A dual role for AMP-activated protein kinase (AMPK) during neonatal hypoxic-ischaemic brain injury in mice.

Perinatal hypoxic-ischaemic encephalopathy (HIE) occurs in 1-2 in every 1000 term infants and the devastating consequences range from cerebral palsy, epilepsy and neurological deficit to death. Cellular damage post insult occurs after a delay and is mediated by a secondary neural energy failure. AMP-activated protein kinase (AMPK) is a sensor of cellular stress resulting from ATP depletion and/or calcium dysregulation, hallmarks of the neuronal cell death observed after HIE. AMPK activation has been implicated in the models of adult ischaemic injury but, as yet, there have been no studies defining its role in neonatal asphyxia. Here, we find that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is pathological activation of the calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß)-AMPKα1 signalling pathway. Pharmacological inhibition of AMPK during the insult promotes neuronal survival but, conversely, inhibiting AMPK activity prior to the insult sensitizes neurons, exacerbating cell death. Our data have pathological relevance for neonatal HIE as prior sensitization such as exposure to bacterial infection (reported to reduce AMPK activity) produces a significant increase in injury. We show that in an in vivo model of neonatal hypoxia-ischaemic and in oxygen/glucose deprivation in neurons, there is a pathological activation of the CaMKKß-AMPKα1 signalling pathway. Inhibiting AMPK during OGD promotes neuronal survival; conversely, inhibiting AMPK prior to OGD exacerbates cell death. Our data have clinical relevance as prior sensitization (e.g. exposure to bacterial infection reducing AMPK activity) increases injury. AMPK, AMP-activated protein kinase; HI, hypoxia-ischaemia; OGD, oxygen-glucose deprivation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling and cell death in the immature central nervous system after hypoxia-ischemia and inflammation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI.

Mitochondria: hub of injury responses in the developing brain.

Progress in the field of mitochondrial biology in the past few years has shown that mitochondrial activities go beyond bioenergetics. These new aspects of mitochondrial physiology and pathophysiology have important implications for the immature brain. A picture emerges in which mitochondrial biogenesis, mitophagy, migration, and morphogenesis are crucial for brain development and synaptic pruning, and play a part in recovery after acute insults. Mitochondria also affect brain susceptibility to injury, and mitochondria-directed interventions can make the immature brain highly resistant to acute injury. Finally, the mitochondrion is a platform for innate immunity, contributes to inflammation in response to infection and acute damage, and participates in antiviral and antibacterial defence. Understanding of these new aspects of mitochondrial function will provide insights into brain development and neurological disease, and enable discovery and development of new strategies for treatment.

Receptor for complement peptide C3a: a therapeutic target for neonatal hypoxic-ischemic brain injury.

Complement is an essential component of inflammation that plays a role in ischemic brain injury. Recent reports demonstrate novel functions of complement in normal and diseased CNS, such as regulation of neurogenesis and synapse elimination. Here, we examined the role of complement-derived peptide C3a in unilateral hypoxia-ischemia (HI), a model of neonatal HI encephalopathy. HI injury was induced at postnatal day 9 (P9), and loss of hippocampal tissue was determined on P31. We compared WT mice with transgenic mice expressing C3a under the control of glial fibrillary acidic protein promoter, which express biologically active C3a only in CNS and without the requirement of a priori complement activation. Further, we injected C3a peptide into the lateral cerebral ventricle of mice lacking the C3a receptor (C3aR) and WT mice and assessed HI-induced memory impairment 41 d later. We found that HI-induced tissue loss in C3a overexpressing mice was reduced by 50% compared with WT mice. C3a peptide injected 1 h after HI protected WT but not C3aR-deficient mice against HI-induced memory impairment. Thus, C3a acting through its canonical receptor ameliorates behavioral deficits after HI injury, and C3aR is a novel therapeutic target for the treatment of neonatal HI encephalopathy.

Maternal exposure to lipopolysaccharide leads to transient motor dysfunction in neonatal rats.

