IntroductionPreclinical work and studies in adults have shown that endogenous regeneration efforts that involve mobilization of progenitor cells take place after brain injury. However, kinetics of endogenous circulating progenitor cells (CPCs) in preterm neonates is not well described, particularly their possible role regarding brain injury and regeneration. We aimed to assess the kinetics of CPCs in neonates with encephalopathy of prematurity in relation to brain injury biomarkers, chemoattractants and relevant antenatal and postanal clinical factors, in an effort to outline the related pathophysiology.Materials and methods47 preterm neonates (of 28–33 weeks GA) were enrolled: 31 newborns with no or minimal brain injury (grade I IVH) and 16 prematures with encephalopathy (grade III or IV IVH, PVL or infarct). Peripheral blood samples obtained on days 1, 3, 9, 18 and 45 after birth were analyzed using flow cytometry, focusing on EPCs (early and late Endothelial Progenitor Cells), HSCs (Hematopoietic Stem Cells) and VSELs (Very Small Embryonic-Like Stem Cells). At the same time-points serum levels of S100B, Neuron-specific Enolase (NSE), Erythropoietin (EPO), Insulin-like growth factor-1 (IGF-1) and SDF-1 were also measured. Neonates were assessed postnatally with brain MRI, and with Bayley III developmental test at 2 years of corrected age.ResultsPreterms with brain injury proved to have significant increase of S100B and NSE, followed by increase of EPO and enhanced mobilization mainly of HSCs, eEPCs and lEPCs. IGF-1 was rather decreased in this group of neonates. IGF-1 and most CPCs were intense decreased in cases of antenatal or postnatal inflammation. S100B and NSE correlated with neuroimaging and language scale in Bayley III test, providing good prognostic ability.ConclusionThe observed pattern of CPCs’ mobilization and its association with neurotrophic factors following preterm brain injury indicate the existence of an endogenous brain regeneration process. Kinetics of different biomarkers and associations with clinical factors contribute to the understanding of the related pathophysiology and might help to early discriminate neonates with adverse outcome. Timely appropriate enhancement of the endogenous regeneration effort, when it is suppressed and insufficient, using neurotrophic factors and exogenous progenitor cells might be a powerful therapeutic strategy in the future to restore brain damage and improve the neurodevelopmental outcome in premature infants with brain injury.

This chapter synopsizes all clinically relevant information found in the literature regarding brain injury in preterm and full-term neonates for the two most studied biomarkers, S100B and NSE. Biomarker’s prognostic ability and levels in cord blood, serum, urine, and saliva, in healthy and diseased infants, that are reported among different studies are brought together and correlated with each other, so that similarities will lead to practical conclusions for the bedside clinician and the future researcher. S100B and NSE seem to be reliable biomarkers for neonatal brain injury in most studies. They correlate well with neuroimaging and long-term neurodevelopmental outcome. S100B is the most studied and appears superior to NSE. Serum is probably the most suitable sample for clinical use. Pitfalls can be avoided when S100B and NSE are used in clinical practice by having control measurements in healthy neonates comparable with ones in bibliography and skipping hemolyzed samples. These two biomarkers are able to help distinguishing neonates of higher risk of adverse long-term outcome in an early phase, so additional neuroprotective strategies and therapeutic protocols can be applied.

OBJECTIVE: Early identification of neonates at risk of neurological impairment is particularly important for the bedside clinician. Clinical value of S100b and neuron-specific enolase in neonates has not been yet established. We investigated their kinetics and possible early clinical utility in neonatal encephalopathy (NE). STUDY DESIGN: 36 full-term neonates (13 with moderate/severe encephalopathy, 11 with mild encephalopathy, 12 controls) were enrolled and studied prospectively. Serum S100b and neuron-specific enolase (NSE) were measured serially on days(d) 1, 3, 9 and 18 of life. Brain MRI and long-term neurodevelopmental outcome were also assessed. RESULT: Neonates with moderate/severe encephalopathy had significantly increased S100b (d1) and NSE levels (d1, d3, d9) compared to controls. Neuron-specific enolase significantly correlated with the degree of encephalopathy, and a cutoff of 38.8 mu g/l (d1) accurately predicted moderate/severe encephalopathy. S100b (d1) cutoff points of 1.6 mu g/l and 11.4 mu g/l prognosticated severe encephalopathy and death/cerebral palsy, respectively. Both biomarkers correlated well with neuroimaging and Bayley-III scores. CONCLUSION: Combined clinical, laboratory, imaging and neurodevelopmental data indicate that serum S100b and NSE can be useful biomarkers for the diagnosis and prognosis of neonatal brain injury, providing useful information to the bedside clinician.

