Olga Romantsik

Exosomes from human mesenchymal stem cells conduct aerobic metabolism in term and preterm newborn infants

Exosomes are secreted nanovesicles that are able to transfer RNA and proteins to target cells. The emerging role of mesenchymal stem cell (MSC) exosomes as promoters of aerobic ATP synthesis restoration in damaged cells, prompted us to assess whether they contain an extramitochondrial aerobic respiration capacity. Exosomes were isolated from culture medium of human MSCs from umbilical cord of ≥37‐wk‐old newborns or between 28‐ to 30‐wk‐old newborns (i.e., term or preterm infants). Characterization of samples was conducted by cytofluorometry. Oxidative phosphorylation capacity was assessed by Western blot analysis, oximetry, and luminometric and fluorometric analyses. MSC exosomes express functional respiratory complexes I, IV, and V, consuming oxygen. ATP synthesis was only detectable in exosomes from term newborns, suggestive of a specific mechanism that is not completed at an early gestational age. Activities are outward facing and comparable to those detected in mitochondria isolated from term MSCs. MSC exosomes display an unsuspected aerobic respiratory ability independent of whole mitochondria. This may be relevant for their ability to rescue cell bioenergetics. The differential oxidative metabolism of preterm vs. term exosomes sheds new light on the preterm newborns clinical vulnerability. A reduced ability to repair damaged tissue and an increased capability to cope with anoxic environment for preterm infants can be envisaged.—Panfoli, I., Ravera, S., Podestà, M., Cossu, C., Santucci, L., Bartolucci, M., Bruschi, M., Calzia, D., Sabatini, F., Bruschettini, M., Ramenghi, L. A., Romantsik, O., Marimpietri, D., Pistoia, V., Ghiggeri, G., Frassoni, F., Candiano, G. Exosomes from human mesenchymal stem cells conduct aerobic metabolism in term and preterm newborn infants. FASEB J. 30, 1416–1424 (2016). www.fasebj.org

High presence of extracellular hemoglobin in the periventricular white matter following preterm intraventricular hemorrhage

Severe cerebral intraventricular hemorrhage (IVH) in preterm infants continues to be a major clinical problem, occurring in about 15–20% of very preterm infants. In contrast to other brain lesions the incidence of IVH has not been reduced over the last decade, but actually slightly increased. Currently over 50% of surviving infants develop post-hemorrhagic ventricular dilatation and about 35% develop severe neurological impairment, mainly cerebral palsy and intellectual disability. To date there is no therapy available to prevent infants from developing either hydrocephalus or serious neurological disability. It is known that blood rapidly accumulates within the ventricles following IVH and this leads to disruption of normal anatomy and increased local pressure. However, the molecular mechanisms causing brain injury following IVH are incompletely understood. We propose that extracellular hemoglobin is central in the pathophysiology of periventricular white matter damage following IVH. Using a preterm rabbit pup model of IVH the distribution of extracellular hemoglobin was characterized at 72 h following hemorrhage. Evaluation of histology, histochemistry, hemoglobin immunolabeling and scanning electron microscopy revealed presence of extensive amounts of extracellular hemoglobin, i.e., not retained within erythrocytes, in the periventricular white matter, widely distributed throughout the brain. Furthermore, double immunolabeling together with the migration and differentiation markers polysialic acid neural cell adhesion molecule (PSA-NCAM) demonstrates that a significant proportion of the extracellular hemoglobin is distributed in areas of the periventricular white matter with high extracellular plasticity. In conclusion, these findings support that extracellular hemoglobin may contribute to the pathophysiological processes that cause irreversible damage to the immature brain following IVH.

Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples.

Background Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups. Methods and Results Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15–4.8) vs 0.3% (0.032–2.23) p = 0.0001 and in neonates <32 weeks of gestational age (GA) compared to those ≥32 wks GA: 0.95% (range 0.18–4.8) and 0.36% (0.15–3.2) respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term) and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP) did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL) resulted higher compared to term (p = 0.004) and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017). The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168). Conclusions We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs) from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144)The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies.

DBS-LC-MS/MS assay for caffeine : Validation and neonatal application

AIM DBS might be an appropriate microsampling technique for therapeutic drug monitoring of caffeine in infants. Nevertheless, its application presents several issues that still limit its use. This paper describes a validated DBS-LC-MS/MS method for caffeine. RESULTS The results of the method validation showed an hematocrit dependence. In the analysis of 96 paired plasma and DBS clinical samples, caffeine levels measured in DBS were statistically significantly lower than in plasma but the observed differences were independent from hematocrit. CONCLUSION These results clearly showed the need for extensive validation with real-life samples for DBS-based methods. DBS-LC-MS/MS can be considered to be a good alternative to traditional methods for therapeutic drug monitoring or PK studies in preterm infants.

