Loss of neuronal GSK3ß reduces dendritic spine stability and attenuates excitatory synaptic transmission via ß-catenin.

Central nervous glycogen synthase kinase 3ß (GSK3ß) is implicated in a number of neuropsychiatric diseases, such as bipolar disorder, depression, schizophrenia, fragile X syndrome or anxiety disorder. Many drugs employed to treat these conditions inhibit GSK3ß either directly or indirectly. We studied how conditional knockout of GSK3ß affected structural synaptic plasticity. Deletion of the GSK3ß gene in a subset of cortical and hippocampal neurons in adult mice led to reduced spine density. In vivo imaging revealed that this was caused by a loss of persistent spines, whereas stabilization of newly formed spines was reduced. In electrophysiological recordings, these structural alterations correlated with a considerable drop in the frequency and amplitude of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-dependent miniature excitatory postsynaptic currents. Expression of constitutively active ß-catenin caused reduction in spine density and electrophysiological alterations similar to GSK3ß knockout, suggesting that the effects of GSK3ß knockout were mediated by the accumulation of ß-catenin. In summary, changes of dendritic spines, both in quantity and in morphology, are correlates of experience-dependent synaptic plasticity; thus, these results may help explain the mechanism of action of psychotropic drugs inhibiting GSK3ß.

Treatment of astrocytoma grade III with Photofrin II as a radiosensitizer. A case report.

INTRODUCTION: Astrocytomas are neoplasms that originate from glial cells. Anaplastic astrocytoma is classified as WHO III, with 27 % of the individuals with grade III astrocytoma living for at least 5 years even after treatment (radiation and chemotherapy). Photofrin II has been demonstrated to serve as a specific and selective radiosensitizing agent in both in vitro and in vivo tumor models. MATERIAL AND METHODS: This case report presents a woman suffering from an inoperable astrocytoma WHO III since 2004. The patient was treated with radiation therapy and Photofrin II as a radiosensitiser. The patient underwent irradiation with 40 + 20 Gy boost. The patient was given a single intravenous dose of 1 mg/kg Photofrin II 24 h prior to the initiation of radiation therapy. RESULTS: The patient is still alive without any significant side effect with a follow up of 106 months. MRI shows no evidence of disease. CONCLUSION: The follow-up results are encouraging regarding the application of Photofrin II as an effective radiosensitizing agent in the treatment of inoperable WHO III astrocytoma.

[To what degree does the NICU environment influence neurodevelopment of preterm infants?]. / Intensivmedizinisches Reizumfeld und Stressoren--welchen Einfluss haben sie auf die Gehirnentwicklung frühgeborener Kinder?

What effect do the various stressing stimuli in a neonatal intensive care unit have on the very sensitive process of synaptogenesis and apoptosis, dendritic growth and neuronal differentiation? To what degree can even minimal changes in the neuronal network of the developing cortex lead to behavioural disorders? And is there any possibility to improve the long term outcome of preterm infants by offering special support within the framework of individualized developmental care? This article combines the results of several studies and discovers interactions to illustrate the complexity and vulnerability of the developing human brain and to discuss the potential benefit of individualized developmental care interventions.

Alteration of antibiotic regimen as an additional control measure in suspected multi-drug-resistant Enterobacter cloacae outbreak in a neonatal intensive care unit.

