Monika Berns

Maturation‐dependent oligodendrocyte apoptosis caused by hyperoxia

In the immature human brain, periventricular leukomalacia (PVL) is the predominant white matter injury underlying the development of cerebral palsy. PVL has its peak incidence during a well‐defined period in human brain development (23–32 weeks postconceptional age) characterized by extensive oligodendrocyte migration and maturation. We hypothesized that the dramatic rise of oxygen tissue tension associated with mammalian birth and additional oxygen exposure of the preterm infant during intensive care may be harmful to immature oligodendrocytes (OLs). We therefore investigated the effects of hyperoxia on rat oligodendroglia cells in vitro and in vivo. Immature OLs (OLN‐93), their progenitors [preoligodendrocytes (pre‐OL)], and mature OLs were subjected to 80% hyperoxia (24–96 hr). Flow cytometry was used to assess cell death. Cell viability was measured by metabolism of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium (MTT). In addition, 6‐day‐old rat pups were subjected to 80% oxygen (24 hr) and then sacrificed, and their brains were processed for immunfluorescence staining. Apoptosis was detected at various stages (annexin‐V, activated caspase‐3) after 24–48 hr of incubation in 80% oxygen in pre‐ and immature OLs. Mature OLs were resistant to oxygen exposure. These results were confirmed by MTT assay. This cell death was blocked by administration of the pan‐caspase inhibitor zVAD‐fmk. Degeneration of OLs was confirmed in 7‐day‐old rat brains by positive staining for activated caspase‐3. Hyperoxia triggers maturation‐dependent apoptosis in immature and pre‐OLs and involves caspase activation. This mechanism may be relevant to the white matter injury observed in infants born preterm.

Transcutaneous Bilirubin After Phototherapy in Term and Preterm Infants

OBJECTIVES: To compare transcutaneous bilirubin (TcB) readings with total serum bilirubin (TSB) after phototherapy, estimating the range of TcB where confirmation through blood sampling can be avoided. METHODS: Preterm and term neonates receiving in-hospital phototherapy underwent TcB measurements (device JM-103, TcB) alongside routine TSB before and after treatment. We calculated time-dependent safety margins for transcutaneous readings to correctly assign 99% of infants not to receive phototherapy. RESULTS: Between August 2011 and December 2012, 86 newborn infants (47 preterm, 39 term) underwent a total of 189 parallel measurements. Mean difference (TcB − TSB) before treatment was −0.6 mg/dL (SD, 1.9 mg/dL). Within the first 8 hours after phototherapy, TcB levels were −2.4 mg/dL (SD, 2.1 mg/dL) below TSB. Thereafter the difference gradually returned to pretreatment values (−1.8 mg/dL in 8–16 hours, −1.1 mg/dL in 16–24 hours, and −0.8 mg/dL after 24 hours), while variations remained stable over time (SD, 1.4–1.8 mg/dL). In the first 8 hours after treatment, TcB levels of −7.3 mg/dL below the individual phototherapy threshold allowed safe rejection of confirmatory blood sampling. After 8 hours, that safety margin was reduced to approximately −5.0 mg/dL. CONCLUSIONS: TcB measurements remain a valuable tool after phototherapy when time-dependent underestimation of TcB is being accounted for.

17β-Estradiol attenuates hyperoxia-induced apoptosis in mouse C8-D1A cell line

In premature infants, oxygen free radicals generated following neonatal resuscitation are associated with subsequent diseases such as retinopathy of prematurity and bronchopulmonary dysplasia. Recent studies in brain tissue samples have shown that nonphysiologic oxygen levels play a key role in induction of apoptosis in the developing brain. Estrogen is a well‐established agent in neuroprotection and, therefore, is thought to be neuroprotective even in the premature brain. Astrocytes appear to have a critical role in protection and survival of neurons in the brain. As one of the glial cell types, they have a great potential for possible involvement in the mediation of estrogen neuroprotective effects. The aim of our study was to analyze whether astrocytes in cell cultures are damaged by hyperoxia and whether 17β‐estradiol (E2) can protect them against apoptosis. Additionally, we investigated the mechanism of the protection by E2, hypothesizing that it is mediated through extracellular signal‐regulated kinase (ERK1/2). Cells underwent eightfold more apoptosis when cultivated in hyperoxia compared with normoxia. Addition of E2 reduced apoptosis in hyperoxia by more than 50%. Levels of ERK1/2 and phosphorylated ERK1/2 were increased after hyperoxia compared with normoxia. Preincubation with E2 prior to exposure to hyperoxia resulted in decreased levels of ERK1/2 and pERK1/2. Hyperoxia induces apoptosis in C8‐D1A cells, and E2 seems to be a protecting factor for astrocytes in hyperoxia. This effect is not mediated through up‐regulation of pERK1/2.