Epidemiological and experimental data implicate maternal infection and inflammation in the etiology of brain white matter injury, which may lead to cerebral palsy in preterm newborns. Our aim was to investigate motor development of the offspring after maternal administration of lipopolysaccharide (LPS). Wistar rats were intraperitoneally injected with Escherichia coli LPS or saline on gestational days 19 and 20. From birth to 3 weeks, pups were tested for neurobehavioral development, neurological signs and reflexes. From 3 to 6 weeks, motor coordination was investigated. At 4 months, animals were tested for locomotion. Brain myelination was assessed by myelin basic protein immunohistochemistry. Days of appearance of several neurological reflexes were significantly delayed, and neonate LPS pups displayed retarded performance in righting, gait and negative geotaxis. At the juvenile stage, LPS animals showed important impairment in coordination. However, although the LPS group performed worse in most tests, they reached vehicle levels by 5 weeks. At 4 months, LPS animals did not show variations in locomotion performances compared to vehicle. No myelination differences have been observed in the brains at adulthood. Maternal LPS administration results in delayed motor development even though these alterations fade to reach control level by 5 weeks. Motor impairments observed at the early stage in this study could be linked to previously reported hypomyelination of the white matter induced by maternal LPS challenge in the neonates. Finally, the normal myelination shown here at adulthood may explain the functional recovery of the animals and suggest either a potential remyelination of the brain or a delayed myelination in LPS pups.

Molecular mechanisms of neonatal brain injury.

Fetal/neonatal brain injury is an important cause of neurological disability. Hypoxia-ischemia and excitotoxicity are considered important insults, and, in spite of their acute nature, brain injury develops over a protracted time period during the primary, secondary, and tertiary phases. The concept that most of the injury develops with a delay after the insult makes it possible to provide effective neuroprotective treatment after the insult. Indeed, hypothermia applied within 6 hours after birth in neonatal encephalopathy reduces neurological disability in clinical trials. In order to develop the next generation of treatment, we need to know more about the pathophysiological mechanism during the secondary and tertiary phases of injury. We review some of the critical molecular events related to mitochondrial dysfunction and apoptosis during the secondary phase and report some recent evidence that intervention may be feasible also days-weeks after the insult.

Mitochondria and perinatal brain injury.

Secondary brain injury after hypoxia-ischemia is associated with delayed loss of high energy phosphates implicating bioenergetic mitochondrial failure at least partly related to deregulation of the energy sensor adenosine monophosphate-activated protein kinase. Furthermore, the toxic intracellular environment (accumulation of reactive oxygen/nitrosative species and intracellular calcium) during post-ischemic reperfusion triggers Bax-dependent mitochondrial permeabilization (MP) leading to activation of caspase-dependent and apoptosis-inducing factor dependent cell death. We still do not understand how MP is induced but some data suggest that mitochondrial fusion/fission as well as migration play a critical role. Mitochondrial dynamics also seem critical for brain development as genetic deficiency of proteins involved in mitochondrial fusion and fission results in malformations including microcephaly, abnormal brain development and dysmyelination. In this brief review, we update the critical role of mitochondria in brain development and the decision of cell fate after hypoxia-ischemia in the immature CNS.

Combined effect of hypothermia and caspase-2 gene deficiency on neonatal hypoxic-ischemic brain injury.

INTRODUCTION: [corrected] Hypoxia-ischemia (HI) injury in term infants develops with a delay during the recovery phase, opening up a therapeutic window after the insult. Hypothermia is currently an established neuroprotective treatment in newborns with neonatal encephalopathy (NE), saving one in nine infants from developing neurological deficits. Caspase-2 is an initiator caspase, a key enzyme in the route to destruction and, therefore, theoretically a potential target for a pharmaceutical strategy to prevent HI brain damage. METHODS: The aim of this study was to explore the neuroprotective efficacy of hypothermia in combination with caspase-2 gene deficiency using the neonatal Rice-Vannucci model of HI injury in mice. RESULTS: HI brain injury was moderately reduced in caspase-2(-/-) mice as compared with wild-type (WT) mice. Five hours of hypothermia (33 °C ) vs. normothermia (36 °C) directly after HI provided additive protection overall (temperature P = 0.0004, caspase-2 genotype P = 0.0029), in the hippocampus and thalamus, but not in other gray matter regions or white matter. Delayed hypothermia initiated 2 h after HI in combination with caspase-2 gene deficiency reduced injury in the hippocampus, but not in other brain areas. DISCUSSION: In conclusion, caspase-2 gene deficiency combined with hypothermia provided enhanced neuroprotection as compared with hypothermia alone.

