Protektive Strategien im Hyperoxie-Schädigungsmodell der neonatalen Ratte

Die Frühgeburtlichkeit ist ein weltweites Problem und betrifft etwa 10-20% der neugeborenen Kinder und ist die häufigste Todesursache bei Kindern unter 5 Jahren. In Deutschland kommt etwa jedes zehnte Kind zu früh auf die Welt, davon etwa 12% mit einem Geburtsgewicht unter 1500 g. Ein geringes Geburtsgewicht und postnatale Komplikationen korrelieren dabei mit einem erhöhten Risiko für Mortalität und Morbidität. Präventive und therapeutische Interventionen haben in den letzten Jahrzehnten die Überlebensrate verbessert, jedoch ist weiterhin insbesondere bei Kindern mit einem Geburtsgewicht unter 1500 g eine hohe Morbiditätsrate zu beklagen. Abgesehen von der systemischen und organischen Unreife des zu früh geborenen Kindes, gibt es eine Vielzahl an exogenen Noxen, die in der Pathogenese frühgeburtlicher Erkrankungen eine Rolle spielen können. Neben der Sauerstofftoxizität, resultierend aus den unphysiologisch hohen Sauerstoffkonzentrationen, denen Frühgeborene aufgrund der Geburt und notwendiger Sauerstoffsupplementierung ausgesetzt sind, sind medikamentöse Insulte, wie Anästhetika, als ebenso kausal anzusehen. Die Unreife bedingt dabei ein Ungleichgewicht physiologischer Prozesse, wie des respiratorischen Systems und des anti-oxidativen Abwehrsysteme. In dieser redox-instabilen Situation können zahlreiche medizinisch notwendige medikamentöse oder therapeutische Insulte modulierend wirken und sind mit Nebenwirkungen für die Kurz- und Langzeitentwicklung behaftet. Oxidativer Stress spielt bei der Pathogenese vieler Erkrankungen der Frühgeborenen eine entscheidende Rolle, wobei die kausalen Pathologien bisher unvollständig verstanden sind. Neben der Klärung der pathologischen Prozesse, ist die Identifizierung möglicher therapeutischer Strategien Ziel der neonatologischen Forschung. Klinische Studien belegen bereits die protektive Wirkung von Koffein, einem in der Neonatologie eingesetztem Medikament zur Behandlung und Prävention von Frühgeborenen-Apnoen, für das Langzeit-Outcome von Frühgeborenen und auch die möglichen neuroprotektiven Effekte von Dexmedetomidin, einem Sedativa mit vielseitigen pharmakologischen Wirkungen, da aber subtile Störungen der neurologischen Entwicklung im Umfeld von Frühgeburtlichkeit, neonatalen Adaptationsstörungen und intensivmedizinische Maßnahmen Einfluss nehmen können, können standardisierte Tierversuche dazu beitragen, potentiell protektive Effekte von Koffein und Dexmedetomidin im sich entwickelnden Gehirn und/oder der unreifen Lunge zu identifizieren. Dazu wurden Untersuchungen der infantilen Ratte, einem neonatalen entwicklungsadäquaten Tiermodell, in einem akuten Hyperoxie-Schädigungsmodell durchgeführt. Ziel der Untersuchungen war, ob die Präkonditionierung mit Koffein oder Dexmedetomidin unter postnataler, akuter Hyperoxie (6, 24 und/oder 48 Stunden), die durch oxidativen Stress assoziierten, zellulären Veränderungen reduzieren kann. Oxidativer Stress im akuten Sauerstoffschädigungsmodell im unreifen Gehirn der Ratte beeinträchtigte die hippocampale Neurogenese und induzierte die oxidativen Stress-Kaskade mit daraus resultierender Neuroinflammation und Neurodegeneration. Des Weiteren scheint die anti-oxidative Abwehrkapazität während der Exposition gegenüber Hyperoxie vermindert zu sein. Eine Präkonditionierung mit einer Einzeldosis Koffein (10 mg/kg KGW) vor dem hyperoxischem Insult verringerte die durch oxidativen Stress ausgelöste Neuroinflammation und Neurodegeneration und verstärkte die anti-oxidative Stressantwort. Anti-inflammatorische Effekte zeigte Koffein auch in der unreifen Lunge mit einer adäquaten Inhibierung der Immunzellinfiltration, einer Reduktion der pro-inflammtorischen Antwort und Verminderung der Chemokin-Expression. Neben der unselektiven Antagonisierung der Adenosinrezeptoren, scheint Koffein per se anti-oxidative Eigenschaften zu vermitteln. Das zur Präkonditionierung eingesetzte Dexmedetomidin (1, 5 und 10 µg/kg KGW) zeigte neuroprotektive Eigenschaften in unserem Modell der oxidativen Stress-Schädigung im sich entwickelnden Rattenhirn, wobei apoptotischer Zelltod und pro-inflammatorische Zytokininsulte inhibiert und die postnatale Neurogenese und neuronale Plastizität erhalten wurden. Dexmedetomidin erhöhte die Proliferationskapazität von mitotischen Zellen, was letztlich zu höheren Zellzahlen von reifen Neuronen führte, ohne die Apoptoserate bei Kontrolltieren zu beeinflussen. Die anti-oxidativen und anti-inflammatorischen Eigenschaften von Dexmedetomidin über die 2-Adrenorezeptor-Agonisierung scheint eine potente Möglichkeit der Neuroprotektion darzustellen. Weiterführende Untersuchungen zu protektiven Medikamenten mit anti-oxidativen Eigenschaften, die bereits in der neonatalen Intensivversorgung als Medikament zugelassen