Ibuprofen Augments Bilirubin Toxicity in Rat Cortical Neuronal Culture

Premature infants are at risk for bilirubin-associated brain damage. In cell cultures bilirubin causes neuronal apoptosis and necrosis. Ibuprofen is used to close the ductus arteriosus, and is often given when hyperbilirubinemia is at its maximum. Ibuprofen is known to interfere with bilirubin–albumin binding. We hypothesized that bilirubin toxicity to cultured rat embryonic cortical neurons is augmented by coincubation with ibuprofen. Incubation with ibuprofen above a concentration of 125 μg/mL reduced cell viability, measured by methylthiazole tetrazolium reduction, to 68% of controls (p < 0.05). Lactate dehydrogenase (LDH) release increased from 29 to 38% (p < 0.01). The vehicle solution did not affect cell viability. Coincubation with 10 μM unconjugated bilirubin (UCB)/human serum albumin in a molar ratio of 3:1 and 250 μg/mL ibuprofen caused additional loss of cell viability and increased LDH release (p < 0.01), DNA fragmentation, and activated caspase-3. Preincubation with the pan-caspase inhibitor z-val-ala-asp-fluoromethyl ketone abolished ibuprofen- and UCB-induced DNA fragmentation. The study demonstrates that bilirubin in low concentration of 10 μM reduces neuron viability and ibuprofen increases this effect. Apoptosis is the underlying cell death mechanism.

Hyperbilirubinämie beim reifen Neugeborenen

ZusammenfassungDer Neugeborenenikterus ist ein aktuelles Thema für jeden Pädiater. Über 60% aller reifen gesunden Neugeborenen werden in den ersten Lebenstagen ikterisch. Nur ein geringer Anteil muss mit Phototherapie oder Austauschtransfusion behandelt werden. Durch sichere Therapiegrenzwerte können neurologische Schäden wie die akute Bilirubinenzephalopathie und der irreversible Kernikterus vermieden werden. Eine Zunahme von Letzterem wird diskutiert. Eine der Hauptursachen, neben unerkannten nichtimmunologischen Risikofaktoren, sind die immer kürzeren stationären Aufenthalte von Mutter und Kind nach der Entbindung und das Überwachungsdefizit in der Folgezeit. Es müssen deshalb sichere Interventionsgrenzen für reife Neugeborenen definiert, Neugeborene mit dem Risiko für eine schwere Hyperbilirubinämie vor der Entlassung erkannt und die Aufmerksamkeit bei Kinderärzten, Hebammen und Eltern für das Problem geschult werden.AbstractNeonatal jaundice is a frequent problem for all pediatricians. Over 60% of all term newborns show clinical signs of jaundice in the first week of life, but only a small number need phototherapy or exchange transfusion. Safe value limits for intervention can reduce bilirubin-induced neurotoxicity such as acute bilirubinencephalopathy and irreversible kernicterus. Recurrence of kernicterus has been described; besides unknown nonimmunological risk factors, early hospital discharge and inappropriate follow-up seem to be responsible. Therefore, early identification of newborns at risk for developing severe hyperbilirubinemia, along with parent education, has become the pediatrician’s aim.

Isoflurane but not Fentanyl Causes Apoptosis in Immature Primary Neuronal Cells

Received: December 29, 2016 Revised: March 03, 2017 Accepted: March 09, 2017 Abstract: Background: Anaesthetics are widely used in new-borns and preterm infants, although it is known that they may adversely affect the developing brain. Objective: We assessed the impact of the volatile anaesthetic, isoflurane, and the intravenous analgesic, fentanyl, on immature and mature embryonic neuronal cells. Methods: Primary neuronal cultures from embryonic rats (E18) cultured for 5 (immature) or 15 days (mature) in vitro (DIV), respectively, were exposed to isoflurane (1.5 Vol.%) or fentanyl (0.8 200 ng/ml) for 24 hours. Experiments were repeated in the presence of the γamino butyric acid-A (GABAA) receptor antagonists, bicuculline or picrotoxin (0.1 mmol/l), or the pancaspase inhibitor zVAD-fmk (20 nmol/l). Cell viability was assessed by methyltetrazolium (MTT) metabolism or lactate dehydrogenase (LDH) release. Results: Isoflurane reduced cell viability significantly in primary neuronal cells cultured for 5 DIV (Δ MTT -28 ±13%, Δ LDH +143 ±15%). Incubation with bicuculline, picrotoxin or zVAD-fmk protected the cells mostly from isoflurane toxicity. After 15 DIV, cell viability was not reduced by isoflurane. Viability of primary neurons cultured for 5 DIV did not change with fentanyl over the wide range of concentrations tested. Conclusion: Immature primary neurons may undergo apoptosis following exposure to isoflurane but are unaffected by fentanyl. Mature primary neurons were not affected by isoflurane exposure.