BACKGROUND: Increased occurrence of a particular species of Gram-negative bacteria (GNB), especially when multi-drug-resistant (MDR), in routine screening surveillance in neonatal intensive care units (NICUs) can be evoked by selection pressure. AIM: To evaluate adaptation of the empiric antibiotic regimen for its usefulness as a control measure in suspected outbreaks in the NICU. METHODS: In a retrospective outbreak analysis, cases between 1st December 2017 and 31st March 2018 were identified through microbiology and hygiene surveillance records. Furthermore, risk factors for MDR-GNB colonization were collected. Whole-genome sequencing (WGS) was performed on all isolates. Control measure documentations and interviews were employed to define interventions. As well as infection control measures, administration of third-generation cephalosporins was avoided and replaced whenever clinically acceptable as part of the intervention bundle. FINDINGS: In total, nine patients were found to have rectal colonization with third-generation cephalosporin-resistant Enterobacter cloacae in routine screening surveillance in the pre-intervention period. After implementation of an infection control bundle, the incidence declined rapidly. WGS analysis revealed that two MDR E. cloacae were transmitted, and the majority were new cases. The incidence density of MDR-GNB colonization was 7.94/1000 patient-days (PD) before the intervention and 1.68/1000 PD during the altered antibiotic regimen. No infections with MDR-GNB occurred during the study. CONCLUSIONS: Altering the antibiotic regimen with regard to selection pressure may be considered as part of an intervention bundle to rapidly control the emergence of MDR-GNB in suspected outbreak situations in the NICU.

[Impact of assisted reproduction on obstetrics and neonatology]. / Einfluss der assistierten Reproduktion auf Geburtshilfe und Neonatologie.

BACKGROUND: In industrialised countries, 1-4% of all children are born as a result of assisted reproductive therapies (ART), such as IVF and ICSI. Possible associations of these ARTs with obstetric and neonatal risk constellations are analysed critically in the context of this review. METHODS: A selective literature search was conducted to examine the influence of ART on obstetric and neonatal aspects. RESULTS: Multiple gestations, occurring more frequently after ART, are of special significance with regard to their associated risks. In comparison to spontaneous pregnancies, singleton gestations after ART are associated with higher rates of complications, such as preeclampsia, prematurity, low birth weight, foetal malformations and a higher rate of Caesarean sections. Although causal associations between extracorporeal fertilisation methods and health risks for mothers and infants in singleton pregnancies cannot be ruled out, these complications are rather attributed to the underlying causes of infertility than to the methods of assisted reproduction themselves. CONCLUSIONS: Pregnancies after ART are to be regarded as risk constellations with a need for closer surveillance during gestation - irrespective of the number of developing foetuses. Couples seeking advice about infertility should be informed in detail before the onset of ART.

Whole blood viscosity of preterm infants - differences to term neonates.

Determination of shear stresses at given shear rates with the LS 300 allows approximation of flow curves by mathematical models and to calculate viscosities of non-Newtonian fluids across the entire physiological shear range. Particular in preterm neonates, which in comparison to term neonates and adults have a substantially lower mean arterial blood pressure (MAP), rheological blood properties play a decisive role to maintain the perfusion. Whole blood viscosity was measured in umbilical cord blood taken from 34 preterm neonates using the LS 300 viscometer. In addition aggregation index, plasma viscosity, hematocrit and red blood cell (RBC) deformability was determined. The highest quality of approximation of the flow curve of whole blood was achieved by the method of Ostwald. Shear stresses of whole blood of preterm neonates were significantly lower compared to term neonates at similar shear rates (velocity range 6.16 s-1-50 s-1). With hematocrit calculated to 0.40 the related exponent (n) of the viscosity of preterm neonates (by Ostwald) showed a significant lower exponent (n) (0.71 ± 0.07) than term neonates (0.76 ± 0.06). The highest quality of approximation of the flow curve of plasma was achieved with the model of Ostwald in preterm neonates as well as in term neonates. The viscosity of plasma determined by Newton was lower for preterm neonates (0.89 ± 0.19) than for term neonates (1.04 ± 0.16). Concurrent to term neonates the whole blood of preterm neonates showed a very low aggregation index (term neonates 2.97 ± 2.10; preterm neonates 2.37 ± 1.32) and preterm neonates showed higher RBC deformability than term neonates. Because of the physiologically lower MAP and the particular viscous properties of neonatal blood special attention should be given when treating neonates with conventional blood products derived from adult donors.

Determination of whole blood and plasma viscosity in term neonates by flow curve analysis with the LS300 viscometer1.