sind, sollten weiterhin im Fokus klinischer und experimenteller Studien stehen, um pharmakokinetische und molekulare Mechanismen besser verstehen zu können und die Effektivität und Sicherheit beim klinischen Einsatz zu erhöhen, um nicht-vermeidbare medizinisch-induzierte Maßnahmen mit einem verbessertem Outcome für frühgeborene Kinder in Einklang zu bringen.Preterm birth is a global problem affecting about 10-20% of newborn children and is the leading cause of death in children under the age of 5 years. In Germany, about one child in ten is born prematurely, of which about 12% have a birth weight of less than 1500 g. Low birth weight and postnatal complications correlate with an increased risk of mortality and morbidity. Preventive and therapeutic interventions have improved survival in recent decades, but are still associated with high morbidity rates, especially in infants with a birth weight below 1500 g. Apart from the systemic and organic immaturity of the prematurely infants, there are a variety of exogenous insults that may play a role in the pathogenesis of prematurity. In addition to the oxygen toxicity resulting from the unphysiological high oxygen concentrations to which premature infants are exposed as a result of birth and the need for oxygen supplementation, medical insults such as anesthetics are also considered causal. Immaturity causes an imbalance of physiological processes, such as the respiratory system and the anti-oxidative defense systems. In this redox-unstable situation, numerous medically necessary medical or therapeutic insults can have a modulating effect and are associated with side effects for short and long term development. Oxidative stress plays a crucial role in the pathogenesis of many premature infant diseases, but causes underlying are incompletely understood. In addition to the clarification of pathological processes, the identification of possible therapeutic strategies is the goal of neonatological research. Clinical trials are already demonstrating the protective effects of caffeine, a neonatal drug for the treatment and prevention of apnea of prematurity, for the long term outcome of preterm infants and the potential neuroprotective effects of dexmedetomidine, a sedative with versatile pharmacological effects. Standardized animal studies may help to identify potential protective effects of caffeine and dexmedetomidine in the developing brain and / or immature lung, as subtle neurodevelopmental disorders may be influenced by premature birth, neonatal adaptation disorders and critical care settings. For this purpose, studies in neonatal rats were designed using an acute model of hyperoxia damage. The aim of the study was to determine whether preconditioning with caffeine or dexmedetomidine under postnatal hyperoxia (6, 24, and/or 48 hours) may reduce cellular changes associated with oxidative stress. As a major result in the immature rat brain, hippocampal neurogenesis was affected and the oxidative stress cascade was induced by hyperoxia, hence resulting in neuroinflammation and neurodegeneration. Furthermore, anti-oxidative defense capacity during exposure to hyperoxia appears to be diminished. Pre-conditioning with a single dose of caffeine (10 mg/kg bodyweight) prior to the hyperoxic insult reduced neuroinflammation and neurodegeneration caused by hyperoxia, and it enhanced the anti-oxidative stress response. Caffeine also revealed anti-inflammatory properties in the immature lung with strong inhibition of immune cell infiltration, inhibition of the pro-inflammatory response, and reduction in chemokine expression. In addition to antagonizing adenosine receptors in a non-selective manner, caffeine moreover seems to mediate anti-oxidative properties. Dexmedetomidine (1, 5, and 10 μg/kg bodyweight) used for preconditioning showed neuroprotective properties in our model of oxidative stress injury in the developing rat brain, inhibiting apoptotic cell death and pro-inflammatory cytokine insults, improving postnatal neurogenesis and neuronal plasticity. Dexmedetomidine increased the proliferation capacity of mitotic cells, ultimately leading to higher cell numbers of mature neurons without affecting the rate of apoptosis in control animals. The anti-oxidative and anti-inflammatory properties of dexmedetomidine mediated via α2 adrenoreceptor agonism seem to provide a potent potential for neuroprotection. Further investigation into protective drugs with anti-oxidant properties already approved as a drug in neonatal intensive care should continue to be the focus of clinical and experimental studies to better understand pharmacokinetic and molecular mechanisms, and to increase the effectiveness and safety of clinical usage to reconcile unavoidable medically-induced interventions with improved outcomes for premature infants

