Induction of Mitochondrial Fragmentation and Mitophagy after Neonatal Hypoxia-Ischemia.

Hypoxia-ischemia (HI) leads to immature brain injury mediated by mitochondrial stress. If damaged mitochondria cannot be repaired, mitochondrial permeabilization ensues, leading to cell death. Non-optimal turnover of mitochondria is critical as it affects short and long term structural and functional recovery and brain development. Therefore, disposal of deficient mitochondria via mitophagy and their replacement through biogenesis is needed. We utilized mt-Keima reporter mice to quantify mitochondrial morphology (fission, fusion) and mitophagy and their mechanisms in primary neurons after Oxygen Glucose Deprivation (OGD) and in brain sections after neonatal HI. Molecular mechanisms of PARK2-dependent and -independent pathways of mitophagy were investigated in vivo by PCR and Western blotting. Mitochondrial morphology and mitophagy were investigated using live cell microscopy. In primary neurons, we found a primary fission wave immediately after OGD with a significant increase in mitophagy followed by a secondary phase of fission at 24 h following recovery. Following HI, mitophagy was upregulated immediately after HI followed by a second wave at 7 days. Western blotting suggests that both PINK1/Parkin-dependent and -independent mechanisms, including NIX and FUNDC1, were upregulated immediately after HI, whereas a PINK1/Parkin mechanism predominated 7 days after HI. We hypothesize that excessive mitophagy in the early phase is a pathologic response which may contribute to secondary energy depletion, whereas secondary mitophagy may be involved in post-HI regeneration and repair.

Lipopolysaccharide-induced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo.

Accumulating evidence suggests that changes in the metabolic signature of microglia underlie their response to inflammation. We sought to increase our knowledge of how pro-inflammatory stimuli induce metabolic changes. Primary microglia exposed to lipopolysaccharide (LPS)-expressed excessive fission leading to more fragmented mitochondria than tubular mitochondria. LPS-mediated Toll-like receptor 4 (TLR4) activation also resulted in metabolic reprogramming from oxidative phosphorylation to glycolysis. Blockade of mitochondrial fission by Mdivi-1, a putative mitochondrial division inhibitor led to the reversal of the metabolic shift. Mdivi-1 treatment also normalized the changes caused by LPS exposure, namely an increase in mitochondrial reactive oxygen species production and mitochondrial membrane potential as well as accumulation of key metabolic intermediate of TCA cycle succinate. Moreover, Mdivi-1 treatment substantially reduced LPS induced cytokine and chemokine production. Finally, we showed that Mdivi-1 treatment attenuated expression of genes related to cytotoxic, repair, and immunomodulatory microglia phenotypes in an in vivo neuroinflammation paradigm. Collectively, our data show that the activation of microglia to a classically pro-inflammatory state is associated with a switch to glycolysis that is mediated by mitochondrial fission, a process which may be a pharmacological target for immunomodulation.

γδT cells but not αßT cells contribute to sepsis-induced white matter injury and motor abnormalities in mice.

BACKGROUND: Infection and sepsis are associated with brain white matter injury in preterm infants and the subsequent development of cerebral palsy. METHODS: In the present study, we used a neonatal mouse sepsis-induced white matter injury model to determine the contribution of different T cell subsets (αßT cells and γδT cells) to white matter injury and consequent behavioral changes. C57BL/6J wild-type (WT), T cell receptor (TCR) δ-deficient (Tcrd -/-, lacking γδT cells), and TCRα-deficient (Tcra -/-, lacking αßT cells) mice were administered with lipopolysaccharide (LPS) at postnatal day (PND) 2. Brain myelination was examined at PNDs 12, 26, and 60. Motor function and anxiety-like behavior were evaluated at PND 26 or 30 using DigiGait analysis and an elevated plus maze. RESULTS: White matter development was normal in Tcrd -/- and Tcrα -/- compared to WT mice. LPS exposure induced reductions in white matter tissue volume in WT and Tcrα -/- mice, but not in the Tcrd -/- mice, compared with the saline-treated groups. Neither LPS administration nor the T cell deficiency affected anxiety behavior in these mice as determined with the elevated plus maze. DigiGait analysis revealed motor function deficiency after LPS-induced sepsis in both WT and Tcrα -/- mice, but no such effect was observed in Tcrd -/- mice. CONCLUSIONS: Our results suggest that γδT cells but not αßT cells contribute to sepsis-induced white matter injury and subsequent motor function abnormalities in early life. Modulating the activity of γδT cells in the early stages of preterm white matter injury might represent a novel therapeutic strategy for the treatment of perinatal brain injury.

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.

Novel Approaches to Neonatal Resuscitation and the Impact on Birth Asphyxia.

