Beyond brain injury biomarkers: chemoattractants and circulating progenitor cells as biomarkers of endogenous rehabilitation effort in preterm neonates with encephalopathy.

Introduction: Preclinical 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 methods: 47 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. Results: Preterms 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. Conclusion: The 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.

Urine metabolomic profile in neonates with hypoxic-ischemic encephalopa-thy.

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.

Mobilization of circulating progenitor cells following brain injury in premature neonates could be indicative of an endogenous repair process. A pilot study.

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.

Therapeutic hypothermia in asphyxiated neonates with hypoxic-ischemic encephalopathy: A single-center experience from its first application in Greece.

BACKGROUND/AIM: Therapeutic hypothermia has become an established therapy in asphyxiated neonates with evidence of moderate/severe hypoxic-ischemic encephalopathy. Herein, we describe our recent experience with total body cooling in asphyxiated neonates, which is the first relevant report in Greece. PATIENTS AND METHODS: The medical records of all asphyxiated newborns treated with therapeutic hypothermia in our center between September 2010 and October 2013 were retrospectively reviewed. We recorded data related to neonatal-perinatal characteristics, whole body cooling and outcome. RESULTS: Twelve asphyxiated neonates [median gestational age 38 weeks (36-40)] received whole body cooling (rectal temperature 33.5 ± 0.5 (o)C for 72 hours) during the study period for moderate (n=3) and severe (n=9) hypoxic-ischemic encephalopathy. Cooling was passive in 4 and active in 8 (66.7%) cases. Therapeutic hypothermia was initiated at the median age of 5 hours (0.5-11) after birth. Seven neonates survived (58.3%) to hospital discharge. On follow-up (7-35 months), neurodevelopment outcome was normal in 1 case, while 3, 1 and 2 subjects had mild, moderate and severe impairment, respectively. CONCLUSIONS: Our initial experience with whole body cooling supports its beneficial effect in asphyxiated neonates. This treatment should be offered in all centers involved in the care of such neonates using either simple means (passive cooling) or automated cooling devices. Hippokratia 2014; 18 (3): 226-230.

Comparable effect of conventional ventilation versus early high-frequency oscillation on serum CC16 and IL-6 levels in preterm neonates.

OBJECTIVE: Clara cell 16 kD protein (CC16) and interleukin (IL)-6 have been used as peripheral blood biomarkers of alveolar leakage and inflammation, respectively. Thus, their measurement in the bloodstream could be used to assess ventilator-induced lung injury. The objective of this study was to evaluate the effect of optimized synchronized intermittent mandatory ventilation (SIMV) and high-frequency oscillatory ventilation (HFOV) on circulating CC16 and IL-6 levels when used as the initial ventilation modes in preterm neonates. STUDY DESIGN: Single center, prospective, randomized clinical study in preterm neonates (gestational age 30 weeks) requiring mechanical ventilation within the first 2 h of life. Serum CC16 and IL-6 were measured on establishment of the assigned ventilation mode after admission, at days 3 and 14 of life as well as at 36 weeks postmenstrual age. Demographic-perinatal data and clinical parameters were also recorded. RESULT: Of the 30 neonates studied, 24 (gestational age 27.1±1.7 weeks, birth weight 942±214 g) were finally analyzed, equally assigned into the SIMV and HFOV groups. Both groups had comparable demographic-perinatal characteristics and clinical parameters. Serum CC16 and IL-6 altered significantly over time (repeated-measures analysis of variance, both P<0.001). However, changes were not affected by the ventilation mode. Post hoc analysis showed a significant decrease in CC16 and IL-6 from birth up to 36 weeks postmenstrual age in both groups. CONCLUSION: In preterm neonates, SIMV and HFOV are associated with comparable circulating CC16 and IL-6 levels. These findings suggest a similar alveolar leakage and systemic inflammation with any of the ventilation modes evaluated when their usage is optimized.

Association of increased maternal ferritin levels with gestational diabetes and intra-uterine growth retardation.

AIM: The objectives of the present study were to determine whether or not increased serum ferritin in women with premature labour is associated with gestational diabetes mellitus (GDM) and intra-uterine growth retardation (IUGR) and, if so, whether or not such increased levels reflect excess maternal iron stores, and have an effect on neonatal iron status and outcome. METHODS: This prospective, single-hospital, observational study involved 63 mothers and their 90 preterm neonates. Full blood counts as well as serum ferritin, soluble transferrin receptor (sTfR) and erythropoietin concentrations were compared across the three study groups based on maternal ferritin levels at the time of delivery. Perinatal history, neonatal morbidity and early outcomes were also assessed. RESULTS: High maternal ferritin levels were significantly associated with higher rates of GDM and IUGR. However, there was no correlation between maternal ferritin and sTfR levels or between maternal and neonatal iron status. CONCLUSION: Elevated maternal ferritin is not a reflection of excess iron stores, but is related to an increased risk of GDM or IUGR. Also, maternal ferritin levels are not associated with either neonatal iron status or neonatal outcomes.