Intraventricular hemorrhage (IVH) is a major cause of morbidity and mortality in preterm neonates. Elucidation of the mechanisms underlying IVH and/or development of disease biomarkers is essential. The aim of the study was to investigate the urine metabolic profile of preterm neonates (gestational age < 32 weeks) IVH and explore the role of metabolomics in understanding pathophysiological mechanisms of the disease from which novel biomarkers could be derived. In this single-center, prospective, case-control study, urine samples were collected from seven preterm infants with early IVH (IVH group) and from 11 preterm ones without IVH (control group) on days 1, 3 and 9 of life. Urine metabolites were evaluated using targeted liquid chromatography-tandem mass spectrometry. Demographic and perinatal-clinical characteristics were recorded. Univariate and multivariate statistical analyses were performed. Orthogonal Partial Least Squares-Discriminant Analysis showed that the study groups differed significantly due to alternation in 20 out of the 40 metabolites detected in the urine. Elevated differentiated metabolites included energy intermediates and other important compounds, whereas reduced ones various amino acids, hypoxanthine and nicotinamide. A set of metabolites showed high performance as indicators of IVH, especially during day 1. As evidenced by metabolomics, preterm neonates with IVH demonstrate significant metabolism perturbations. Potentially, a selected panel of metabolites could be used as urine biomarkers of IVH development and/or progression in high-risk preterm infants.

Background: Hypoxic-ischemic encephalopathy (HIE), a serious complication of perinatal asphyxia, is commonly associated with an unfavorable outcome. In-depth research is important not only for the interpretation of the underlying biological alternations but may also provide the basis for the development of novel diagnostic and therapeutic tools. The application of metabolomics in perinatal asphyxia/HIE is a relatively new approach. Methods: We performed a narrative, non-systematic review in the literature of metabolomic studies involving newborn animals and humans exposed to hypoxia-ischemia or developing perinatal asphyxia/HIE. Results: Fifteen animal studies, nine studies in human neonates, and two review articles were evaluated. Changes in the metabolomic profile of newborn animals exposed to hypoxia-ischemia and of asphyxiated neonates with HIE are presented in relation to the underlying pathophysiology. The clinical relevance of these findings is further discussed in a comprehensible to the bedside clinician manner. Conclusions: Metabolomics may provide an explanation for the various metabolic alternations occurring in perinatal asphyxia/HIE, elucidate the biological background of the applied therapeutic interventions and promote the development of novel diagnostic-prognostic biomarkers of the disease.

BACKGROUND Metabolomics could provide valuable insights into hypoxemic-ischemic encephalopathy (HIE) revealing new disease-associated biochemical derangements. The study aimed to investigate urine metabolic changes in neonates with HIE compared to healthy controls, using targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS). PATIENTS AND METHODS In this prospective, single-center study we enrolled neonates born at ≥ 36 weeks gestation with HIE (HIE group) and healthy controls (control group). We collected urine samples for metabolomic analysis on days one, three, and nine of life. RESULTS Twenty-one full-term newborns were studied, 13 in the HIE group and eight in the control group. Six of the affected neonates had moderate/severe HIE and seven mild HIE. Therapeutic hypothermia was applied only in four neonates with moderate/severe HIE. Multivariate and univariate statistical analysis showed a clear separation between the HIE and the control groups. Discriminant metabolites involved pyruvic acid, amino acids, acylcarnitines, inositol, kynurenine, hippuric acid, and vitamins. CONCLUSIONS We have identified a specific metabolic profile in neonates with HIE, adding to the existing knowledge on the disease biochemistry that may potentially help in biomarker development. HIPPOKRATIA 2017, 21(2): 80-84.