Severe neonatal infection secondary to prenatal transmembranous ascending vaginal candidiasis

Abstract Fungal neonatal infection with Candida is rare, despite a prevalence of vaginal mycosis of up to 30% during pregnancy. Although there are no recommendations to treat asymptomatic vaginal colonization with candida in healthy pregnant women, this case report highlights that asymptomatic colonization with Candida can lead to chorioamnionitis and systemic neonatal infection with leukemoid reaction. Treatment of asymptomatic candida colonization in women at risk of preterm delivery should be considered.

Effects of progesterone on hyperoxia‐induced damage in mouse C8‐D1A astrocytes

The birth of most mammals features a dramatic increase in oxygen while placenta‐derived hormones such as β‐estradiol and progesterone plummet. In experimental newborn animals, transiently elevated oxygen concentrations cause death of neurons, astrocytes, and oligodendrocyte precursors. High oxygen has been associated with cerebral palsy in human preterm infants while progesterone is being used to prevent preterm delivery and investigated as a neuroprotective agent.

Acral necrosis and upper brachial plexus palsy after prenatal fetal thrombosis

Abstract Intrauterine fetal thrombosis is an extremely rare event with a devastating outcome. The etiology of the condition often remains unclear. A 29-year-old 2nd gravida 1st para presented with mild preeclampsia at 34 weeks of gestation with pathological cardiotocography (CTG), but normal resistance indices in the umbilical and the uterine arteries. The fetal middle cerebral artery (MCA) displayed a significantly reduced pulsatility index (PI). An immediate cesarean section was performed and a male neonate with a birth weight of 2300 g was delivered. Immediately after birth, a progressive necrosis of the neonate’s 4th right final finger link and a palsy of the right brachial plexus were observed. MRI and ultrasound imaging did not display mechanical injuries. The Doppler ultrasound showed a thrombus in the right common carotid and subclavian artery with poststenotic decreased blood flow of the right middle cerebral artery. Further analysis did not reveal inherited or acquired thrombophilias of the neonate. The puzzling prenatal finding of a markedly decreased PI in the MCA as well as the initially suspected postnatal diagnosis of traumatic plexus injury were clarified by the diagnosis of the thrombosis.

143 Estrogen Attenuates Oligodendrocyte Apoptosis Caused by Hyperoxia

Background: In the developing human brain, periventricular leukomalacia (PVL) is the predominant white matter injury underlying the development of cerebral palsy. Our group recently showed that a rise of oxygen tissue tension is a powerful trigger to initiate apoptosis in premature and immature, but not in mature oligodendrocytes (OL). Estrogen plays an important role in the development and function of the cerebral nervous system, and estrogen receptors show a specific distribution with high density arround the third ventricle, the region where OL progenitors are generated. We therefore examined the effects of estrogen on hyperoxia-induced cell death in cultured rat immature oligodendroglia cells.Methods: OLN-93 cells, derived from spontaneously transformed cells of 5- to 10-day-old primary rat brain oligodendrocytes were subjected to 24–72 h of hyperoxia (80% O2) in the presence or absence of estrogen (17beta-estradiol) at various concentrations. These immature oligodendrocytes resemble an intermediate stage between oligodendrocyte precursors and mature oligodendrocytes. Flow cytometry was used to assess apoptosis via annexin-V, anti-activated caspase-3 antibody, and propidium iodide staining. Oligodendrocyte maturation status and caspase-3 activation were confirmed by immunochemistry.Results: In immature rat oligodendrocytes (OLN-93), apoptosis was detected at various stages (early: annexin-V, effector: caspase-3) after 24–72 h of hyperoxia (80% O2). Cell death was reduced up to 30% by preincubation with estrogen at a concentration optimum of 10–7 M estrogen.Conclusion: Estrogen reduces apoptosis in immature oligodendrocytes exposed to hyperoxia. The sudden drop in circulating estrogen of placental origin after mammalian birth may aggravate the susceptibility of immature oligodendrocytes to increased oxygen tension. Supported by grants from the German Federal Department of Education and Research (BMBF, # 01 ZZ 0101), Bonn, and the Ernst Schering Research Foundation, Berlin.