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

Introduction 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. Methods In this study, we investigated the effects of hyperoxia (80% O2 for 24, 48, and 72 h) on cultured C8-D1A astrocytes in the presence or absence of progesterone at concentrations ranging from 10−9 to 10−5 mol/L. Results Hyperoxia measured by methytetrazolium assay (MTT) reduced cell viability, increased release of lactate dehydrogenase (LDH), reduced carboxyfluorescein diacetate succinimidyl ester (CFSE)-assessed cell proliferation, and downregulated Cylin D2 expression. Progesterone did not affect any of these hyperoxia-mediated indicators of cell death or malfunctioning. Real-time PCR analysis showed that hyperoxia caused downregulation of the progesterone receptors PR-AB und PR-B. Conclusions Our experiments showed that there was no protective effect of progesterone on hyperoxia-inducted cell damage on mouse C8-D1A astrocytes. Down regulation of the progesterone receptors might be linked to the lack of protective effects

Neuroinflammation after traumatic injury to the developing brain

PURPOSES: Mechanical trauma to the developing rodent brain induces a diffuse secondary neuroapoptosis associated with infiltration of immune cells, local and systemic increased levels of proinflammatory mediators. Our aim was to study their expression, cellular localization, distribution pattern and time course in various brain regions. MATERIALS AND METHODS: 7-day-old Wistar rats and C57/BL6 mice were subjected to cortical trauma. Animals were sacrificed at defined time points - from 2 h to 14 days following trauma. Brain tissues were processed for molecular analyses, single or double indirect peroxidase/fluorescence immunohistochemistry for apoptotic cell death, microglia and interleukin (IL)-1ß/IL-18. RESULTS: Apoptotic neuronal cell death detected by TUNEL was found at distant regions to trauma site mainly ipsilateral from 6 h to 5 days later. A substantial activation of ED1+ microglia occurred at the site of primary and secondary damages. It was first evident at 12 h, peaked at 36-48 h and decreased significantly after 5 days. A marked increase of mRNA, protein levels and imunohistochemical expression of two pro-inflammatory cytokines, interleukin (IL)-1ß and IL-18, was found from 2 h to 5 days following trauma. Mice deficient in IL-18 (IL-18−/−) were protected against post-traumatic brain damage. CONCLUSIONS: Brain trauma leads to neuroinflammation expressed by microglial activation and an increase in IL-1ß and IL-18. Activated microglia are one of the main cellular sources of elevated levels for both cytokines. They are probably involved in and help sustain apoptotic neurodegeneration over several days after trauma. This finding might define microglia and IL-1ß/IL-18 as potential post-traumatic therapeutic targets

Oxygen impairs oligodendroglial development via oxidative stress and reduced expression of HIF-1α