Pharmacological pain and sedation interventions for the prevention of intraventricular hemorrhage in preterm infants on assisted ventilation ‐ an overview of systematic reviews

This is a protocol for a Cochrane Review (Overview). The objectives are as follows: To summarize the evidence from systematic reviews regarding the effects and safety of pharmacological interventions related to pain and sedation management in order to prevent GMH-IVH in ventilated preterm infants.

Behavioral testing and litter effects in the rabbit

Background: Behavioral testing provides an essential approach in further developing our understanding of brain structure and function. The aim of our study was to outline a more expanded approach to cognition‐ and anxiety‐related behavior in the rabbit. Methods: Twenty‐one 70‐day old rabbits (13 female, 8 male) were exposed to open field test, dark‐light box test and object recognition testing with variations in inter‐trial‐interval, olfactory recognition and object location testing. Independent T‐tests were used to compare data by individual baseline characteristics, i.e. birth weight, weight at testing, sex, litter #, litter size. Results: In the open field test, median time spent in the center was 3.64s (0.84–41.36) for the 9 rabbits who entered the center; median distance moved in the arena was 874.42cm (54.20–3444.83). In the dark light box test, 12 rabbits entered the light compartment. In the object recognition task, rabbits spent significantly less time exploring the familiar object compared to the novel (0.40s [0–2.8] vs. 3.17s [1.30–32.69]; P=0.003) when using a 30‐min inter‐trial interval, as well with a 90‐min inter‐trial interval: 0.87s [0–7.8] vs. 7.65s [0–37.6] (P=0.008). However, recognition was lost when using a 24‐h inter‐trial interval (time spent exploring the familiar object: 3.33 [0–10.90]; novel object:3.87 [1.15–48.53]; n.s). In the object location task and in olfactory object recognition task, median discrimination indexes were 0.69 (−1 to 1) and 0.37 (−0.38 to 0.78) respectively, higher than level expected by chance (P<0.001). Litter size >3 during the neonatal period was associated with increased explorative behavior in the dark light box test (P=0.046) and in the visual object recognition task (P=0.005), whereas body weight and sex were not. Conclusions: Settings and outcome measures for multiple behavioral tests, providing reference values and considerations for future developmental studies are reported. Discrimination and memory in the rabbit appear to relate to litter characteristics, although a larger sample size is needed to confirm our findings. HIGHLIGHTSA comprehensive approach to behavioral testing in the adult rabbit is described.The rabbits show intact object recognition memory longer than previously reported.Exploration and memory performance are dependent partly upon litter characteristics.

Pathophysiology of extracellular haemoglobin : use of animal models to translate molecular mechanisms into clinical significance

The bloods major gas exchange is carried out by haemoglobin, a haeme protein that binds iron and oxygen and can have potentially dangerous side‐effects due to redox reactions. Haemoglobin is a very abundant molecule with a concentration of 150 g/l in whole blood, resulting in almost one kg haemoglobin in an adult human body. Normal turnover of red blood cells results in significant haemoglobin release, and pathological conditions that involve haemolysis can lead to massive haemoglobin levels. To control for the potential threat of extracellular haemoglobin, several protective defence systems have evolved. Many pathological conditions, diseases as well as iatrogenic conditions, such as infusion of haemoglobin‐based oxygen carriers, cerebral intraventricular haemorrhage, extracorporeal circulation and the pregnancy complication pre‐eclampsia, involve abnormal levels of haemolysis and extracellular haemoglobin. Although quite different aetiology, the haemoglobin‐induced damage often causes similar clinical sequelae and symptoms. Here, we will give an overview of the pathophysiological mechanisms of extracellular haemoglobin and its metabolites. Furthermore, we will highlight the use of animal models in advancing the understanding of these mechanisms and discuss how to utilize the knowledge in the development of new and better pharmaceutical therapies.

Clonidine for neonates receiving mechanical ventilation

© 2016 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.This is a protocol for a Cochrane Review (Intervention). The objectives are as follows: To assess whether clonidine infusion in term and preterm newborn infants receiving mechanical ventilation reduces the rate of mortality and morbidity. The intervention will be compared to placebo, no treatment and dexmedetomidine. In addition, the safety of clonidine infusion will be assessed for potential harms. We will perform subgroup analyses according to gestational age; birth weight; dose, duration and route of clonidine administration; and presence of pharmacological sedation as co-intervention.