Introduction: Various populations of adult stem cells have been described in bonne marrow and other tissues. It has been reported that in acute stress or disease conditions adult stem cells may be released in the circulation and home to the region of the lesion aiming to limit the tissue damage and eventually contribute in the regeneration of the affected tissue. However, in case of a severe neonatal brain damage this endogenous process is not sufficient to heal the patient resulting in long term neurodevelopmental consequences.

BACKGROUND:Preclinical data and adult studies have showed an endogenous regeneration process following brain damage that involves mobilization of progenitor cells. This process is not well described in preterm neonates. The present study aims to investigate the mobilization of Circulating Progenitor Cells (CPCs) and their relation to biomarkers of brain injury in preterm neonates.METHODS:This is a prospective cohort study of preterm infants with gestational age (GA) <34 weeks. Serial cranial ultrasounds scans were performed in all neonates. Brain injury was defined by the presence of intraventricular hemorrhage grade III/IV, cystic periventricular leukomalacia or infarct. Peripheral blood samples were collected from all neonates on days(d) 1, 3, 9, 18 and 45 of life for the measurement of levels of CPCs [early and late Endothelial Progenitor Cells (EPCs), Haematopoietic Stem Cells (HSCs) and Very Small Embryonic-Like Stem Cells (VSELs)], Neuron-Specific Enolase (NSE), S100b, Erythropoietin (EPO) and Stromal Cell-Derived Factor-1 (SDF-1) .RESULTS:Ten out of the 23 preterm infants included in the study developed brain injury; the remaining thirteen infants served as controls. In the brain injury group a significant increase of HSCs (d9, d45), early EPCs (d3, d9, d18) and late EPCs (d1, d3, d9, d18, d45) was observed compared to controls. VSELs on d45 were significantly higher in controls. S100b on d1, EPO on d1, SDF-1 on d3 and NSE on d18 were significantly increased in the brain injury group. Moreover, CPCs were significantly related to S100b, NSE, EPO and SDF-1 levels at multiple time points.CONCLUSIONS:The observed pattern of CPCs mobilization and its association with biomarkers following brain injury in preterm neonates indicate the existence of an endogenous brain regeneration process. Enhancement of this process with exogenous progenitor cell transplantation might be a powerful therapeutic strategy to restore brain damage and improve the neurodevelopmental outcome in premature infants. Hippokratia 2015; 19 (2):141-147.

Background and Aims The assumption that the circulating progenitor cells participate in a chemotactic way in the endogenous regeneration effort after damage of body tissues is controversial and the exact type of the cells involved remains unknown, especially when referring to damages of the Central Nervous System (CNS). We assume that preterm neonates who undergo CNS injury respond in a similar way, and we investigate the progenitor cell populations that might be related to the devastating event. Methods 23 preterm newborns were enrolled (gestation age ≤32weeks). 10 of them undergo severe perinatal stress with metabolic acidosis and developed CNS injury (IVH III or higher, PVL or infarct), whereas 13 of them were assumed as controls with no obvious CNS injury. Peripheral blood was collected at days 1, 3, 9, 18 and 45 after birth and analyzed using flow cytometry. Cell populations of interest were EPCs (Endothelial progenitor cells, CD34+/CD133+/CD184+), HSCs (Hematopoietic stem cells, CD34+/CD184+/CD45+) and VSELs (Very Small Embryonic-Like SCs, CD34+/CD184+/CD45-). Results EPCs were significantly increased in the group with CNS injury at days 1.9 and 18 and VSELs were marginally increased at day 1 and significantly at day 9. HSCs showed no specific variation. Conclusion Circulating progenitor cells seem to play a role in the endogenous regeneration effort. Enhancing this effort might prove to be a good therapeutic practice in the future, whereas delineation of the timeframe of this effort would be essential. Larger studies are needed, as well as correlation with chemoattractants and longterm outcome is necessary.