OBJECTIVE: The purpose of this study was to evaluate the efficacy and safety of erythropoietin in neonatal hypoxic-ischemic encephalopathy (HIE), by using a randomized, prospective study design. METHODS: A total of 167 term infants with moderate/severe HIE were assigned randomly to receive either erythropoietin (N = 83) or conventional treatment (N = 84). Recombinant human erythropoietin, at either 300 U/kg (N = 52) or 500 U/kg (N = 31), was administered every other day for 2 weeks, starting <48 hours after birth. The primary outcome was death or disability. Neurodevelopmental outcomes were assessed at 18 months of age. RESULTS: Complete outcome data were available for 153 infants. Nine patients dropped out during treatment, and 5 patients were lost to follow-up monitoring. Death or moderate/severe disability occurred for 35 (43.8%) of 80 infants in the control group and 18 (24.6%) of 73 infants in the erythropoietin group (P = .017) at 18 months. The primary outcomes were not different between the 2 erythropoietin doses. Subgroup analyses indicated that erythropoietin improved long-term outcomes only for infants with moderate HIE (P = .001) and not those with severe HIE (P = .227). No negative hematopoietic side effects were observed. CONCLUSION: Repeated, low-dose, recombinant human erythropoietin treatment reduced the risk of disability for infants with moderate HIE, without apparent side effects.

Isoflurane and related anesthetics are widely used to anesthetize children, ranging from premature babies to adolescents. Concerns have been raised about the safety of these anesthetics in pediatric patients, particularly regarding possible negative effects on cognition. The purpose of this study was to investigate the effects of repeated isoflurane exposure of juvenile and mature animals on cognition and neurogenesis. Postnatal day 14 (P14) rats and mice, as well as adult (P60) rats, were anesthetized with isoflurane for 35 mins daily for four successive days. Object recognition, place learning and reversal learning as well as cell death and cytogenesis were evaluated. Object recognition and reversal learning were significantly impaired in isoflurane-treated young rats and mice, whereas adult animals were unaffected, and these deficits became more pronounced as the animals grew older. The memory deficit was paralleled by a decrease in the hippocampal stem cell pool and persistently reduced neurogenesis, subsequently causing a reduction in the number of dentate gyrus granule cell neurons in isoflurane-treated rats. There were no signs of increased cell death of progenitors or neurons in the hippocampus. These findings show a previously unknown mechanism of neurotoxicity, causing cognitive deficits in a clearly age-dependent manner.

Sex‐related brain injury was evaluated after unilateral hypoxia–ischaemia (HI) in C57/BL6 mice on postnatal day (P) 5, 9, 21 or 60, corresponding developmentally to premature, term, juvenile and adult human brains. There was no sex difference in brain injury when the insult was severe, as evaluated by pathological scoring or tissue loss, but when the insult was moderate, adult (P60) females displayed less injury. In the immature (P9) male brains, neurones displayed a more pronounced translocation of apoptosis‐inducing factor (AIF) (loss of AIF from the mitochondrial fraction and increase in nuclear AIF) after HI, whereas the female brain neurones displayed a stronger activation of caspase 3 (more pronounced loss of pro‐caspase 3, increase in cleaved caspase 3 and increase in caspase 3 enzymatic activity). Two other mechanisms of injury, peroxynitrite‐induced formation of nitrotyrosine and autophagy, were no different between males and females at P9. These data show that the CNS is more resistant to HI in adult females compared with males, whereas no sex differences were found in the extent of injury in neonatal mice. However, critical sex‐dependent differences were demonstrated in vivo with regard to cellular, apoptosis‐related mechanisms.

Upon cerebral hypoxia-ischemia (HI), apoptosis-inducing factor (AIF) can move from mitochondria to nuclei, participate in chromatinolysis, and contribute to the execution of cell death. Previous work (Cande, C., N. Vahsen, I. Kouranti, E. Schmitt, E. Daugas, C. Spahr, J. Luban, R.T. Kroemer, F. Giordanetto, C. Garrido, et al. 2004. Oncogene. 23:1514–1521) performed in vitro suggests that AIF must interact with cyclophilin A (CypA) to form a proapoptotic DNA degradation complex. We addressed the question as to whether elimination of CypA may afford neuroprotection in vivo. 9-d-old wild-type (WT), CypA^+/−, or CypA^−/− mice were subjected to unilateral cerebral HI. The infarct volume after HI was reduced by 47% (P = 0.0089) in CypA^−/− mice compared with their WT littermates. Importantly, CypA^−/− neurons failed to manifest the HI-induced nuclear translocation of AIF that was observed in WT neurons. Conversely, CypA accumulated within the nuclei of damaged neurons after HI, and this nuclear translocation of CypA was suppressed in AIF-deficient harlequin mice. Immunoprecipitation of AIF revealed coprecipitation of CypA, but only in injured, ischemic tissue. Surface plasmon resonance revealed direct molecular interactions between recombinant AIF and CypA. These data indicate that the lethal translocation of AIF to the nucleus requires interaction with CypA, suggesting a model in which two proteins that normally reside in separate cytoplasmic compartments acquire novel properties when moving together to the nucleus.

Nine-day-old harlequin (Hq) mice carrying the hypomorphic apoptosis-inducing factor (AIF)^Hq mutation expressed 60% less AIF, 18% less respiratory chain complex I and 30% less catalase than their wild-type (Wt) littermates. Compared with Wt, the infarct volume after hypoxia-ischemia (HI) was reduced by 53 and 43% in male (YX^Hq) and female (X^HqX^Hq) mice, respectively (P<0.001). The Hq mutation did not inhibit HI-induced mitochondrial release of cytochrome c or activation of calpain and caspase-3. The broad-spectrum caspase inhibitor quinoline-Val-Asp(OMe)-CH₂-PH (Q-VD-OPh) decreased the activation of all detectable caspases after HI, both in Wt and Hq mice. Q-VD-OPh reduced the infarct volume equally in Hq and in Wt mice, and the combination of Hq mutation and Q-VD-OPh treatment showed an additive neuroprotective effect. Oxidative stress leading to nitrosylation and lipid peroxidation was more pronounced in ischemic brain areas from Hq than Wt mice. The antioxidant edaravone decreased oxidative stress in damaged brains, more pronounced in the Hq mice, and further reduced brain injury in Hq but not in Wt mice. Thus, two distinct strategies can enhance the neuroprotection conferred by the Hq mutation, antioxidants, presumably compensating for a defect in AIF-dependent redox detoxification, and caspase inhibitors, presumably interrupting a parallel pathway leading to cellular demise.