Hydrogen peroxide signaling modulates neuronal differentiation via microglial polarization and Wnt/ß-catenin pathway.

OBJECTIVE: Reactive oxygen species (ROS) are generated within the cell and serve as second messengers in fundamental cellular processes under physiologic conditions. Although the deleterious effects of high-level ROS associated with oxidative stress are well established, it is unclear how the developing brain reacts to redox changes. Our aim is to investigate how redox alteration affects neurogenesis and the mechanism that underlies it. MATERIALS AND METHODS: We investigated in vivo microglial polarization and neurogenesis in zebrafish after hydrogen peroxide (H2O2) incubation. To quantify intracellular H2O2 levels in vivo, a transgenic zebrafish line that expresses Hyper and termed Tg(actb2:hyper3)ka8 was used. Then, in vitro studies with N9 microglial cells, 3-dimensional neural stem cell (NSC)-microglia coculture, and conditioned medium experiments are carried out to comprehend the mechanism underlying the changes in neurogenesis upon redox modulation. RESULTS: In zebrafish, exposure to H2O2 altered embryonic neurogenesis, induced M1 polarization in microglia, and triggered the Wnt/ß-catenin pathway. N9 microglial cell culture experiments revealed that exposure to H2O2 resulted in M1 polarization in microglial cells, and this polarization was mediated by the Wnt/ß-catenin pathway. Redox modulation of microglia interfered with NSC differentiation in coculture experiments. Neuronal differentiation was significantly higher in NSCs cocultured with H2O2-treated microglia when compared to control microglia. Wnt inhibition prevented the effects of H2O2-treated microglia on NSCs. No significant alterations were observed in conditioned medium experiments. CONCLUSIONS: Our findings point to a robust interplay between microglia and neural progenitors influenced by the redox state. Intracellular H2O2 levels can interfere with neurogenesis by altering the phenotypic state of the microglia via the Wnt/ß-catenin system.

Impact of postnatal nutrition on neurodevelopmental outcome in rat model of intrauterine growth restriction.

OBJECTIVE: There are limited data on nutritional management of infants with intrauterine growth restriction (IUGR). Postnatal protein supplementation for promoting growth is a common clinical practice in neonatology. The present study aims to investigate the consequences of protein supplementation on long-term growth, brain and body weight, brain histology and behavioral outcome in a rat model of IUGR. MATERIALS AND METHODS: Twenty-four IUGR-formed rat puppies and 12 healthy puppies were included in the study. IUGR model was established by low (10%) protein diet throughout pregnancy together with intraperitoneal injection of lipopolysaccharide (LPS). Pups were started to be fed with either standard protein (SP), or high protein (HP) diet until postnatal day (PN) 35. Puppies in the control group were given SP diet for 35 days. Six pups from each group were sacrificed at PN7, remaining six were evaluated by Morris water maze test between PN 30 to 35 days and then sacrificed at PN35. Histologic evaluation of brain tissue was performed at PN7 and PN35. RESULTS: IUGR group displayed lower body and brain weights at PN7 when compared with control. At PN35, SP group achieved similar brain/body weight ratios with control, whereas HP group displayed lowest brain/body weight ratio. The number of TUNEL positive cells was significantly higher and myelin basic protein and oligodendrocyte marker O4 immunoreactivity were significantly lower in HP group when compared with SP at PN35. Neuronal density in prefrontal cortex and hippocampus at PN7 were similar among SP and HP groups, but significantly lower in HP group when compared with SP at PN35. SP group displayed better results in the Morris water maze test when compared with HP group. CONCLUSIONS: Although postnatal HP support is associated with increase in body weight at PN35, it did not result in better brain/body weight ratios in the rat model of IUGR. In IUGR rats, HP diet was associated with increased apoptosis in brain tissue with lower neuronal density and decreased myelination when compared to SP. Furthermore, better neurodevelopmental scores were achieved by SP diet rather than HP support in IUGR.

