Monika Marcinkowski

Glutaric Acid and Its Metabolites Cause Apoptosis in Immature Oligodendrocytes: A Novel Mechanism of White Matter Degeneration in Glutaryl-CoA Dehydrogenase Deficiency

Glutaryl-CoA dehydrogenase deficiency is an inherited metabolic disease characterized by elevated concentrations of glutaric acid (GA) and its metabolites glutaconic acid (GC) and 3-hydroxy-glutaric acid (3-OH-GA). Its hallmarks are striatal and cortical degeneration, which have been linked to excitotoxic neuronal cell death. However, magnetic resonance imaging studies have also revealed widespread white matter disease. Correspondingly, we decided to investigate the effects of GA, GC, and 3-OH-GA on the rat immature oligodendroglia cell line, OLN-93. For comparison, we also exposed the neuroblastoma line SH-SY5Y and the microglia line BV-2 to GA, GC, and 3-OH-GA. Cell viability was measured by metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium. Flow cytometry was used to assess apoptosis via annexin-V, anti-active caspase-3 antibody, and propidium iodide staining. GA, GC, and 3-OH-GA reduced OLN-93 oligodendroglia cell viability in a dose-dependent manner. Toxicity of GA, GC, and 3-OH-GA was abrogated by preincubation with the pan-caspase inhibitor z-VAD-fmk. Apoptosis but not necrosis was detected at various stages (early: annexin-V; effector: caspase-3) after 24–48 h of incubation with GA, GC, or 3-OH-GA in OLN-93 but not in neuroblastoma or microglia cells. OLN-93 lacked expression of N-methyl-d-aspartate receptors, making classical glutamatergic excitotoxicity an unlikely explanation for the selective toxicity of GA, GC, and 3-OH-GA for OLN-93 cells. GA, GC, and 3-OH-GA directly initiate the apoptotic cascade in oligodendroglia cells. This mechanism may contribute to the white matter damage observed in glutaryl-CoA dehydrogenase deficiency.

Glial fibrillary acidic protein and RNA expression in adult rat hippocampus following low-level lead exposure during development

The astroglial cytoskeletal element, glial fibrillary acidie protein (GFAP), is a generally accepted sensitive indicator for neurotoxic effects in the mature brain. We used GFAP as a marker for structural changes in rat hippocampus related to chronic low level lead exposure during different developmental periods. Four groups of rats were investigated: a control group, a perinatal group, which was exposed during brain development (EO-P16), a permanent group, exposed during and after brain development (E0-P100), and a postweaning group, exposed after brain development (P16–P100). Sections were processed for light microscopy (hematoxylin-eosin, Nissl, periodic acid Schiff (PAS) and GFAP-specific immunohistology), for electron microscopy, and for in-situ hybridization (GFAP). Sections were prepared from animals tested for active avoidance learning (AAL) and long-term potentiation (LTP). Chronic lead exposure did not affect glial and neuronal functions, as assessed by LTP and AAL, when lead exposure started after brain development (postweaning group). In this group, astrocytes displayed increased GFAP and GFAP gene transcript levels. However, lead exposure affected neuronal and glial function when the intoxication fell into the developmental period of the brain (perinatal and permanent groups). In these groups, LTP and AAL were impaired, and astrocytes failed to react to the toxic exposure with an adequate increase of GFAP and GFAP gene transcripts. Although GFAP is an accepted marker for neurotoxicity, our data suggest the marker function of GFAP to be restricted to postnatal toxic insult.

Leukocyte and endothelial activation in a laboratory model of extracorporeal membrane oxygenation (ECMO).

An inflammatory response and a capillary leak syndrome frequently develop during the treatment of neonatal respiratory failure by extracorporeal membrane oxygenation (ECMO). The present study was performed to investigate leukocyte activation and endothelial cell dysfunction that are associated with prolonged contact of blood components with synthetic surfaces. Laboratory ECMO was performed with fresh human blood at 37°C for 8 h (n = 6). Leukocyte activation was measured by l-selectin (CD62L) and CD18 integrin surface expression and by neutrophil-derived elastase release. To monitor endothelial activation, endothelial cell ICAM-1 (CD54) expression was measured in cultured endothelial cells from human umbilical veins (HUVEC) after incubation with plasma from the ECMO experiments. CD18 integrin expression was found significantly up-regulated on polymorphonuclear neutrophils and monocytes after 2–4 h of laboratory ECMO. l-selectin was reduced on both cell types during the total duration of the experiments. Soluble l-selectin (sCD62L) and total and differential leukocyte counts remained unchanged during the experiment. Neutrophil-derived elastase content was maximal after 8 h of ECMO. Plasma from the ECMO experiments did not induce ICAM-1 expression of cultured HUVEC. We conclude that prolonged contact with synthetic surfaces during ECMO activates phagocytes, which may contribute to the inflammatory response seen in ECMO-treated patients. Activated phagocytes do not accumulate in the extracorporeal system nor release humoral factors inducing ICAM-1 expression on endothelial cells.