Determination of shear stresses at given shear rates allow approximation of flow curves by mathematical models and to calculate viscosities of non-Newtonian fluids. In term neonates, the mean arterial blood pressure (MAP) is markedly below that of adults, therefore rheological properties of blood play an important role in maintaining perfusion. Whole blood viscosity was measured in umbilical cord blood taken from 62 term neonates using the LS 300 viscometer. Individual parameters that influence the viscosity of whole blood were measured: red blood cell (RBC) aggregation, plasma viscosity, hematocrit, and RBC deformability. The flow curve of whole blood of neonates was approximated by the method of Ostwald with the highest quality whereas in adults the best approximation was found by the method of Casson. With hematocrits of 0.40, the viscosity of whole blood in newborns approximated by Ostwald (9.84 ± 5.12 mPa·s) was significantly lower than that of adults (15.34 ± 3.01 mPa·s). The aggregation index of the blood of newborns was markedly lower (2.98 ± 2.12) than in adults (14.63 ± 3.50) whereas RBC deformability was higher in neonates. The viscosity of plasma determined by Ostwald revealed a lower exponent (n) in neonates (0.94 ± 022) compared to adults (1.01 ± 0.12) and the viscosity determined by Newton was lower in neonates (1.04 ± 0.16 mPa·s) than in adults (1.19 ± 0.07 mPa·s). The flow curve of neonatal blood which is best approximated by the model of Ostwald emphasizes its important viscous properties necessary for conditions with physiologically low blood pressure.

Group B Streptococcus impairs erythrocyte deformability in neonates more than in adults.

Group B beta-haemolytic Streptococcus (GBS) may cause severe septic shock and death in neonates, whereas this is rarely the case in adults. As impaired red blood cell (RBC) deformability might disturb microcirculation in septic shock, the in vitro effects of GBS (1.7 x 10(8) cfu/ml) on RBC deformation (rheoscope) and haemolysis were studied in blood from preterm infants, term neonates, and adults. Furthermore, RBC deformation was studied in term neonates with GBS sepsis. RBC deformation at a shear stress of 4 Pa decreased significantly within 5 minutes of GBS incubation in preterm infants (-13%) and term neonates (-9%). In adults RBC deformation did not change during the first 15 minutes, but decreased significantly after 30 (-10%) and 60 minutes (-13%). In the term infants there was little further decrease in RBC deformation between 5 and 60 minutes of GBS incubation; RBC deformation in preterm infants decreased by 19% after 60 minutes compared with the preincubation values. RBC deformation in septic neonates was significantly decreased at shear stresses of 1, 2, and 3 Pa (-19%, -18%, and -9%). Sixty minutes of incubation of RBC from adults and neonates with GBS and without GBS resulted in haemolysis below 4%. It is concluded that neither neonatal nor adult RBC are haemolysed by GBS. In vitro, neonatal RBC deformability is more impaired than that in adults. This may contribute to the high risk of neonates for compromised microcirculation and circulatory shock as a result of GBS sepsis.

Effects of dietary supplementation of saturated fatty acids and of n-6 or n-3 polyunsaturated fatty acids on plasma and red blood cell membrane phospholipids and deformability in weanling guinea pigs.

The fatty acid composition of plasma cholesteryl esters, plasma phospholipids, red blood cell (RBC) membrane phosphatidylcholine (corresponding to the outer membrane leaflet), and phosphatidylethanolamine (corresponding to the inner membrane leaflet) was investigated in weanling guinea pigs fed with diets of cacao (saturated fatty acids), sunflower oil [n-6 polyunsaturated fatty acids (PUFA)] or fish oil (n-3 PUFA) for 20 wk. RBC deformation was measured by means of a cell-transit analyzer (filtration) and a cone-plate rheoscope. The contents of saturated fatty acids in plasma phospholipids and RBC membrane leaflets were similar in all three groups. Diets with sunflower oil resulted in a high content of linoleic acid in plasma cholesteryl esters and in the outer leaflet of RBC membranes. Fatty acids of fish oil were mainly incorporated in plasma phospholipids and in the inner leaflet of RBC membranes. The arachidonic acid content was high in all groups in the plasma phospholipids and in the inner leaflet. The n-6 and n-3 PUFA were mainly incorporated in the inner leaflet. In all groups the polyunsaturated/saturated fatty acid ratio and the total PUFA content were similar in the inner RBC membrane. The RBC filtration times and the RBC deformation indices were not affected by the dietary treatment.

