Mitochondrial Optic Atrophy (OPA) 1 Processing Is Altered in Response to Neonatal Hypoxic-Ischemic Brain Injury

Perturbation of mitochondrial function and subsequent induction of cell death pathways are key hallmarks in neonatal hypoxic-ischemic (HI) injury, both in animal models and in term infants. Mitoprotective therapies therefore offer a new avenue for intervention for the babies who suffer life-long disabilities as a result of birth asphyxia. Here we show that after oxygen-glucose deprivation in primary neurons or in a mouse model of HI, mitochondrial protein homeostasis is altered, manifesting as a change in mitochondrial morphology and functional impairment. Furthermore we find that the mitochondrial fusion and cristae regulatory protein, OPA1, is aberrantly cleaved to shorter forms. OPA1 cleavage is normally regulated by a balanced action of the proteases Yme1L and Oma1. However, in primary neurons or after HI in vivo, protein expression of YmelL is also reduced, whereas no change is observed in Oma1 expression. Our data strongly suggest that alterations in mitochondria-shaping proteins are an early event in the pathogenesis of neonatal HI injury

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

Dysmaturation of somatostatin interneurons following umbilical cord occlusion in preterm fetal sheep

IntroductionCerebral white matter injury is the most common neuropathology observed in preterm infants. However, there is increasing evidence that gray matter development also contributes to neurodevelopmental abnormalities. Fetal cerebral ischemia can lead to both neuronal and non-neuronal structural-functional abnormalities, but less is known about the specific effects on interneurons.ObjectiveIn this study we used a well-established animal model of fetal asphyxia in preterm fetal sheep to study neuropathological outcome. We used comprehensive stereological methods to investigate the total number of oligodendrocytes, neurons and somatostatin (STT) positive interneurons as well as 3D morphological analysis of STT cells 14 days following umbilical cord occlusion (UCO) in fetal sheep.Materials and MethodsInduction of asphyxia was performed by 25 min of complete UCO in five preterm fetal sheep (98–100 days gestational age). Seven, non-occluded twins served as controls. Quantification of the number of neurons (NeuN), STT interneurons and oligodendrocytes (Olig2, CNPase) was performed on fetal brain regions by applying optical fractionator method. A 3D morphological analysis of STT interneurons was performed using IMARIS software.ResultsThe number of Olig2, NeuN, and STT positive cells were reduced in IGWM, caudate and putamen in UCO animals compared to controls. There were also fewer STT interneurons in the ventral part of the hippocampus, the subiculum and the entorhinal cortex in UCO group, while other parts of cortex were virtually unaffected (p > 0.05). Morphologically, STT positive interneurons showed a markedly immature structure, with shorter dendritic length and fewer dendritic branches in cortex, caudate, putamen, and subiculum in the UCO group compared with control group (p < 0.05).ConclusionThe significant reduction in the total number of neurons and oligodendrocytes in several brain regions confirm previous studies showing susceptibility of both neuronal and non-neuronal cells following fetal asphyxia. However, in the cerebral cortex significant dysmaturation of STT positive neurons occurred in the absence of cell loss. This suggests an abnormal maturation pattern of GABAergic interneurons in the cerebral cortex, which might contribute to neurodevelopmental impairment in preterm infants and could implicate a novel target for neuroprotective therapies

An investigation into the effect of in utero hypoxia on cerebral blood vessels and brain activity in late gestation fetal sheep.

Fetal hypoxia contributes significantly to the pathogenesis of permanent perinatal brain injury. Cerebrovascular pathology following hypoxia can lead to devastating and debilitating brain injury, such as that seen following stroke and haemorrhage, but the effect of in utero fetal hypoxia on the developing cerebrovasculature is not well understood. In the adult brain, robust up-regulation of vascular endothelial growth factor (VEGF) occurs following hypoxia and cerebral ischemia which results in the formation of new blood vessels, as an adaptive response to protect and repair the brain from hypoxic injury. However, newly-formed blood vessels are fragile and prone to rupture. The aims of the studies described in this thesis were to determine how a single brief, but severe episode of fetal hypoxia in late gestation in sheep affects VEGF expression and the cerebral vasculature. We also determined the effects of this global hypoxia on fetal and neonatal behaviour to determine if the fetal brain can repair itself following a single brief hypoxic insult. The results show that 48 h after an in utero hypoxic insult, produced by 10 min of complete umbilical cord occlusion, the distribution of mean blood vessel size is greater in white matter but not gray matter, and there was greater blood vessel-associated VEGF expression white matter compared to gray matter. This was associated with disruption in blood brain barrier (BBB) permeability seen by increased extravasation of plasma albumin in white matter. When the fetuses were left in utero (5 – 10 days following UCO) until the onset of labour, increased VEGFR-2 expression was again seen in white, not gray matter. These studies highlight the importance of assessing VEGF/VEGFR system following in utero hypoxia in the developing fetal brain. Studies assessing both fetal and newborn behaviour following in utero global hypoxia showed that fetal electrocortical (ECoG) activity and breathing movements were disturbed throughout late gestation, and after birth there were delays in the newborn lamb achieving important behavioural milestones such as the ability to stand, use all four legs, and find the udder and suckle. Therefore, it is concluded that an antepartum hypoxic event late in gestation does not result in the fetal brain being able to recover fully, resulting in persistent and profound changes in brain structure and pre- and post-natal behaviour