Pitfalls in the quest of neuroprotectants for the perinatal brain.

Sick preterm and term newborns are highly vulnerable to neural injury, and thus there has been a major search for new, safe and efficacious neuroprotective interventions in recent decades. Preclinical studies are essential to select candidate drugs for clinical trials in humans. This article focuses on 'negative' preclinical studies, i.e. studies where significant differences cannot be detected. Such findings are critical to inform both clinical and preclinical investigators, but historically they have been difficult to publish. A significant amount of time and resources is lost when negative results or nonpromising therapeutics are replicated in separate laboratories because these negative results were not shared with the research community in an open and accessible format. In this article, we discuss approaches to strengthen conclusions from negative preclinical studies and, conversely, to reduce false-negative preclinical evaluations of potential therapeutic compounds. Without being exhaustive, we address three major issues in conducting and interpreting preclinical experiments, including: (a) the choice of animal models, (b) the experimental design, and (c) issues concerning statistical analyses of the experiments. This general introduction is followed by synopses of negative data obtained from studies of three potential therapeutics for perinatal brain injury: (1) the somatostatin analog octreotide, (2) an AMPA/kainate receptor antagonist, topiramate, and (3) a pyruvate derivative, ethyl pyruvate.

Genetic inhibition of caspase-2 reduces hypoxic-ischemic and excitotoxic neonatal brain injury.

OBJECTIVE: Perinatal brain injury is a major cause of neurodevelopmental handicaps. Multiple pathways of oxidant stress, inflammation, and excitotoxicity lead to cell damage and death, including caspase-dependent apoptosis. Caspase-2 (Casp2; Nedd-2, Ich-1) is a developmentally regulated initiator caspase, which poorly cleaves other caspases but can initiate mitochondrial outer membrane permeabilization. We have investigated if Casp2 could mediate perinatal ischemic brain damage. METHODS: Casp2 expression in human neonatal brains and developmental patterns in rats and mice were evaluated. Casp2-deficient (Casp2(-/-)), wild-type (WT), and heterozygous (Casp2(+/-)) newborn C57BL/6 mice were subjected to hypoxia-ischemia (unilateral carotid occlusion + exposure to 10% oxygen for 50 minutes) or intracerebral injection of the excitotoxic N-methyl-D-aspartate-receptor agonist ibotenate. In addition, Casp2 specific siRNAs were preinjected into the brain of WT newborn mice 24 hours before ibotenate treatment. Brain tissues were examined by immunohistochemical staining (cresyl violet, MAP2, NF68, Casp2, Casp3) and Western blotting. Lesion volumes and injury in the cortical plates and white matter were quantified together with activated Casp3. RESULTS: Casp2 is highly expressed in the neonatal brain. Casp2-deficient mice subjected to hypoxia-ischemia at postnatal day 9 present significantly lower cerebral infarction, reduced white matter injury, and reduced Casp3 activation in the thalamus and hippocampus. Both Casp2(-/-) mice and siRNA-administered WT mice conferred reduction of gray and white matter injury after excitotoxic insult at postnatal day 5. Casp3 activation was also found reduced in Casp2-deficient mice subjected to excitotoxicity. INTERPRETATION: These data suggest for the first time a role of Casp2 in neonatal brain damage.

Attenuation of reactive gliosis does not affect infarct volume in neonatal hypoxic-ischemic brain injury in mice.