Formation and relaxation of erythrocyte membrane tethers in micropipettes.

The red blood cell (RBC) membrane forms tethers in response to shear forces acting on tiny membrane points. Tether formation depends on viscous and elastic membrane properties and has been used as indicator of membrane fragility. A micropipette technique was used to study time dependent tether formation and tether relaxation of individual RBC. Point attached RBC were aspirated at a negative pressure of -5 mm H2O into a micropipette with an internal diameter of 7.8 microm. If tether formation occurred and the tether reached a length of approximately 16 microm, the pipette was carefully pulled back. The RBC left the orifice of the micropipette and the tether relaxed and pulled the main body of the RBC back to the attachment point. The relaxation of the tethers was exponential. The time constant for tether relaxation was 0.144 s which is similar to the time constant for recovery of entire RBC from extensional elastic deformation. Repeated tether formation and relaxation of the same RBC led to an earlier begin of tether formation and changed the behavior of tether growth, although the relaxation time did not change. We conclude that repeated tether formation decreases the resistance of the RBC membrane to form tethers upon given shear forces. Weakening of the membrane due to repeated plastic deformation may play a role in the membrane loss of circulating RBC during aging.

The NO-donor FK409 improves mechanical properties of activated neonatal PMN.

BACKGROUND: Activated polymorphonuclear neutrophils (PMN) play an important role in the microcirculation. Nitric oxide (NO) reduces the sequestration of PMN in the narrow vessels of various organs and, therefore, may reduce organ injury during inflammation. OBJECTIVES: Since PMN of term neonates show various functional differences compared to PMN in adults (decreased chemotaxis, decreased intracellular killing, decreased adhesion), we studied the influence of the semi-synthetical NO-donor FK-409 (4-Ethyl-2-hydroxyimino-5-nitro-3-hexenamide) on the deformability of IL-8 activated PMN in term neonates and adults. METHODS: A cell transit analyzer (CTA) was used to study transit times of individual PMN through 8 µm filter pores, neutrophil elastase concentrations were determined by enzyme-immunoessay and activation of PMN was classified by mircroscopic evaluation. RESULTS: The transit times of PMN activated by IL-8 in adults were 9.3 ± 2.9 s, in term neonates 10.7 ± 3.3 s. FK-409 improved the transit time of activated PMN in adults (5.4 ± 1.6 s) and in term neonates (5.6 ± 1.1 s). Despite of the functional differences of PMN in term neonates and adults, the improvement of the transit times by FK-409 was not different between the two groups. The NO donor decreased the neutrophil elastase concentrations and the morphological signs of activation in neonates and adults. CONCLUSIONS: We conclude that the NO-donor FK-409 improves the microcirculation by increasing the deformability of IL-8 activated PMN. NO may reduce in neonates tissue damage by reduced PMN sequestration due to decreased PMN rigidity.

Shear stress and force required for tether formation of neonatal and adult erythrocytes.

Red blood cells (RBC) of neonates have a shorter survival time and they are more susceptible to mechanical alterations than RBC of adults. Irreversible alteration of the membrane of RBC of preterm neonates, term neonates and adults due to tether formation was studied by means of a micropipette technique. Shear stress and forces were applied with this technique in an axisymmetric configuration and were calculated with an approximation method. The applied shear stress and forces that induced tether formation were lowest for RBC of preterm neonates (1.5 Pa, 1.8 pN), in between for RBC of neonates (1.7 Pa, 2.1 pN) and highest for RBC in adults (1.9 Pa, 2.6 pN). Decreased mechanical stability of the membrane of neonatal RBC may in part cause the shorter life span, the greater tendency to endocytosis and the higher amount of irregularly shaped cells of neonatal RBC compared to RBC in adults.

