth by 5% and 10% in WT and TG, respectively (P < 0.001). The extent of mechanical recovery [rate-pressure product (RPP) = beats/min x mmHg] of FO-fed WT and TG hearts was similar (50 +/- 7% vs. 45 +/- 12%, 30 min reperfusion), and this was not significantly different from CTR-fed WT or TG. To evaluate whether systemic estrogen was masking a protective effect of the FO diet, the responses of ovariectomized (OVX) WT and TG mice to FO dietary intervention were assessed. The extent of mechanical recovery of FO-fed OVX WT and TG (RPP, 50 +/- 4% vs. 64 +/- 8%) was not enhanced compared with CTR-fed mice (RPP, 60 +/- 11% vs. 80 +/- 8%, P = 0.335). Dietary FO did not suppress the incidence of reperfusion arrhythmias in WT or TG hearts (ovary-intact mice or OVX). Our findings indicate a lack of cardioprotective effect of dietary FO in females, determined by assessment of mechanical and arrhythmic activity postischemia in a murine ex vivo heart model.

Effect of moderate hyperventilation and induced hypertension on cerebral tissue oxygenation after cardiac arrest and therapeutic hypothermia.

AIM: Improving cerebral perfusion is an essential component of post-resuscitation care after cardiac arrest (CA), however precise recommendations in this setting are limited. We aimed to examine the effect of moderate hyperventilation (HV) and induced hypertension (IH) on non-invasive cerebral tissue oxygenation (SctO2) in patients with coma after CA monitored with near-infrared spectroscopy (NIRS) during therapeutic hypothermia (TH). METHODS: Prospective pilot study including comatose patients successfully resuscitated from out-of-hospital CA treated with TH, monitored with NIRS. Dynamic changes of SctO2 upon HV and IH were analyzed during the stable TH maintenance phase. HV was induced by decreasing PaCO2 from ∼40 to ∼30 mmHg, at stable mean arterial blood pressure (MAP∼70 mmHg). IH was obtained by increasing MAP from ∼70 to ∼90 mmHg with noradrenaline. RESULTS: Ten patients (mean age 69 years; mean time to ROSC 19 min) were studied. Following HV, a significant reduction of SctO2 was observed (baseline 74.7±4.3% vs. 69.0±4.2% at the end of HV test, p<0.001, paired t-test). In contrast, IH was not associated with changes in SctO2 (baseline 73.6±3.5% vs. 74.1±3.8% at the end of IH test, p=0.24). CONCLUSIONS: Moderate hyperventilation was associated with a significant reduction in SctO2, while increasing MAP to supra-normal levels with vasopressors had no effect on cerebral tissue oxygenation. Our study suggests that maintenance of strictly normal PaCO2 levels and MAP targets of 70mmHg may provide optimal cerebral perfusion during TH in comatose CA patients.

Arrhythmogenesis in the developing heart during anoxia-reoxygenation and hypothermia-rewarming: an in vitro model.

INTRODUCTION: The spatio-temporal pattern of arrhythmias in the embryonic/fetal heart subjected to a transient hypoxic or hypothermic stress remains to be established. METHODS AND RESULTS: Spontaneously beating hearts or isolated atria, ventricles, and conotruncus from 4-day-old chick embryos were subjected in vitro to 30-minute anoxia and 60-minute reoxygenation. Hearts were also submitted to 30-minute hypothermia (0-4 degrees C) and 60-minute rewarming. ECG disturbances and alterations of atrial and ventricular electromechanical delay (EMD) were systematically investigated. Baseline functional parameters were stable during at least 2 hours. Anoxia induced tachycardia, followed by bradycardia, atrial ectopy, first-, second-, and third-degree atrio-ventricular blocks and, finally, transient electromechanical arrest after 6.8 minutes, interquartile ranges (IQR) 3.1-16.2 (n = 8). Reoxygenation triggered also Wenckebach phenomenon and ventricular escape beats. At the onset of reoxygenation QT, PR, and ventricular EMD increased by 68%, 70%, and 250%, respectively, whereas atrial EMD was not altered. No fibrillations, no ventricular ectopic beats, and no electromechanical dissociation were observed. Arrhythmic activity of the isolated atria persisted throughout anoxia and upon reoxygenation, whereas activity of the isolated ventricles abruptly ceased after 5 minutes of anoxia and resumed after 5 minutes of reoxygenation. During hypothermia-rewarming, cardiac activity stopped at 17.9 degrees C, IQR 16.2-20.6 (n = 4) and resumed at the same temperature with no arrhythmias. All preparations fully recovered after 40 minutes of reoxygenation or rewarming. CONCLUSION: In the embryonic heart, arrhythmias mainly originated in the sinoatrial tissue and resembled those observed in the adult heart. Furthermore, oxygen readmission was by far more arrhythmogenic than rewarming and the chronotropic, dromotropic, and inotropic effects were fully reversible.

Body temperature regulation and outcome after cardiac arrest and therapeutic hypothermia.