Complement activation by in vivo neonatal and in vitro extracorporeal membrane oxygenation.

Complment activation during extracorporeal membrane oxygenation (ECMO) in newborns can be caused by both the underlying disease processes and by blood contact with the ECMO circuit. We investigated the relative importance of these mechanisms by measuring C3a, C5a and sC5b-9 before, during and after neonatal ECMO in six consecutive newborn patients using enzyme-linked immunoassay. In addition complement activation during in vitro ECMO with repeated flow of the same blood volume was measured using blood from healthy adult donors. C3a increased significantly in vivo after 1 h (from 1035+/-193 to 1865+/-419 microg/l) and in vitro ECMO (from 314+/-75 to 1962+/-1062 microg/l). C5a increased during ECMO without significant differences between in vivo and in vitro activation. In neonatal patients, sC5b-9 rose faster than in vitro, but the rapid increase was also significant for in vitro experiments (in vivo: from 328+/-63 to 1623+/-387 microg/l after 2 h; and in vitro: from 78+/-32 to 453+/-179 microg/l after 8 h). After this initial peak at 1-2 h, complement activation decreased gradually until 2-3 days after the initiation of ECMO. We conclude that in newborns the rapid activation of the complement system after the start of ECMO is predominantly caused by contact with artificial surfaces rather than the patients underlying disease.

Postnatal changes of extracellular volume, atrial natriuretic factor, and diuresis in a randomized controlled trial of high-frequency oscillatory ventilation versus intermittent positive-pressure ventilation in premature infants <30 weeks gestation

Objectives High-frequency oscillatory ventilation (HFOV) with a high lung volume strategy is an experimental mode of ventilating preterm infants aimed at achieving maximal alveolar recruitment. Higher mean airway pressures are used during HFOV than during intermittent positive-pressure ventilation (IPPV), and the intrathoracic volume increase is relatively constant. Both factors increase the risk to depress organ blood flow and diuresis. Our objective was to test the hypothesis that high lung volume HFOV attenuates the postnatal reduction of extracellular volume in preterm infants by reducing plasma atrial natriuretic factor and diuresis. Design Prospective, randomized, controlled clinical trial. Setting University hospital, Level III neonatal intensive care unit. Patients Premature infants <30 wks gestation requiring intubation for respiratory distress syndrome within the first 6 hrs of life; 15 infants (gestational age, 26 [24–29] wks, birth weight 814 [452–1340] g) were randomized to HFOV, 19 infants (gestational age 27 [24–39] wks, birth weight 930 [644–1490] g) to IPPV. Interventions The randomized mode of ventilation was assigned within 1 hr after intubation. During HFOV mean airway pressure was increased as long as oxygenation improved and no lung overinflation was seen on chest radiograph. IPPV rates were ≥60/min. Measurements and Main Results We measured extracellular volume (sucrose dilution) and atrial natriuretic factor on Day 1 and Day 3. Mean airway pressure, body weight, diuresis, and fluid intake were measured daily. During HFOV mean airway pressure was higher at 12 hrs (median 7 cm H2O vs. 4 cm H2O;p = .001) and 24 hrs (median 6 cm H2O vs. 3 cm H2O;p = .01). In both groups, extracellular volume decreased between Day 1 and Day 3 (HFOV from 428 ± 126 mL to 344 ± 145 mL [p = .003], IPPV from 466 ± 108 mL to 414 ± 124 mL [p = .01]) and diuresis increased (HFOV, from 2.5 ± 1.7 to 4.6 ± 0.9 mL/kg/hr [p = .001]; IPPV, from 2.8 ± 1.6 to 4.2 ± 1.0 mL/kg/hr [p = .01]). Plasma atrial natriuretic factor was not decreased in the HFOV group. Conclusions High lung volume HFOV as primary mode of ventilation in preterm infants <30 wks gestation did not result in unwanted fluid retention and a decrease in diuresis in the first days of life.

