Vassiliki Soubasi

Erythropoietin prevents long-term sensorimotor deficits and brain injury following neonatal hypoxia-ischemia in rats

Perinatal asphyxia accounts for behavioral dysfunctions that often manifest as sensorimotor, learning or memory disabilities throughout development and into maturity. Erythropoietin (Epo) has been shown to exert neuroprotective effects in different models of brain injury including experimental models of perinatal asphyxia. However, the effect of Epo on functional abilities following cerebral hypoxia-ischemia (HI) in neonatal rats is not known. The aim of the present study is to investigate the effect of Epo on sensorimotor deficits and brain injury induced by hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia. Epo was administered as a single dose immediately after the hypoxic insult (2000 U/kg). The neuroprotective effect of Epo was evaluated at postnatal day 42 by using a battery of behavioral tests and histological analysis. The results of the present study suggest that Epo treatment immediately after HI insult significantly facilitated recovery of sensorimotor function. Consistently, histopathological evaluation demonstrated that Epo significantly attenuated brain injury and preserved the integrity of cerebral cortex. These findings indicate that long-term neuroprotective effect of Epo on neonatal HI-induced brain injury might be associated with the preservation of sensorimotor functions.

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.

ABSTRACT: 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. Although stimulation of erythropoiesis was apparent in both uncomplicated and complicated neonates, the end-result of increased need for transfusion in complicated neonates was related to altered indication of transfusion. These infants probably require further or longer EPO administration after weaning from ventilation and improvement of clinical condition.

Hypoxia-ischemia affects erythropoietin and erythropoietin receptor expression pattern in the neonatal rat brain

Erythropoietin (EPO), known for its role in erythroid differentiation, has been suggested to have non-hematopoietic functions in the brain, especially during development. In the present study, we investigated the expression of erythropoietin and erythropoietin receptor (EPOR) in the developing rat brain following hypoxia-ischemia. Seven-day-old rats underwent unilateral, permanent carotid artery ligation followed by 1 h of hypoxia, and their brains were examined immediately, 24 h or 4 days after hypoxia-ischemia. RT-PCR and Western blot analysis revealed that hypoxia-ischemia only marginally affected EPO expression. Immunohistochemical study of brains 4 days after hypoxia showed that 60 min of hypoxia (resulting in cortical infarction and severe neuronal loss in other regions) led to the increased EPO immunoreactivity, especially in the boundaries of the damaged cerebral cortex, associated with astrocytosis. In contrast, EPOR was dramatically upregulated within 24 h after hypoxia-ischemia. These results suggest that there is a rapid response of EPOR to the hypoxic-ischemic stimulus, which seems to precede that of EPO, leading to the hypothesis that the EPO/EPOR system is implicated in the processes of neuroprotection from hypoxia-ischemia.

Resveratrol ameliorates hypoxia/ischemia-induced behavioral deficits and brain injury in the neonatal rat brain

Hypoxia-ischemia (HI) induced injury of the neonatal brain accounts for behavioral deficits concerning mainly neurological reflexes, sensorimotor functions and learning/memory disabilities that may evolve throughout development. The positive biological effects of resveratrol, a natural compound with anti-oxidant/anti-inflammatory properties found mainly in red wine have been indicated recently. Aim of this study was to investigate the delayed outcome of early administration of resveratrol in an experimental model of hypoxic-ischemic encephalopathy, by means of behavioral analysis and late neuropathological examination. Seven-day-old (P7) rats were separated into 3 groups: Group 1 underwent HI and treated with resveratrol. Group 2 (HI-treated) was subjected to HI and received same volume of saline. Group 3 (sham-operated) was the control group. A battery of behavioral tests was performed from days P8-P66, during which early reflexes (righting reflex, gait, geotaxis), sensorimotor (rope suspension, beam walking, rotarod) and learning/memory function (passive avoidance, Morris water-maze) were examined. Significant difference among the groups was observed in righting reflex, rotarod and water maze tests in which the resveratrol group almost reached the performance of the control animals. The other behavioral tests showed that control and resveratrol groups were better compared to HI, although not significant. Neuropathology study revealed a remarkable reduction of the infarct and preservation of myelination after resveratrol treatment, which was in most cases correlated with the better performance of the resveratrol group. These findings indicate that long-term neuroprotective effect of resveratrol on neonatal HI-induced gray and white matter damage might be associated with the preservation of behavioral functions.

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.

Neuroprotective effect of long-term MgSO4 administration after cerebral hypoxia-ischemia in newborn rats is related to the severity of brain damage.