The premature increase of oxygen tension may contribute to oligodendroglial precursor cell (OPC) damage in preterm infants. Fetal OPCs are exposed to low oxygen tissue tensions not matched when cells are cultured in room air. Maturation (A2B5, O4, O1, MBP, CNP, arborization), oxidative stress (nitrotyrosine Western blot, NRF2 and SOD2 expression), apoptosis (TUNEL), proliferation (Ki67), and expression of transcription factors regulated by Hypoxia-Inducible-Factor-1-alpha (Hif-1α) expressed in OPCs (Olig1, Olig2, Sox9, Sox10) were assessed in rat OPCs and OLN93 cells cultured at 5% O2 and 21% O2. Influences of Hif-1α were investigated by Hif-1α luciferase reporter assays and Hif-1α-knockdown experiments. At 21% O2, cell proliferation was decreased and process arborization of OPCs was reduced. Expression of MBP, CNP, Olig1, Sox9 and Sox10 was lower at 21% O2, while Nrf2, SOD2, nitrotyrosine were increased. Apoptosis was unchanged. Luciferease reporter assay in OLN93 cells indicated increased Hif-1α activity at 5% O2. In OLN93 cells at 5% O2, Hif-1α knockdown decreased the expression of MBP and CNP, similar to that observed at 21% O2. These data indicate that culturing OPCs at 21% O2 negatively affects development and maturation. Both enhanced oxidative stress and reduced expression of Hif-1α-regulated genes contribute to these hyperoxia-induced changes

Dexmedetomidine Prevents Lipopolysaccharide-Induced MicroRNA Expression in the Adult Rat Brain

During surgery or infection, peripheral inflammation can lead to neuroinflammation, which is associated with cognitive impairment, neurodegeneration, and several neurodegenerative diseases. Dexmedetomidine, an α-2-adrenoceptor agonist, is known to exert anti-inflammatory and neuroprotective properties and reduces the incidence of postoperative cognitive impairments. However, on the whole the molecular mechanisms are poorly understood. This study aims to explore whether dexmedetomidine influences microRNAs (miRNAs) in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation. Adult Wistar rats were injected with 1 mg/kg LPS intraperitoneal (i.p.) in the presence or absence of 5 µg/kg dexmedetomidine. After 6 h, 24 h, and 7 days, gene expressions of interleukin 1-β (IL1-β), tumor necrosis factor-α (TNF-α), and microRNA expressions of miR 124, 132, 134, and 155 were measured in the hippocampus, cortex, and plasma. Dexmedetomidine decreased the LPS-induced neuroinflammation in the hippocampus and cortex via significant reduction of the IL1-β and TNF-α gene expressions after 24 h. Moreover, the LPS-mediated increased expressions of miR 124, 132, 134, and 155 were significantly decreased after dexmedetomidine treatment in both brain regions. In plasma, dexmedetomidine significantly reduced LPS- induced miR 155 after 6 h. Furthermore, there is evidence that miR 132 and 134 may be suitable as potential biomarkers for the detection of neuroinflammation. View Full-Tex

Postnatal myelination of the immature rat cingulum is regulated by GABAB receptor activity

Myelination of axons in the neonatal brain is a highly complex process primarily achieved by oligodendroglial cells (OLs). OLs express receptors for gamma-aminobutyric acid (GABA) which is released from cortical interneurons on a basal level, while glial cells can be a source of GABA, too. We investigated GABA-induced oligodendroglial maturation, proliferation, apoptosis, and myelin production after pharmacological inhibition of GABA(A) and GABA(B) in the neonatal rat brain. Daily injections of the reverse GABA(A) receptor agonist (DMCM) and the GABA(B) receptor antagonist (CGP35348) were performed from postnatal day 6 (P6) to P11. MBP expression was examined by Western blots and immunohistochemistry. Furthermore, we determined the number of CC1(+)OLIG2(+) and CNP(+)OLIG2(+) cells to assess maturation, the number of PCNA(+)OLIG2(+) oligodendrocytes to assess proliferation, the number of oligodendrocyte precursor cells (PDGFR alpha(+)OLIG2(+)), and apoptosis of OLs (CASP3A(+)OLIG2(+)) as well as apoptotic cells in total (CASP3A(+)DAPI(+)) at P11 and P15. In addition, we analyzed the expression Pdgfr alpha and CNP. MBP expression was significantly reduced after CGP treatment at P15. In the same animal group, CNP expression and CNP(+)OLIG2(+) cells decreased temporarily at P11. At P15, the proliferation of PCNA(+)OLIG2(+) cells and the number of PDGFR alpha(+)OLIG2(+) cells increased after GABA(B) receptor antagonization whereas no significant differences were visible in the Pdgfr alpha gene expression. No changes in apoptotic cell death were observed. CGP treatment induced a transient maturational delay at P11 and deficits in myelin expression at P15 with increased oligodendroglial proliferation. Our in vivo study indicates GABA(B) receptor activity as a potential modulator of oligodendroglial development

In vitro P38MAPK inhibition in aged astrocytes decreases reactive astrocytes, inflammation and increases nutritive capacity after oxygen-glucose deprivation

