Frances J. Northington

Neuronal cell death in neonatal hypoxia-ischemia.

Perinatal hypoxic‐ischemic encephalopathy (HIE) is a significant cause of mortality and morbidity in infants and young children. Therapeutic opportunities are very limited for neonatal and pediatric HIE. Specific neural systems and populations of cells are selectively vulnerable in HIE; however, the mechanisms of degeneration are unresolved. These mechanisms involve oxidative stress, excitotoxicity, inflammation, and the activation of several different cell death pathways. Decades ago the structural and mechanistic basis of the cellular degeneration in HIE was thought to be necrosis. Subsequently, largely due to advances in cell biology and to experimental animal studies, emphasis has been switched to apoptosis or autophagy mediated by programmed cell death (PCD) mechanisms as important forms of degeneration in HIE. We have conceptualized based on morphological and biochemical data that this degeneration is better classified according to an apoptosis‐necrosis cell death continuum and that programmed cell necrosis has prominent contribution in the neurodegeneration of HIE in animal models. It is likely that neonatal HIE evolves through many cell death chreodes influenced by the dynamic injury landscape. The relevant injury mechanisms remain to be determined in human neonatal HIE, though preliminary work suggests a complexity in the cell death mechanisms greater than that anticipated from experimental animal models. The accurate identification of the various cell death chreodes and their mechanisms unfolding within the immature brain matrix could provide fresh insight for developing meaningful therapies for neonatal and pediatric HIE. Ann Neurol 2011;69:743–758

Treatment advances in neonatal neuroprotection and neurointensive care

Knowledge of the nature, prognosis, and ways to treat brain lesions in neonatal infants has increased remarkably. Neonatal hypoxic-ischaemic encephalopathy (HIE) in term infants, mirrors a progressive cascade of excito-oxidative events that unfold in the brain after an asphyxial insult. In the laboratory, this cascade can be blocked to protect brain tissue through the process of neuroprotection. However, proof of a clinical effect was lacking until the publication of three positive randomised controlled trials of moderate hypothermia for term infants with HIE. These results have greatly improved treatment prospects for babies with asphyxia and altered understanding of the theory of neuroprotection. The studies show that moderate hypothermia within 6 h of asphyxia improves survival without cerebral palsy or other disability by about 40% and reduces death or neurological disability by nearly 30%. The search is on to discover adjuvant treatments that can further enhance the effects of hypothermia.

Necrostatin decreases oxidative damage, inflammation, and injury after neonatal HI

Necrostatin-1 inhibits receptor-interacting protein (RIP)-1 kinase and programmed necrosis and is neuroprotective in adult rodent models. Owing to the prominence of necrosis and continuum cell death in neonatal hypoxia–ischemia (HI), we tested whether necrostatin was neuroprotective in the developing brain. Postnatal day (P)7 mice were exposed to HI and injected intracerebroventricularly with 0.1 μL of 80 μmol necrostatin, Nec-1, 5-(1H-Indol-3-ylmethyl)-(2-thio-3-methyl) hydantoin, or vehicle. Necrostatin significantly decreased injury in the forebrain and thalamus at P11 and P28. There was specific neuroprotection in necrostatin-treated males. Necrostatin treatment decreased necrotic cell death and increased apoptotic cell death. Hypoxia–ischemia enforced RIP1–RIP3 complex formation and inhibited RIP3–FADD (Fas-associated protein with death domain) interaction, and these effects were blocked by necrostatin. Necrostatin also decreased HI-induced oxidative damage to proteins and attenuated markers of inflammation coincidental with decreased nuclear factor-κB and caspase 1 activation, and FLIP ((Fas-associated death-domain-like IL-1β-converting enzyme)-inhibitory protein) gene and protein expression. In this model of severe neonatal brain injury, we find that cellular necrosis can be managed therapeutically by a single dose of necrostatin, administered after HI, possibly by interrupting RIP1–RIP3-driven oxidative injury and inflammation. The effects of necrostatin treatment after HI reflect the importance of necrosis in the delayed phases of neonatal brain injury and represent a new direction for therapy of neonatal HI.