Previous studies have shown contradictory results regarding magnesium-mediated neuroprotection in animal models of perinatal asphyxia. The aim of this study is to investigate the e fects of MgSO4 postasphyxial treatment on hypoxia-ischemia (HI)—induced brain injury in neonatal rats and the possibility that this e fect is related to the severity of brain damage. Seven-day-old rats underwent unilateral carotid artery ligation followed by 1 or 2 hours of hypoxia (8% O2) and MgSO4 administration. Adenosine triphosphate/phosphocreatine and glutamate/glutamine measurements and neuropathological evaluation of the hippocampus were used to assess the e fects of HI and MgSO4. HI caused time-dependent changes in energy stores, amino acid concentrations, and brain damage. Administration of MgSO4 after 1 hour but not after 2 hours of hypoxia resulted in significant prevention of HI-induced brain injury. MgSO4 administration results in a significant protection against moderate HI-induced brain damage, whereas it fails to offer a similar effect against severe brain damage.

Evaluation of Long-Lasting Sensorimotor Consequences following Neonatal Hypoxic-Ischemic Brain Injury in Rats: The Neuroprotective Role of MgSO4

Perinatal asphyxia (PA) is a major determinant for long-term sensorimotor and locomotor deficits. The model of neonatal hypoxia-ischemia (HI) in 7-day-old rats produces sensorimotor cortex, thalamus and striatum injury, which are all critical for the maintenance of sensory motor function. The aim of this study was to evaluate the long-term neurodevelopmental disturbances in the above experimental model and to assess the neuroprotective effect of MgSO4 in terms of long-term behavioral and morphological changes. Seven-day-old rats were separated into three groups: A (control), neither ligated nor exposed to hypoxia; B (HI/MgSO4) ligated, exposed to hypoxia and treated with MgSO4 (2 g/kg b.w., i.p.), and C (HI) ligated and exposed to hypoxia. At the age of 42 days, the behavior of the rats was evaluated using 5 sensorimotor tests. Muscle power, motor coordination, reflexes, and limb placing were tested to different sensory stimuli. The study was completed with the histopathological evaluation of brain tissue damage. In all individual tests the HI-treated rats performed significantly worse than the control and MgSO4-treated rats and this difference was more pronounced in the limb placing tests. Additionally, neonatal HI resulted in extensive neuronal damage that was limited after MgSO4 administration. Behavioral alterations represent a useful endpoint for studying the consequences of a perinatal HI insult and the efficacy of potential neuroprotective treatments. MgSO4 administration resulted in prevention of HI-induced sensorimotor deficits and brain injury.

Erythropoietin prevents hypoxia/ischemia-induced DNA fragmentation in an experimental model of perinatal asphyxia

Erythropoietin (EPO) prevents neuronal damage following ischemic, metabolic and excitotoxic stress. Recent studies have shown that EPO plays a significant role in the developing brain. The present study investigates the effect of EPO administration on hypoxic-ischemic brain injury and the possibility that its neuroprotective action may be associated with anti-apoptotic activity. Seven-day-old rats were treated with EPO (2000 U/kg) and subjected to a modified Levine procedure. EPO administration before the hypoxic-ischemic insult significantly reduces the severity of brain damage and improved the short-term functional brain recovery. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling and DNA electrophoresis displayed no evidence of DNA fragmentation in EPO-treated animals. These results suggest that EPO might protect the neonatal rat brain by anti-apoptotic mechanisms.

Early abnormal amplitude-integrated electroencephalography (aEEG) is associated with adverse short-term outcome in premature infants.

BACKGROUND In preterm infants with IVH the electrocortical background activity is affected and there is a correlation between the severity of cerebral injury to the degree of depression, however the usefulness of the early aEEG recordings has hardly been determined. AIM To identify early aEEG features that could be used as prognostic markers for severe brain injury in prematures. METHODS In 115 infants, 25-32 wk GA, aEEG recordings during the first 72 h of life were correlated with head ultrasound findings. Continuity (Co), sleep-wake cycling (Cy) and amplitude of the lower border (LB) of the aEEG were evaluated by semi-quantitative analysis. RESULTS The infants were divided into four groups based on head ultrasound findings: A (n=72, normal), B [n=16, grades 1-2 intraventricular hemorrhage (IVH)], C (n=21, grades 3-4 IVH) and D (n=6, periventricular leukomalacia). 18 infants (16 of group C and 2 of group D) died during hospitalization. Significantly lower values of all aEEG features were found in group C infants. The presence of pathological tracings (burst-suppression, continuous low-voltage, flat trace) or discontinuous low-voltage (DLV), the absence of Cy and LB<3 μV in the initial aEEG displayed a sensitivity of 88.9%, 63% and 51.9% respectively, for severe brain injury. Logistic regression of aEEG features and GA to the presence or absence of severe injury revealed that only Co was significantly correlated to outcome. Using this feature 83.19% of cases were correctly classified. CONCLUSION Pathological tracings or DLV in the initial aEEG is predictive for poor short-term outcome in premature neonates.