An investigation into the effect of in utero hypoxia on cerebral blood vessels and brain activity in late gestation fetal sheep.

Fetal hypoxia contributes significantly to the pathogenesis of permanent perinatal brain injury. Cerebrovascular pathology following hypoxia can lead to devastating and debilitating brain injury, such as that seen following stroke and haemorrhage, but the effect of in utero fetal hypoxia on the developing cerebrovasculature is not well understood. In the adult brain, robust up-regulation of vascular endothelial growth factor (VEGF) occurs following hypoxia and cerebral ischemia which results in the formation of new blood vessels, as an adaptive response to protect and repair the brain from hypoxic injury. However, newly-formed blood vessels are fragile and prone to rupture. The aims of the studies described in this thesis were to determine how a single brief, but severe episode of fetal hypoxia in late gestation in sheep affects VEGF expression and the cerebral vasculature. We also determined the effects of this global hypoxia on fetal and neonatal behaviour to determine if the fetal brain can repair itself following a single brief hypoxic insult. The results show that 48 h after an in utero hypoxic insult, produced by 10 min of complete umbilical cord occlusion, the distribution of mean blood vessel size is greater in white matter but not gray matter, and there was greater blood vessel-associated VEGF expression white matter compared to gray matter. This was associated with disruption in blood brain barrier (BBB) permeability seen by increased extravasation of plasma albumin in white matter. When the fetuses were left in utero (5 – 10 days following UCO) until the onset of labour, increased VEGFR-2 expression was again seen in white, not gray matter. These studies highlight the importance of assessing VEGF/VEGFR system following in utero hypoxia in the developing fetal brain. Studies assessing both fetal and newborn behaviour following in utero global hypoxia showed that fetal electrocortical (ECoG) activity and breathing movements were disturbed throughout late gestation, and after birth there were delays in the newborn lamb achieving important behavioural milestones such as the ability to stand, use all four legs, and find the udder and suckle. Therefore, it is concluded that an antepartum hypoxic event late in gestation does not result in the fetal brain being able to recover fully, resulting in persistent and profound changes in brain structure and pre- and post-natal behaviour

Effect of Trp53 gene deficiency on brain injury after neonatal hypoxia-ischemia

Hypoxia-ischemia (HI) can result in permanent life-long injuries such as motor and cognitive deficits. In response to cellular stressors such as hypoxia, tumor suppressor protein p53 is activated, potently initiating apoptosis and promoting Bax-dependent mitochondrial outer membrane permeabilization. The aim of this study was to investigate the effect of Trp53 genetic inhibition on injury development in the immature brain following HI. HI (50 min or 60 min) was induced at postnatal day 9 (PND9) in Trp53 heterozygote (het) and wild type (WT) mice. Utilizing Cre-LoxP technology, CaMK2α-Cre mice were bred with Trp53-Lox mice, resulting in knockdown of Trp53 in CaMK2α neurons. HI was induced at PND12 (50 min) and PND28 (40 min). Extent of brain injury was assessed 7 days following HI. Following 50 min HI at PND9, Trp53 het mice showed protection in the posterior hippocampus and thalamus. No difference was seen between WT or Trp53 het mice following a severe, 60 min HI. Cre-Lox mice that were subjected to HI at PND12 showed no difference in injury, however we determined that neuronal specific CaMK2α-Cre recombinase activity was strongly expressed by PND28. Concomitantly, Trp53 was reduced at 6 weeks of age in KO-Lox Trp53 mice. Cre-Lox mice subjected to HI at PND28 showed no significant difference in brain injury. These data suggest that p53 has a limited contribution to the development of injury in the immature/juvenile brain following HI. Further studies are required to determine the effect of p53 on downstream targets