Use of erythropoietin and its effects on blood lactate and 2, 3-diphosphoglycerate in premature neonates.

The aim of this study was to investigate the effect of recombinant human erythropoietin (rHu-EPO) on oxygen affinity and adequate oxygen delivery to the tissues of stable premature infants. 36 very-low-birth-weight infants were randomly assigned to either receive rHu-EPO (200 units/kg every other day) or not, and both groups were supplemented with iron, folic acid and vitamin E. Arterial blood gases, oxygen saturation, complete blood counts, fetal haemoglobin, 2,3-diphosphoglycerate (2,3-DPG) and blood lactate were analysed weekly, from the 1st week till discharge. Patients in the two groups were comparable. There was a trend in increasing lactate values towards the 4th to 5th weeks of life, which did not reach statistical significance. There was no correlation between lactate values and the studied variables (pH, BE, oxygen saturation). In 35 transfusions, pre- and 24 h post-transfusion blood lactate status was studied. In 23 of them, a decrease in post-transfusion lactate was noticed, whilst an increased post-transfusion level was shown in 10 cases and no change in 2 cases. The mean pre-transfusion lactate value was significantly higher than the post-transfusion one (24.04 +/- 11.9 mg/dl before and 16.27 +/- 8.5 mg/dl after transfusion; p = 0.0025). In both groups there was a steady rise in 2,3-DPG concentration over the period of study, and the 2,3-DPG values at the end of our study were significantly increased in the rHu-EPO group (rHu-EPO 5.98 +/- 0.9, control 4.84 +/- 0.7; p = 0.04). In conclusion, the use of rHu-EPO did not affect blood lactate levels compared to the control group. Regarding oxygen affinity, it seems that rHu-EPO causes a shift of the oxy-haemoglobin dissociation curve to the right. This is a previously unreported effect of rHu-EPO and its clinical use may, thus, confer to preterm babies an added advantage.

Influence of recombinant human erythropoietin on neutrophil function in premature neonates.

The in vivo influence of recombinant human erythropoietin (rhEpo) and iron on human neutrophil (PMN) antimicrobial function was assessed. A total of 21 preterm infants were randomized to receive either 200 U/kg/other day of rHuEPO+12 mg/kg/day of iron (EPO+high Fe, seven infants) or 200 U/kg/other day of rhEPO+4 mg/kg/day of iron (EPO+standard Fe, 9 infants) or 4 mg/kg/day of iron only (standard Fe, five infants). PMNs were isolated from blood of these infants 60+/-5 days after birth and from eight healthy adults. No differences between infants and adults were found in PMN random migration and chemotactic activity to N-formylmethionyl leucyl phenylalanine (FMLP), superoxide anion production in response to FMLP and phagocytosis of Staphylococcus aureus. In contrast, percentage phagocytosis was significantly lower in EPO+standard Fe as compared to both EPO+high Fe and standard Fe groups (P<0.01). This modest impairment of phagocytic activity of neonatal PMNs found in association with administration of rhEPO and standard iron may be related to consumption of iron during rhEPO-enhanced erythropoiesis.

Effect of ketamine on hypoxic-ischemic brain damage in newborn rats.

The present study tests the hypothesis that ketamine, a dissociative anesthetic known to be a non-competitive antagonist of the NMDA receptor, will attenuate hypoxic-ischemic damage in neonatal rat brain. Studies were performed in 7-day-old rat pups which were divided into four groups. Animals of the first group, neither ligated nor exposed to hypoxia, served as controls. The second group was exposed to hypoxic-ischemic conditions and sacrificed immediately afterwards. Animals of the third and fourth groups were treated either with saline or ketamine (20 mg/kg, i.p.) in four doses following hypoxia. Hypoxic-ischemic injury to the left cerebral hemisphere was induced by ligation of the left common carotid artery followed by 1 h of hypoxia with 8% oxygen. Measurements of high energy phosphates (ATP and phosphocreatine) and amino acids (glutamate and glutamine) and neuropathological evaluation of the hippocampal formation were used to assess the effects of hypoxia-ischemia. The combination of common carotid artery ligation and exposure to an hypoxic environment caused major alterations in the ipsilateral hemisphere. In contrast, minor alterations in amino acid concentrations were observed after the end of hypoxia in the contralateral hemisphere. These alterations were restored during the early recovery period. Post-treatment with ketamine was associated with partial restoration of energy stores and amino acid content of the left cerebral hemisphere. Limited attenuation of the damage to the hippocampal formation as demonstrated by a reduction in the number of damaged neurons was also observed. These findings demonstrate that systemically administered ketamine after hypoxia offers partial protection to the newborn rat brain against hypoxic-ischemic injury.