BACKGROUND: Astroglial cells are activated following injury and up-regulate the expression of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. Adult mice lacking the intermediate filament proteins GFAP and vimentin (GFAP(-/-)Vim(-/-)) show attenuated reactive gliosis, reduced glial scar formation and improved regeneration of neuronal synapses after neurotrauma. GFAP(-/-)Vim(-/-) mice exhibit larger brain infarcts after middle cerebral artery occlusion suggesting protective role of reactive gliosis after adult focal brain ischemia. However, the role of astrocyte activation and reactive gliosis in the injured developing brain is unknown. METHODOLOGY/PRINCIPAL FINDINGS: We subjected GFAP(-/-)Vim(-/-) and wild-type mice to unilateral hypoxia-ischemia (HI) at postnatal day 9 (P9). Bromodeoxyuridine (BrdU; 25 mg/kg) was injected intraperitoneally twice daily from P9 to P12. On P12 and P31, the animals were perfused intracardially. Immunohistochemistry with MAP-2, BrdU, NeuN, and S100 antibodies was performed on coronal sections. We found no difference in the hemisphere or infarct volume between GFAP(-/-)Vim(-/-) and wild-type mice at P12 and P31, i.e. 3 and 22 days after HI. At P31, the number of NeuN(+) neurons in the ischemic and contralateral hemisphere was comparable between GFAP(-/-)Vim(-/-) and wild-type mice. In wild-type mice, the number of S100(+) astrocytes was lower in the ipsilateral compared to contralateral hemisphere (65.0+/-50.1 vs. 85.6+/-34.0, p<0.05). In the GFAP(-/-)Vim(-/-) mice, the number of S100(+) astrocytes did not differ between the ischemic and contralateral hemisphere at P31. At P31, GFAP(-/-)Vim(-/-) mice showed an increase in NeuN(+)BrdU(+) (surviving newly born) neurons in the ischemic cortex compared to wild-type mice (6.7+/-7.7; n = 29 versus 2.9+/-3.6; n = 28, respectively, p<0.05), but a comparable number of S100(+)BrdU(+) (surviving newly born) astrocytes. CONCLUSIONS/SIGNIFICANCE: Our results suggest that attenuation of reactive gliosis in the developing brain does not affect the hemisphere or infarct volume after HI, but increases the number of surviving newborn neurons.

Apoptotic mechanisms in the immature brain: involvement of mitochondria.

Brain injury after hypoxic-ischemic encephalopathy often develops with delayed appearance, opening a therapeutic window. Clinical studies in newborns show that post-hypoxic-ischemic hypothermia improves outcome. This has generated renewed interest in the molecular mechanisms of hypoxic-ischemic brain injury. In this brief review, we propose that mitochondrial permeabilization is crucial for injury to advance beyond the point of no return. We suggest that excitatory amino acids, nitric oxide, inflammation, trophic factor withdrawal, and an increased pro- versus antiapoptotic Bcl-2 protein ratio will trigger Bax-dependent mitochondrial outer membrane permeabilization. Mitochondrial outer membrane permeabilization, in turn, elicits mitochondrial release of cytochrome C, apoptosis-inducing factor, second mitochondria-derived activator of caspase/Diablo, and HtrA2/Omi. Cytochrome C efflux activates caspase-9/-3, leading to DNA fragmentation. Apoptosis-inducing factor interacts with cyclophilin A and induces chromatinolysis. Blockage of mitochondrial outer membrane permeabilization holds promise as a strategy for perinatal brain protection.

Developmental shift of cyclophilin D contribution to hypoxic-ischemic brain injury.

Cyclophilin D (CypD), a regulator of the mitochondrial membrane permeability transition pore (PTP), enhances Ca(2+)-induced mitochondrial permeabilization and cell death in the brain. However, the role of CypD in hypoxic-ischemic (HI) brain injury at different developmental ages is unknown. At postnatal day (P) 9 or P60, littermates of CypD-deficient [knock-out (KO)], wild-type (WT), and heterozygous mice were subjected to HI, and brain injury was evaluated 7 d after HI. CypD deficiency resulted in a significant reduction of HI brain injury at P60 but worsened injury at P9. After HI, caspase-dependent and -independent cell death pathways were more induced in P9 CypD KO mice than in WT controls, and apoptotic activation was minimal at P60. The PTP had a considerably higher induction threshold and lower sensitivity to cyclosporin A in neonatal versus adult mice. On the contrary, Bax inhibition markedly reduced caspase activation and brain injury in immature mice but was ineffective in the adult brain. Our findings suggest that CypD/PTP is critical for the development of brain injury in the adult, whereas Bax-dependent mechanisms prevail in the immature brain. The role of CypD in HI shifts from a predominantly prosurvival protein in the immature to a cell death mediator in the adult brain.