Historically, recommendations for neonatal resuscitation were largely based on dogma, but there is renewed interest in performing resuscitation studies at birth. The emphasis for resuscitation following birth asphyxia is administering effective ventilation, as adequate lung aeration leads not only to an increase in oxygenation but also increased pulmonary blood flow and heart rate. To aerate the lung, an initial sustained inflation can increase heart rate, oxygenation, and blood pressure recovery much faster when compared with standard ventilation. Hyperoxia should be avoided, and extra oxygen given to restore cardiac function and spontaneous breathing should be titrated based on oxygen saturations.

Correction: Single Sustained Inflation followed by Ventilation Leads to Rapid Cardiorespiratory Recovery but Causes Cerebral Vascular Leakage in Asphyxiated Near-Term Lambs.

[This corrects the article DOI: 10.1371/journal.pone.0146574.].

The cardiovascular response to birth asphyxia is altered by the surrounding environment.

BACKGROUND: A sustained bradycardia is used as a major indicator of severe perinatal asphyxia. However, lambs asphyxiated ex utero do not exhibit the same bradycardic response as lambs asphyxiated in utero. It is possible that the local in utero environment may influence the initial cardiovascular response to asphyxia. We assessed the effect of facial immersion in water on the cardiovascular response to birth asphyxia. METHODS: Pregnant ewes (138±1 days gestation) were anaesthetised and fetuses were exteriorised and instrumented for measurement of cardiopulmonary haemodynamics. The lamb's head either remained in air (n=5) or was placed in water that was either warm (40±1°C; n=5) or at room temperature (21±1°C; n=5) before the umbilical cord was clamped to induce asphyxia. RESULTS: Heart rate after bradycardia onset was reduced in lambs asphyxiated with their head in cool water (-34±2%) and warm water (-25±4%) compared with those in air (-11±5%; p<0.05). Similarly, the decrease in blood pressure was faster in lambs with water around the face compared with those in air. From 75 s after asphyxia onset, mean and end-diastolic carotid blood flow was higher in the group asphyxiated in air (25±4 mL/kg/min), compared with the groups in water (13±3 mL/kg/min, warm water; 16±2 mL/kg/min, cool water; p<0.05). CONCLUSIONS: The cardiovascular response to birth asphyxia is altered by the presence and temperature of water surrounding the head. The previous understanding of the vagally mediated bradycardia associated with birth asphyxia may include components of the diving reflex.

Single Sustained Inflation followed by Ventilation Leads to Rapid Cardiorespiratory Recovery but Causes Cerebral Vascular Leakage in Asphyxiated Near-Term Lambs.

BACKGROUND: A sustained inflation (SI) rapidly restores cardiac function in asphyxic, bradycardic newborns but its effects on cerebral haemodynamics and brain injury are unknown. We determined the effect of different SI strategies on carotid blood flow (CaBF) and cerebral vascular integrity in asphyxiated near-term lambs. METHODS: Lambs were instrumented and delivered at 139 ± 2 d gestation and asphyxia was induced by delaying ventilation onset. Lambs were randomised to receive 5 consecutive 3 s SI (multiple SI; n = 6), a single 30 s SI (single SI; n = 6) or conventional ventilation (no SI; n = 6). Ventilation continued for 30 min in all lambs while CaBF and respiratory function parameters were recorded. Brains were assessed for gross histopathology and vascular leakage. RESULTS: CaBF increased more rapidly and to a greater extent during a single SI (p = 0.01), which then decreased below both other groups by 10 min, due to a higher cerebral oxygen delivery (p = 0.01). Blood brain barrier disruption was increased in single SI lambs as indicated by increased numbers of blood vessel profiles with plasma protein extravasation (p = 0.001) in the cerebral cortex. There were no differences in CaBF or cerebral oxygen delivery between the multiple SI and no SI lambs. CONCLUSIONS: Ventilation with an initial single 30 s SI improves circulatory recovery, but is associated with greater disruption of blood brain barrier function, which may exacerbate brain injury suffered by asphyxiated newborns. This injury may occur as a direct result of the initial SI or to the higher tidal volumes delivered during subsequent ventilation.

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.

Effects of chest compressions on cardiovascular and cerebral hemodynamics in asphyxiated near-term lambs.

BACKGROUND: Chest compressions (CC) and adrenaline administration are recommended in asphyxiated newborns with persistent bradycardia despite effective ventilation. The effects of CC on cerebral blood flow in newborns at birth are unknown. Our aim was to determine the effects of CC, with or without adrenaline administration, on the return of spontaneous circulation, carotid blood flow (CBF), and carotid arterial pressure (CAP) in asphyxiated near-term lambs. METHODS: Asphyxia was induced in near-term lambs by clamping the umbilical cord and delaying ventilation onset until spontaneous circulation ceased. Lambs were then resuscitated by positive pressure ventilation along with CC followed by adrenaline administration. CAP and CBF were continuously recorded. RESULTS: Mean CAP did not increase significantly during CC and only increased following adrenaline administration. CC did not increase mean CBF but increased CBF amplitude due to increased peak flow and the onset of retrograde flow during diastole. Adrenaline increased mean CBF from 1 ± 2 to 15 ± 5 ml/kg/min and abolished retrograde diastolic CBF, leading to the return in spontaneous circulation. CONCLUSION: We conclude that CC with adrenaline administration was required to increase CBF and restore spontaneous circulation in asphyxiated lambs. Low CBF and retrograde diastolic CBF during CC indicate hypoperfusion to the brain.