Envelliment; Astròcits; P38MAPKEnvejecimiento; Astrocitos; P38MAPKAgeing; Atrocytes; P38MAPKProper astroglial functioning is essential for the development and survival of neurons and oligodendroglia under physiologic and pathological circumstances. Indeed, malfunctioning of astrocytes represents an important factor contributing to brain injury. However, the molecular pathways of this astroglial dysfunction are poorly defined. In this work we show that aging itself can drastically perturb astrocyte viability with an increase of inflammation, cell death and astrogliosis. Moreover, we demonstrate that oxygen glucose deprivation (OGD) has a higher impact on nutritive loss in aged astrocytes compared to young ones, whereas aged astrocytes have a higher activity of the anti-oxidant systems. P38MAPK signaling has been identified to be upregulated in neurons, astrocytes and microglia after ischemic stroke. By using a pharmacological p38α specific inhibitor (PH-797804), we show that p38MAPK pathway has an important role in aged astrocytes for inflammatory and oxidative stress responses with the subsequent cell death that occurs after OGD.Deutsche Forschungsgemeinschaft (SCHE 2078/2-1). Förderverein für frühgeborene Kinder an der Charité e.V. Basque Government Postdoc (2017_1_0095)

In Vitro P38MAPK Inhibition in Aged Astrocytes Decreases Reactive Astrocytes, Inflammation and Increases Nutritive Capacity After Oxygen-Glucose Deprivation

Proper astroglial functioning is essential for the development and survival of neurons and oligodendroglia under physiologic and pathological circumstances. Indeed, malfunctioning of astrocytes represents an important factor contributing to brain injury. However, the molecular pathways of this astroglial dysfunction are poorly defined. In this work we show that aging itself can drastically perturb astrocyte viability with an increase of inflammation, cell death and astrogliosis. Moreover, we demonstrate that oxygen glucose deprivation (OGD) has a higher impact on nutritive loss in aged astrocytes compared to young ones, whereas aged astrocytes have a higher activity of the anti-oxidant systems. P38MAPK signaling has been identified to be upregulated in neurons, astrocytes and microglia after ischemic stroke. By using a pharmacological p38 alpha specific inhibitor (PH-797804), we show that p38MAPK pathway has an important role in aged astrocytes for inflammatory and oxidative stress responses with the subsequent cell death that occurs after OGD.Deutsche Forschungsgemeinschaft (SCHE 2078/2-1). Forderverein fur fruhgeborene Kinder an der Charite e.V. Basque Government Postdoc (2017_1_0095

Dexmedetomidine Restores Autophagic Flux, Modulates Associated microRNAs and the Cholinergic Anti-inflammatory Pathway upon LPS-Treatment in Rats

Infections and perioperative stress can lead to neuroinflammation, which in turn is linked to cognitive impairments such as postoperative delirium or postoperative cognitive dysfunctions. The alpha 2-adrenoceptor agonist dexmedetomidine (DEX) prevents cognitive impairments and has organo-protective and anti-inflammatory properties. Macroautophagy (autophagy) regulates many biological processes, but its role in DEX-mediated anti-inflammation and the underlying mechanism of DEX remains largely unclear. We were interested how a pretreatment with DEX protects against lipopolysaccharide (LPS)-induced inflammation in adult male Wistar rats. We used Western blot and activity assays to study how DEX modulated autophagy- and apoptosis-associated proteins as well as molecules of the cholinergic anti-inflammatory pathway, and qPCR to analyse the expression of autophagy and inflammation-associated microRNAs (miRNA) in the spleen, cortex and hippocampus at different time points (6 h, 24 h, 7 d). We showed that a DEX pretreatment prevents LPS-induced impairments in autophagic flux and attenuates the LPS-induced increase in the apoptosis-associated protein cleaved poly(ADP-ribose)-polymerase (PARP) in the spleen. Both, DEX and LPS altered miRNA expression and molecules of the cholinergic anti-inflammatory pathway in the spleen and brain. While only a certain set of miRNAs was up- and/or downregulated by LPS in each tissue, which was prevented or attenuated by a DEX pretreatment in the spleen and hippocampus, all miRNAs were up- and/or downregulated by DEX itself - independent of whether or not they were altered by LPS. Our results indicate that the organo-protective effect of DEX may be mediated by autophagy, possibly by acting on associated miRNAs, and the cholinergic anti-inflammatory pathway