OBJECTIVE: Therapeutic temperature modulation is recommended after cardiac arrest (CA). However, body temperature (BT) regulation has not been extensively studied in this setting. We investigated BT variation in CA patients treated with therapeutic hypothermia (TH) and analyzed its impact on outcome. METHODS: A prospective cohort of comatose CA patients treated with TH (32-34°C, 24h) at the medical/surgical intensive care unit of the Lausanne University Hospital was studied. Spontaneous BT was recorded on hospital admission. The following variables were measured during and after TH: time to target temperature (TTT=time from hospital admission to induced BT target <34°C), cooling rate (spontaneous BT-induced BT target/TTT) and time of passive rewarming to normothermia. Associations of spontaneous and induced BT with in-hospital mortality were examined. RESULTS: A total of 177 patients (median age 61 years; median time to ROSC 25 min) were studied. Non-survivors (N=90, 51%) had lower spontaneous admission BT than survivors (median 34.5 [interquartile range 33.7-35.9]°C vs. 35.1 [34.4-35.8]°C, p=0.04). Accordingly, time to target temperature was shorter among non-survivors (200 [25-363]min vs. 270 [158-375]min, p=0.03); however, when adjusting for admission BT, cooling rates were comparable between the two outcome groups (0.4 [0.2-0.5]°C/h vs. 0.3 [0.2-0.4]°C/h, p=0.65). Longer duration of passive rewarming (600 [464-744]min vs. 479 [360-600]min, p<0.001) was associated with mortality. CONCLUSIONS: Lower spontaneous admission BT and longer time of passive rewarming were associated with in-hospital mortality after CA and TH. Impaired thermoregulation may be an important physiologic determinant of post-resuscitation disease and CA prognosis. When assessing the benefit of early cooling on outcome, future trials should adjust for patient admission temperature and use the cooling rate rather than the time to target temperature.

Deep accidental hypothermia with core temperature below 24°c presenting with vital signs.

Abstract Pasquier, Mathieu, Noemi Zurron, Barbara Weith, Pierre Turini, Fabrice Dami, Pierre-Nicolas Carron, and Peter Paal. Deep accidental hypothermia with core temperature below 24°C presenting with vital signs. High Alt Med Biol. 15:58-63, 2014.-Background: According to the Swiss hypothermia clinical staging, patients with stage III are unconscious with preserved vital signs, with core temperature usually between 24° and 28°C. With stage IV, vital signs are absent with core temperature <24°C. Aims: To describe a patient presenting with HT stage III with vital signs but a core temperature of <24°C, and to search for similar patients in the medical literature. Materials and methods: MEDLINE was used to search for cases of deep accidental hypothermia (<24°C) and preserved vital signs. Results: We found 22 cases in addition to our case (n=23). Median age was 44 years (IQR 36; range 4-83) and median core temperature 22°C (IQR 1.7; 17-23.8). Vital signs were often minimal. Seven patients developed ventricular fibrillation (VF). Twenty patients survived with excellent neurological outcome. Conclusions: Vital signs can be present in hypothermic patients with core temperature <24°C. In deeply hypothermic patients, a careful check and prolonged check of vital functions should be made, as vital signs may be minimal. The clinical Swiss staging remains valuable in the prehospital evaluation of hypothermic patients; its correlation with core temperature should be better defined.

Postischemic treatment of neonatal cerebral ischemia should target autophagy.

OBJECTIVE: To evaluate the contributions of autophagic, necrotic, and apoptotic cell death mechanisms after neonatal cerebral ischemia and hence define the most appropriate neuroprotective approach for postischemic therapy. METHODS: Rats were exposed to transient focal cerebral ischemia on postnatal day 12. Some rats were treated by postischemic administration of pan-caspase or autophagy inhibitors. The ischemic brain tissue was studied histologically, biochemically, and ultrastructurally for autophagic, apoptotic, and necrotic markers. RESULTS: Lysosomal and autophagic activities were increased in neurons in the ischemic area from 6 to 24 hours postinjury, as shown by immunohistochemistry against lysosomal-associated membrane protein 1 and cathepsin D, by acid phosphatase histochemistry, by increased expression of autophagosome-specific LC3-II and by punctate LC3 staining. Electron microscopy confirmed the presence of large autolysosomes and putative autophagosomes in neurons. The increases in lysosomal activity and autophagosome formation together demonstrate increased autophagy, which occurred mainly in the border of the lesion, suggesting its involvement in delayed cell death. We also provide evidence for necrosis near the center of the lesion and apoptotic-like cell death in its border, but in nonautophagic cells. Postischemic intracerebroventricular injections of autophagy inhibitor 3-methyladenine strongly reduced the lesion volume (by 46%) even when given >4 hours after the beginning of the ischemia, whereas pan-caspase inhibitors, carbobenzoxy-valyl-alanyl-aspartyl(OMe)-fluoromethylketone and quinoline-val-asp(OMe)-Ch2-O-phenoxy, provided no protection. INTERPRETATION: The prominence of autophagic neuronal death in the ischemic penumbra and the neuroprotective efficacy of postischemic autophagy inhibition indicate that autophagy should be a primary target in the treatment of neonatal cerebral ischemia.