Expression of BARHL1 in medulloblastoma is associated with prolonged survival in mice and humans.

Medulloblastoma is the most common malignant brain tumor in childhood, and development of targeted therapies is highly desired. Although the molecular mechanisms of malignant transformation are not fully understood, it is known that medulloblastomas may arise from cerebellar granule neuron precursors. The homeodomain transcription factor Barhl1 is known to regulate migration and survival of granule cell precursors, but its functional role in medulloblastoma is unknown. We show here that the expression of BARHL1 is significantly upregulated during human cerebellar development and in human medulloblastoma samples as compared with the normal adult cerebellum. We also detected high levels of Barhl1 expression in medulloblastomas of Math1-cre:SmoM2 mice, a mouse model for Sonic hedgehog-associated medulloblastomas that we developed previously. To investigate Barhl1 function in vivo during tumor development, we generated Barhl1(-/-)Math1-cre:SmoM2 mice. Interestingly, tumors that developed in these mice displayed increased mitotic activity and decreased neuronal differentiation. Moreover, survival of these mice was significantly decreased. Similarly, low expression of BARHL1 in human medulloblastoma cases was associated with a less favorable prognosis for patients. These results suggest that the expression of Barhl1 decelerates tumor growth both in human and in murine medulloblastomas and should be further investigated with respect to potential implications for individualized therapeutic strategies.

Effects of phosphodiesterase (III/IV)-inhibitors and cytokines on mechanical properties of neutrophilic granulocytes in neonates and adults.

Sequestration of activated PMN and enrichment in tissues play a key role in tissue damage during septicaemia and after ischemia/reperfusion. Since polymorphonuclear neutrophilic granulocytes (PMN) of term neonates show various functional differences compared to PMN in adults (decreased chemotaxis, decreased intracellular killing, decreased adhesion) we studied the influence of interleukin 8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) on the reduction of deformability of PMN in neonates and adults. The following phosphodiesterase (PDE)-inhibitors were applied to ameliorate the reduction in deformability when the PMN were stimulated with fMLP or IL-8: Enoximone, Milrinone (PDE-III-inhibitors), Pentoxifylline (PTX) and Piclamilast (PDE-IV-inhibitors). The micropipette technique and the cell transit analyzer (CTA) were used and compared. Aspiration times into micropipettes with an internal diameter of 5 microm, transit times through 8 microm filter pores and neutrophil elastase concentrations were determined. Despite of the functional differences of PMN in neonates compared to adults the significant decrease of deformability of PMN activated with cytokines compared to passive PMN was not different in both groups. The neutrophil elastase concentrations reflect the activation of the PMN: highest concentrations during activation, decreased concentrations due to PDE-inhibitors, and PMN in a passive state. The neutrophil elastase concentrations were not different from PMN of neonates and adults. These PDE-inhibitors significantly increased the deformability of activated PMN but significant differences between the deformability of PMN in neonates and adults were not found. Despite the functional differences of PMN in neonates PDE-III/IV-inhibitors lead to similar improvement of mechanical properties of activated PMN in neonates and adults. These drugs may ameliorate impaired microcirculation also in neonates during inflammation.

Gamma-hydroxybutyric acid sedation in neonates and children undergoing MR imaging.

BACKGROUND: Insufficient sedation in pediatric magnetic resonance imaging (MRI) results in prolonged examination time. To describe the efficacy and side effects of sedation with Phenobarbital short-time infusion followed by continuous gamma-hydroxybutyric acid (GHB) infusion in neonates and children for MRI examinations in a retrospective study. PATIENTS: With Institutional Review Board approval 94 children (Group I: 1-4 weeks; Group II: >1 to 6 months; Group III: >6 months) were sedated with phenobarbital 10 mg/kg (maximum 200 mg) intravenously 30 min prior to examination. Than intravenous sedation was maintained with GHB 10 mg/kg/h after a priming dose of 30 mg/kg in 20 min. RESULTS: In group 1 all neonates (n=8) were well sedated without side effect. One of 21 infants in group 2 showed restlessness and the MRI failed. Two of 65 patients of group 3 were not sufficiently sedated and one of them vomited. CONCLUSIONS: Non-invasive diagnostic procedures in neonates and children may be managed by phenobarbital and GHB sedation with side effects or failure of 3%.