Antenatal bacterial endotoxin sensitizes the immature rat brain to postnatal excitotoxic injury.

Intracerebral injection of ibotenate in newborn rodents produces brain damage that mimics that of infants with cerebral palsy. Because maternal infection may contribute to brain injury in preterm infants, we investigated brain damage after maternal inflammation and postnatal ibotenate treatment in a rat model of cerebral palsy. Pregnant rats were injected intraperitoneally with lipopolysaccharide at Days 19 and 20 of gestation. Neonates were given intracerebral injections of ibotenate at postnatal Day 4 and were then killed at Day 9. Lesion sizes were measured by cresyl violet staining, and microglial activation, astrogliosis, and myelination were evaluated by immunohistochemistry. The lipopolysaccharide groups had larger cortical and white matter lesions than the control group; they also had significantly greater microglial activation and astrogliosis and less white matter myelination in the lesioned hemispheres compared with the controls. Thus, maternal endotoxin exposure may affect prenatal development of the offspring and modulate the subsequent development of excitotoxic brain lesions. These results demonstrate the critical influence of prenatal immune events on neonatal central nervous system vulnerability and provide a model for studying the pathophysiology of cerebral damage in preterm infants and, specifically, the interplay between brain inflammation and excitotoxicity.

Lipopolysaccharide-induced inflammation and perinatal brain injury.

Both energy failure and infections are important risk factors for brain injury in term and preterm infants. In this review we focus on recent experimental studies that have examined the effects of lipopolysaccharide (LPS) exposure to the fetus or neonate and the interaction of LPS with other events. Intracerebral LPS injections induce a marked cerebral cytokine response and prominent white matter lesions. LPS administered intravenously to the fetus also induces gross lesions, which are mainly localised to the white matter and are accompanied by activation of inflammatory cells. Cerebral effects following fetal LPS exposure via more distant routes, such as intracervical, intrauterine or maternal LPS administration, are characterised by reductions in oligodendrocyte or myelin markers without macroscopic lesions being evident. Both antenatal and neonatal LPS exposures increase the sensitivity of the brain to subsequent hypoxic/ischaemic events, even in adulthood. These studies suggest that fetal inflammation is the strongest predictor of brain lesions.

Maternal exposure to LPS induces hypomyelination in the internal capsule and programmed cell death in the deep gray matter in newborn rats.

Epidemiologic and experimental findings implicate maternal infection in the etiology of injury to brain white matter, which may lead to cerebral palsy in preterm newborns. In the present study, inflammation and brain damage in 1- and 7-d-old rats were investigated after maternal inflammation. Intraperitoneal injection of 300 microg/kg of Escherichia coli lipopolysaccharide was administered to pregnant Wistar rats at d 19 and 20 of gestation (LPS group). Control females received a saline injection. Proinflammatory cytokines IL-1beta, tumor necrosis factor-alpha, and IL-6 expression in the fetal brain were determined by reverse transcription quantitative polymerase chain reaction. Brain injury was examined in 16-mum coronal brain sections by GFAP, MBP, caspase-3 immunohistochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Expression of IL-1beta was significantly increased 3 d after maternal administration (P1). A significant increase in cell death occurred at P1 and P7 in specific brain areas, i.e. in the subventricular striatal zone at P1, and in 1) the periventricular striatum, 2) the periventricular white matter, and 3) the germinative ventricular zone at P7. We also observed typical astrogliosis and strong hypomyelination in the external and internal capsule in the LPS group at P7. These results demonstrate that maternal LPS treatment induces persistent fetal inflammatory reactions associated with significant white matter injury in progeny at P1 and P7. This model should be relevant for the study of the pathophysiological mechanisms involved in cerebral white matter damage in preterm human newborns and in the development of therapeutic strategies.