Follow-up of very low birth weight infants after erythropoietin treatment to prevent anemia of prematurity.

OBJECTIVE: Treatment with recombinant human erythropoietin (rHuEPO) stimulates erythropoiesis and reduces the need for transfusions in hospitalized preterm infants. The aim of our study was to follow very low birth weight infants after the initial 6 weeks of rHuEPO treatment. DESIGN AND METHODS: We randomly assigned 97 very low birth weight infants with a gestational age of 31 weeks or less and birth weight of 1500 gm or less to receive rHuEPO, 300 units/kg per week (erythropoietin (EPO) 300, n = 33), rHuEPO, 750 units/kg per week (EPO 750; n = 28), or no treatment (control, n = 36). The rHuEPO was administered from the first week of life for 6 weeks. After EPO therapy was discontinued, 75 neonates were followed weekly until discharge and at 3, 6, and 12 months of age. RESULTS: Mean numbers (+/- SD) of packed erythrocyte transfusions per patient from the time rHuEPO therapy was discontinued until discharge were 0.38 +/- 0.64 (EPO 300), 0.23 +/- 0.52 (EPO 750), 0.9 +/- 1.1 (control) (p < 0.05 in both EPO groups vs control). Mean reticulocyte counts at the sixth week were 6% +/- 2.2% (EPO 300), 6.9% +/- 2.2% (EPO 750), and 3.1% +/- 2.6% (control) in the three groups (p < 0.01 in both EPO groups vs control), and at the eighth week were 4.7% +/- 2.8% (EPO 300), 5.4% +/- 2.7% (EPO 750), and 2.6% +/- 2.2% (control) (p < 0.01 in both EPO groups vs control). Serum ferritin levels were significantly higher at the sixth week, and the percentage of hemoglobin F was significantly lower at 6, 8, and 10 weeks in the control group versus EPO groups. At 3, 6, and 12 months of age, there were no differences in reticulocytes, ferritin, HbF, and growth among groups. CONCLUSION: Preterm infants who received rHuEPO had a normal pattern of erythropoiesis after the drug was discontinued. These data provide strong evidence that the anemia of prematurity is the result of a transient developmental abnormality in EPO production.

In which neonates does early recombinant human erythropoietin treatment prevent anemia of prematurity? Results of a randomized, controlled study.

To assess whether erythropoietin (EPO) treatment is safe and reduces the need for transfusion, we randomized 44 preterm infants to an EPO group and a comparable control (CON) group. EPO 150 U/kg was given s.c. twice weekly for 6 wk from the 1st wk of life. Hematologic parameters, transfusion requirements, and growth were followed during therapy and for 6 mo thereafter. To better assess in which neonates EPO treatment was effective, we classified retrospectively the EPO and CON groups into uncomplicated neonates (EPO A: n = 9, birth weight = 1247 +/- 126 g, gestational age = 29.8 +/- 1.5 wk; CON A: n = 7, birth weight = 1217 +/- 145 g, gestational age = 29.9 +/- 1.5 wk) and neonates requiring artificial ventilation (EPO B: n = 16, birth weight = 1169 +/- 249 g, gestational age = 28.1 +/- 2 wk; CON B: n = 12, birth weight = 1173 +/- 215 g, gestational age = 28.3 +/- 2 wk). There were significant differences in reticulocytes between both uncomplicated and ventilated neonates in the EPO group compared with respective control groups. However, the need for transfusion was significantly less in the uncomplicated EPO group (EPO A: 0.44 +/- 0.73 versus CON A: 1.28 +/- 0.75, p < 0.05) but not in the neonates on ventilation (EPO B: 8.25 +/- 5 versus CON B: 7.75 +/- 3.7). In conclusion, early EPO administration reduces the need for transfusion in uncomplicated premature neonates.(ABSTRACT TRUNCATED AT 250 WORDS)