Circulatory responses to asphyxia differ if the asphyxia occurs in utero or ex utero in near-term lambs.

BACKGROUND: A cornerstone of neonatal resuscitation teaching suggests that a rapid vagal-mediated bradycardia is one of the first signs of perinatal compromise. As this understanding is based primarily on fetal studies, we investigated whether the heart rate and blood pressure response to total asphyxia is influenced by whether the animal is in utero or ex utero. METHODS: Fetal sheep were instrumented at ∼ 139 days of gestation and then asphyxiated by umbilical cord occlusion until mean arterial blood pressure decreased to ∼ 20 mmHg. Lambs were either completely submerged in amniotic fluid (in utero; n = 8) throughout the asphyxia or were delivered and then remained ex utero (ex utero; n = 8) throughout the asphyxia. Heart rate and arterial blood pressure were continuously recorded. RESULTS: Heart rate was higher in ex utero lambs than in utero lambs. Heart rates in in utero lambs rapidly decreased, while heart rates in ex utero lambs initially increased following cord occlusion (for ∼ 1.5 min) before they started to decrease. Mean arterial pressure initially increased then decreased in both groups. CONCLUSIONS: Heart rate response to asphyxia was markedly different depending upon whether the lamb was in utero or ex utero. This indicates that the cardiovascular responses to perinatal asphyxia are significantly influenced by the newborn's local environment. As such, based solely on heart rate, the stage and severity of a perinatal asphyxic event may not be as accurate as previously assumed.

Effect of sustained inflation duration; resuscitation of near-term asphyxiated lambs.

OBJECTIVE: The 2010 ILCOR neonatal resuscitation guidelines do not specify appropriate inflation times for the initial lung inflations in apnoeic newborn infants. The authors compared three ventilation strategies immediately after delivery in asphyxiated newborn lambs. DESIGN: Experimental animal study. SETTING: Facility for animal research. SUBJECTS: Eighteen near-term lambs (weight 3.5-3.9 kg) delivered by caesarean section. INTERVENTIONS: Asphyxia was induced by occluding the umbilical cord and delaying ventilation onset (10-11 min) until mean carotid blood pressure (CBP) was ≤22 mm Hg. Animals were divided into three groups (n=6) and ventilation started with: (1) inflation times of 0.5 s at a ventilation rate 60/min, (2) five 3 s inflations or (3) a single 30 s inflation. Subsequent ventilation used inflations at 0.5 s at 60/min for all groups. MAIN OUTCOME MEASURES: Times to reach a heart rate (HR) of 120 bpm and a mean CBP of 40 mm Hg. Secondary outcome was change in lung compliance. RESULTS: Median time to reach HR 120 bpm and mean CBP 40 mm Hg was significantly shorter in the single 30 s inflation group (8 s and 74 s) versus the 5×3 s inflation group (38 s and 466 s) and the conventional ventilation group (64 s and 264 s). Lung compliance was significantly better in the single 30 s inflation group. CONCLUSION: A single sustained inflation of 30 s immediately after birth improved speed of circulatory recovery and lung compliance in near-term asphyxiated lambs. This approach for neonatal resuscitation merits further investigation.

An initial sustained inflation improves the respiratory and cardiovascular transition at birth in preterm lambs.

A sustained inflation (SI) facilitates lung aeration after birth but may impair the neonatal cardiovascular transition. We aimed to determine the effect of an initial SI on pulmonary arterial and carotid blood flow (PBF and CBF) after preterm birth. Fetal sheep were instrumented at ∼ 122 d of gestation (d). Lambs were delivered at ∼ 127 d and received either an initial SI (40 cm H2O for 1 min or until a volume of 20 mL/kg was administered) followed by ventilation for 30 min (SI; n = 7) or ventilation for 30 min (non-SI; n = 6). At 10 min after ventilation onset, inspired O2 content increased from 21 to 100% for 10 min. PBF, CBF, pulmonary arterial and carotid pressures, tidal volume, and inspiratory pressures were recorded. PBF was greater during the SI (p < 0.05) but thereafter was similar between groups. Non-SI lambs were hypoxemic and had higher CBF than SI lambs (p < 0.05). Cerebral oxygen delivery was constant in SI lambs but increased ∼ 4-fold in non-SI lambs during ventilation with 100% O2 (p < 0.05). Lung compliance and respiratory status were better in SI than non-SI lambs (p < 0.05). A SI improved lung function without adverse circulatory effects, seemed to stabilize neonatal cerebral O2 delivery, and may protect against cerebral hyperoxia.