Hypothalamic Necroses and Arterial Hypotension in a Preterm Infant with Asphyxia

We report on a preterm infant with Beckwith-Wiedemann syndrome with asphyxia in whom hypotension began 4 h after birth and who was resistant to volume infusion and even high doses of catecholamines. Cerebral ultrasound did not show any intracranial bleeding during the first week. The infant died at the age of 9 days because of congestive heart failure and severe therapy-resistant arterial hypotension. Autopsy disclosed unifocal symmetrical necroses of the lateral hypothalamus in the area of blood pressure regulation. It is postulated that the necrotic areas are the hypothalamic centres responsible for blood pressure regulation.

177 Differentiation-Dependant Sensitivity Of Neuronal Precursor Cells Towards Oxidative Stress

Background: Apoptosis is an endogenous cell suicide mechanism triggered in response to biological factors and genotoxic stimuli often resulting from oxidative stress. Neuronal apoptosis especially in the developing brain may cause long-term brain dysfunction. Cognitve deficits in the later childhood are frequent in preterm infants. Preterm infants are exposed to high oxygen due to their premature lungs. At the same time their nutritional regimens are deficient in antioxidant precursors. In cell cultures a relation between oxidative stress and cell death could be found. This study tested the hypothesis that differentiated rat adrenal medullary pheochromocytoma PC12 cells are less sensitive to hyperoxia than undifferentiated cells.Methods: PC12 cells differentiated with nerve growth factor protein (NGF) and undifferentiated cells were exposed to an atmosphere of 80% oxygen and treated with buthionine sulfoximine (BSO), a glutathione synthesis inhibitor. Control cells did not receive BSO. Cell viability was tested by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide). Apoptosis was measured by flow cytometry (annexin/ propidiumjodid, caspase-3-activity).Results: Undifferentiated cells treated with BSO exposed to hyperoxia showed an increase in apoptosis (cytometry) and a significant decrease in viability (BSO+normoxia 81±5,1%, hyperoxia 95±3,1%, BSO+hyperoxia 22±3,0%, means ± S.E.M. for 4– 6 experiments, p< 0,05, cell viability assay). Differentiated cells showed reduced sensibility towards BSO and hyperoxia (BSO+normoxia 101±3,3%, hyperoxia 97±1,9%, BSO+hyperoxia 87,6±2,5%, cell viability assay). BSO alone did not induce apoptosis.Conclusion: We conclude that exposure to high oxygen in combination with limited antioxidant protection is responsible for increased cell death via apoptosis in undifferentiated neurons. This finding may contribute to longterm cognitive deficits in preterm infants exposed to high oxygen.

Second Symposium on Normal and Abnormal Development of the Human Fetal Brain

Neuroembryology 2002;1:191–193 DOI: 10.1159/000066269 Second Symposium on Normal and Abnormal Development of the Human Fetal Brain The University of Rostock, Neuroembryonic Research Laboratory, Department of Anatomy, Rostock, Germany

Postnatal changes of atrial natriuretic peptide (ANP), extracellular volume(ECV), and diuresis in a randomised controlled trial of high frequency oscillation (HFO) vs conventional ventilation in preterm infants <30 weeks GA † 962

Postnatal changes of atrial natriuretic peptide (ANP), extracellular volume(ECV), and diuresis in a randomised controlled trial of high frequency oscillation (HFO) vs conventional ventilation in preterm infants <30 weeks GA † 962

Randomised controlled trial of high frequency oscillation (HFO) vs conventional ventilation (CV): effects on extracellular volume (ECV) and fluid balance

Background. Higher mean airway pressures (MAD) are used in HFO(high volume strategy) than in CV. High MADs reduce cardiac output and diuresis. Hypothesis. HFO inhibits the postnatal decrease in ECV by reducing diuresis. Methods. Ventilated preterm infants (GA<30 wks) were randomised to HFO (Infant Star ™) or CV (Sechrist™ model IV-100B). We measured ECV (sucrose dilution), urine output, fluid intake and MAD. Results. HFO-infants had higher MADs for 24 hours (median 6.7 vs 3.7 cmH2O; p=0.01). Postnatal urine output increased and ECV decreased in both groups. Conclusion. HFO did not prevent the postnatal adaptation of extracellular volume. Table