Increased morbidity in severe early intrauterine growth restriction.

OBJECTIVE: To determine the relative frequencies of complications in severe early intrauterine growth-restricted (IUGR) infants.METHODS: All infants 32 weeks gestation or less with birth weight less than the fifth percentile admitted from January 1991 to December 1998 were identified retrospectively. Two infants were identified for each IUGR case: the subsequent admission with birth weight ±100 g of the case, and the subsequent admission with the same gestational age. Infants with multiple congenital anomalies, congenital infections or admission after 48 hours of age were excluded. Maternal and neonatal demographic data, neonatal morbidity and mortality until discharge were gathered by chart review.RESULTS: A total of 39 IUGR identified infants met criteria, with 41 gestational age infants and 33 birth weight infants. Mean birth weights and gestational ages for the IUGR group, gestational age group, and birth weight group were 744 g and 29.6 weeks, 1370 g and 29.7 weeks, and 781 g and 25.5 weeks respectively. Mortality was higher for IUGR infants than gestational age infants (20.5 vs 0%), but less than the birth weight infants (30%). In surviving infants, total ventilator days, total oxygen days, days to full feeds, and patent ductus arteriosis, were higher for IUGR infants than gestational age infants, but less than birth weight infants. Hypoglycemia, direct hyperbilirubinemia, necrotizing enterocolitis (NEC), thrombo-cytopenia, chronic lung disease and feeding difficulties occurred more frequently in IUGR infants than in both other groups. Length of stay for survivors and incidence of retinopathy of prematurity (ROP) was similar for the IUGR and birth weight infants.CONCLUSIONS: Infants born prematurely who are also severely IUGR have higher neonatal morbidity and mortality when compared to infants of similar gestational age. The surviving IUGR infants had less intraventricular hemorrhage and periventricular leukomalacia than less mature infants of comparable birth weight, but a similar incidence of ROP and length of stay. They had a higher incidence of NEC, direct hyperbilirubinemia and chronic lung disease, probably due to end-organ damage in utero from chronic placental insufficiency. These findings highlight the unique pattern of mortality and morbidity seen in infants with severe early IUGR.

Which Neuroprotective Agents are Ready for Bench to Bedside Translation in the Newborn Infant

Neonatal encephalopathy caused by perinatal hypoxiaischemia in term newborn infants occurs in 1 to 3 per 1000 births1 and leads to high mortality and morbidity rates with life-long chronic disabilities.2,3 Although therapeutic hypothermia is a significant advance in the developed world and improves outcome,4,5 hypothermia offers just 11% reduction in risk of death or disability, from 58% to 47%. Therefore, there still is an urgent need for other treatment options. Further, there are currently no clinically established interventions that can be given antenatally to ameliorate brain injury after fetal distress. One of the major limitations to progress is what may be called “the curse of choice.” A large number of possible neuroprotective therapies have shown promise in pre-clinical studies.6,7 How should we select from them? There is no consensus at present on which drugs have a high chance of success for either antenatal or postnatal treatment. There are insufficient societal resources available to test them all. Thus, it is imperative to marshal finite resources and prioritize potential therapies for investigation. The authors believe that facilitating discussion of strategy and findings in “competing” laboratories is critical to facilitate efficient progress toward optimizing neuroprotection after hypoxia-ischemia. Few studies have examined possible interactions of medications with hypothermia and whether combination therapies augment neuroprotection. The timing of the administration of medications may be critical to optimize benefit and avoid neurotoxicity (eg, early acute treatments targeted at amelioration of the neurotoxic cascade compared with subacute treatment that can promote regeneration and repair). Intervention early on in the cascade of neural injury is likely to achieve more optimal neuroprotection8,9; however, there is frequently little warning of impending perinatal hypoxia-ischemia episodes. Sensitizing factors such as maternal pyrexia,10 maternal/fetal infection,11,12 and poor fetal growth13 are well recognized and contribute to the heterogeneity of the fetal response and outcome in neonatal encephalopathy. We include potential antenatal therapy medications in the scoring process; however, electronic fetal monitoring has a low positive predictive value (3%–18%) for identifying intrapartum asphyxia.14–18 At present, therefore, any antenatal intervention potentially involves treatment of many cases that do not need treatment in order to benefit a few at risk of brain injury. In January 2008, investigators from research institutions with a special interest in neuroprotection of the newborn appraised published evidence about medications that have been used in pre-clinical animal models, pilot clinical studies, or both as treatments for: (1) antenatal therapy for fetuses with a diagnosis of antenatal fetal distress at term; and (2) postnatal therapy of infants with moderate to severe neonatal encephalopathy. The aims of this study were to: (1) prioritize potential treatments for antenatal and postnatal therapy; and (2) provide a balanced reference for further discussions in the perinatal neuroscience community for future research and clinical translation of novel neuroprotective treatments of the newborn.