Deformability of passive and activated neutrophils in children with Gram-negative septicemia.

Animal experiments suggest that obstruction of small vessels by polymorphonuclear neutrophils (PMNs) may contribute to the disturbed microcirculation in septic shock. The purpose of this investigation was to study deformability and volume of passive and activated PMNs and immature neutrophils in 15 children with Gram-negative septicemia and in 20 healthy children. Membrane cytoplasm tongues of neutrophils were aspirated by means of a micropipette system into 2.5-microm (diameter) pipettes for 60 s. Tongue growth was used as an indicator of deformability and the cell volume was calculated from the cell circumference before aspiration. Septicemic children showed higher percentages of immature neutrophils (38+/-13%) and activated PMN (12+/-5%) than healthy children (3+/-2% and 3+/-2%). In septicemic children, cellular volume of passive neutrophils decreased progressively with increasing maturity from myeloblasts (493+/-105 fL) to mature PMNs (346+/-29 fL) and the final tongue length in the micropipette increased from 2.7+/-1.1 to 8.5+/-1.8 microm during maturation. The final tongue length of activated PMNs was decreased by 60% compared with that of passive PMNs. The increased number of rigid activated and immature neutrophils may contribute to impaired microcirculation in septicemic patients.

Effect of lipid A on the deformability, membrane rigidity and geometry of human adult red blood cells.

Lipid A is responsible for the activities of endotoxin and may cause circulatory failure and haemolysis. This study evaluated the effects of different lipid A concentrations on red blood cell (RBC) deformation (rheoscope), the aspiration pressure required to aspirate RBC into 3.3 microns pipettes, the membrane shear elastic modulus (i.e. membrane rigidity) and cellular geometry (micropipette system) after 15 min of incubation. Lipid A concentrations of 10 and 100 micrograms ml-1 of RBCs decreased RBC deformability by 26% and 39%, respectively. The aspiration pressure for RBCs into a 3.3 microns micropipette increased by 235% at a lipid A concentration of 10 micrograms ml-1 and by 586% at a concentration of 100 micrograms ml-1. The elastic shear modulus almost doubled at a lipid A concentration of 10 micrograms ml-1 and tripled at 100 micrograms ml-1. At a lipid A concentration of 100 micrograms ml-1, 37% of RBCs showed spicules. These echinocytes were less deformable than discocytes. Mean corpuscular volume, RBC volume and surface area were not affected by lipid A. We conclude that lipid A causes marked reduction of RBC deformability due to increasing membrane rigidity.

Lipid A binding to neonatal and adult red blood cells.

Lipid A is responsible for the endotoxic activities of gram-negative bacteria. Binding of lipid A (50 micrograms/ml) to RBC was studied using a passive hemolysis test. RBC from adults, cord and venous RBC from full-term infants and RBC from preterm infants were studied. Lipid A sensitized RBC were hemolysed with anti-lipid A and guinea pig complement. Hemolysis was expressed as hemoglobin concentration (absorbance at 546 nm) in the supernatant after centrifugation. 50 micrograms/ml lipid A did not increase spontaneous hemolysis (< 3%) after 60 min of incubation in any of the four groups. The passive hemolysis test did not result in additional hemolysis (5%) of umbilical cord RBC. RBC of preterm infants showed a significant increase in hemolysis (24%) after 60 min of incubation. In RBC of full-term neonates, increased hemolysis (14 and 46%) was detected after 30 and 60 min of incubation. Adult RBC hemolysed stronger (26 and 64%) after 30 and 60 min than neonatal RBC. We conclude that lipid A binds less to neonatal RBC compared to adults.