Cell therapy for neonatal hypoxia–ischemia and cerebral palsy

Perinatal hypoxic–ischemic brain injury remains a major cause of cerebral palsy. Although therapeutic hypothermia is now established to improve recovery from hypoxia–ischemia (HI) at term, many infants continue to survive with disability, and hypothermia has not yet been tested in preterm infants. There is increasing evidence from in vitro and in vivo preclinical studies that stem/progenitor cells may have multiple beneficial effects on outcome after hypoxic–ischemic injury. Stem/progenitor cells have shown great promise in animal studies in decreasing neurological impairment; however, the mechanisms of action of stem cells, and the optimal type, dose, and method of administration remain surprisingly unclear, and some studies have found no benefit. Although cell‐based interventions after completion of the majority of secondary cell death appear to have potential to improve functional outcome for neonates after HI, further rigorous testing in translational animal models is required before randomized controlled trials should be considered. ANN NEUROL 2012;

Neurodegeneration in the thalamus following neonatal hypoxia-ischemia is programmed cell death

We studied neuronal cell body, axonal, and terminal degeneration in brains from 7-day-old rat pups recovered for 0, 1.5, 3, 6, 24, 48, 72 h, and 6 days following hypoxia-ischemia and identified proteins involved in the delayed neurodegeneration in the thalamus. We found that injury is biphasic with initial necrosis in the ipsilateral forebrain by 3 h following hypoxia-ischemia, in contrast to more delayed and apoptotic-like injury in the ventral-basal thalamus, brainstem, and other remote non-forebrain regions. Prior to the appearance of large numbers of apoptotic profiles in the ventral-basal thalamus, expression of Fas death receptor protein, activated forms of caspase 8 and caspase 3, and pro-apoptotic Bcl-2 proteins are increased. This manuscript combines our data on hypoxic-ischemic injury in the developing brain and presents evidence for at least two forms of neurodegeneration, namely, acute necrosis in the forebrain and delayed neurodegeneration in the thalamus, which is death-receptor-mediated programmed cell death.

White Matter Injury Correlates with Hypertonia in an Animal Model of Cerebral Palsy

Hypertonia and postural deficits are observed in cerebral palsy and similar abnormalities are observed in postnatal rabbits after antenatal hypoxia–ischemia. To explain why some kits become hypertonic, we hypothesized that white matter injury was responsible for the hypertonia. We compared newborn kits at postnatal day 1 (P1) with and without hypertonia after in vivo global fetal hypoxia–ischemia in pregnant rabbits at 70% gestation. The aim was to examine white matter injury by diffusion tensor magnetic resonance imaging indices, including fractional anisotropy (FA). At P1, FA and area of white matter were significantly lower in corpus callosum, internal capsule, and corona radiata of the hypertonic kits (n = 32) than that of controls (n = 19) while nonhypertonic kits (n = 20) were not different from controls. The decrease in FA correlated with decrease in area only in hypertonia. A threshold of FA combined with area identified only hypertonic kits. A reduction in volume and loss of phosphorylated neurofilaments in corpus callosum and internal capsule were observed on immunostaining. Concomitant hypertonia with ventriculomegaly resulted in a further decrease of FA from P1 to P5 while those without ventriculomegaly had a similar increase of FA as controls. Thus, hypertonia is associated with white matter injury, and a population of hypertonia can be identified by magnetic resonance imaging variables. The white matter injury manifests as a decrease in the number and density of fiber tracts causing the decrease in FA and volume. Furthermore, the dynamic response of FA may be a good indicator of the plasticity and repair of the postnatal developing brain.

Necrostatin-1 attenuates mitochondrial dysfunction in neurons and astrocytes following neonatal hypoxia–ischemia

Receptor interacting protein (RIP)-1 kinase activity mediates a novel pathway that signals for regulated necrosis, a form of cell death prominent in traumatic and ischemic brain injury. Recently, we showed that an allosteric inhibitor of RIP-1 kinase activity, necrostatin-1 (Nec-1), provides neuroprotection in the forebrain following neonatal hypoxia-ischemia (HI). Because Nec-1 also prevents early oxidative injury, we hypothesized that mechanisms involved in this neuroprotection may involve preservation of mitochondrial function and prevention of secondary energy failure. Therefore, our objective was to determine if Nec-1 treatment following neonatal HI attenuates oxidative stress and mitochondrial injury. Postnatal day (p) 7 mice exposed to HI were injected intracerebroventricularly with 0.1 μL (80 μmol) of Nec-1 or vehicle. Nec-1 treatment prevented nitric oxide (NO•), inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine increase, and attenuated glutathione oxidation that was found in vehicle-treated mice at 3h following HI. Similarly, Nec-1 following HI prevented: (i) up-regulation of hypoxia inducible factor-1 alpha (HIF-1α) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) expression, (ii) decline in mitochondrial complex-I activity, (iii) decrease in ATP levels, and (iv) mitochondrial structural pathology in astrocytes and in neurons. Up-regulation of glial fibrillary acidic protein (GFAP) following HI was also prevented by Nec-1 treatment. No differences by gender were observed. We conclude that Nec-1 immediately after HI, is strongly mitoprotective and prevents secondary energy failure by blocking early NO• accumulation, glutathione oxidation and attenuating mitochondrial dysfunction.

Reduction of Caspase-8 and -9 Cleavage Is Associated With Increased c-FLIP and Increased Binding of Apaf-1 and Hsp70 After Neonatal Hypoxic/Ischemic Injury in Mice Overexpressing Hsp70

BACKGROUND AND PURPOSE Caspase-8 and caspase-9 are essential proteases of the extrinsic and intrinsic apoptotic pathways, respectively. We investigated whether neuroprotection associated with overexpression of heat-shock protein 70 (Hsp70), a natural cellular antiapoptotic protein, is mediated by caspase-8 and caspase-9 signaling in the neonatal mouse brain after hypoxia/ischemia (H/I) injury. METHODS Postnatal day 7 transgenic mice overexpressing rat Hsp70 (Hsp70 Tg) and their wild-type (Wt) littermates underwent unilateral common carotid artery ligation followed by 30 minutes of exposure to 8% O2. The expression of apoptotic proteins was quantified by Western blot analysis, and the specific interaction between Hsp70 and apoptotic protease activating factor 1 (Apaf-1) was determined by coimmunoprecipitation. RESULTS Hsp70 overexpression reduced cytosolic translocation of cytochrome c without affecting the levels of Apaf-1 and pro-caspase-9 24 hours after H/I. The expression of these apoptotic proteins in the naïve neonatal brains was also not affected by Hsp70 overexpression. Reduced caspase-9 cleavage occurred in Hsp70 Tg mice compared with Wt littermates 24 hours after H/I and correlated with increased binding of Hsp70 and Apaf-1. Increased cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) expression and decreased caspase-8 cleavage were also observed in Hsp70 Tg compared with Wt mice 24 hours after H/I. CONCLUSIONS Our results suggest that the extrinsic and intrinsic apoptotic pathways mediate the neuroprotective effects of Hsp70 overexpression in neonatal H/I, specifically by upregulating FLIP and sequestering Apaf-1, leading to reduced cleavage of caspase-8 and caspase-9.