Topiramate and darbepoetin alpha ameliorate bilirubin-induced neuronal cell damage.

OBJECTIVE: The aim of this study was to determine whether prophylactic darbepoetin alpha and/or topiramate administration could prevent bilirubin neurotoxicity (BNTx) in experimental model of kernicterus. MATERIALS AND METHODS: A total of 60 Wistar albino rat puppies with experimental kernicterus model were included in the study. The Kernicterus was established administering a bilirubin injection via a cisterna magna puncture 30 minutes after ip drug injection. The puppies were divided into five groups with 12 in each group as shown below: a control group, bilirubin group, darbepoetin alpha group, topiramate group and darbepoetin alpha+ topiramate group. Darbepoetin alpha and/or topiramate were administered on day 5 intraperitoneally (ip). At the 6th and 24th hours, bilirubin induced neurological dysfunction (BIND) score was used to assess behavioral changes. Hearing functions were evaluated on days 10 and 28. On day 30, the Water Maze water tank test was implemented to evaluate spatial memory. The rats were sacrificed on days 6 and 34 and apoptosis in the globus pallidus and hippocampus was examined. RESULTS: The BIND score was improved following darbepoetin alpha treatment. Neither darbepoetin alpha nor topiramate therapy ameliorate spatial memory. There were no significant differences between groups in terms of the auditory brainstem response (ABR). The combined use of darbepoetin alpha and topiramate lead to slight decrease in apoptosis. CONCLUSIONS: Darbepoetin alpha or topiramate administration ameliorates bilirubin induced neurological dysfunction in experimental model of kernicterus.

Beneficial effects of carnosine and carnosine plus vitamin E treatments on doxorubicin-induced oxidative stress and cardiac, hepatic, and renal toxicity in rats.

OBJECTIVE: Oxidative stress plays an important role in doxorubicin (DOX)-induced toxicity. Carnosine (CAR) is a dipeptide with antioxidant properties. The aim of this study was to evaluate the decreasing or preventive effect of CAR alone or combination with vitamin E (CAR + Vit E) on DOX-induced toxicity in heart, liver, and brain of rats. METHODS: Rats were treated with CAR (250 mg kg(-1) day(-1); intraperitoneally (i.p.)) or CAR + Vit E (equals 200 mg kg(-1) α-tocopherol; once every 3 days; intramuscularly) for 12 consecutive days. On the 8th day of treatment, rats were injected with a single dose of DOX (30 mg kg(-1), i.p.). Serum cardiac troponin I (cTnI), urea, and creatinine levels; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities; and oxidative stress parameters in tissues were measured. We also determined thiobarbituric acid reactive substances, diene conjugate, protein carbonyl (PC), and glutathione levels and antioxidant enzyme activities. RESULTS: DOX resulted in increased serum cTnI, ALT, AST, urea, and creatinine levels and increased lipid peroxide and PC levels in tissues. CAR or CAR + Vit E treatments led to decreases in serum cTnI levels and ALT and AST activities. These treatments reduced prooxidant status and ameloriated histopathologic findings in the examined tissues. CONCLUSION: Our results may indicate that CAR alone, especially in combination with Vit E, protect against DOX-induced toxicity in heart, liver, and kidney tissues of rats. This was evidenced by improved cardiac, hepatic, and renal markers and restoration of the prooxidant state and amelioration of histopathologic changes.

Prolonged unconjugated hyperbilirubinaemia associated with the haem oxygenase-1 gene promoter polymorphism.

AIM: To elucidate the genetic factors causing hyperbilirubinaemia in prolonged jaundice of the newborns, we investigated whether the HO-1 gene promoter polymorphism is a cause in unexplained pathological or prolonged jaundice. METHODS: Three groups were defined: healthy newborns with no clinical jaundice, newborns hospitalized for jaundice without any identifiable pathological cause and newborns with prolonged jaundice associated with breast milk. Genomic DNA was extracted from the white blood cells and the promoter region of the HO-1 gene was amplified using PCR and their allelic repeats were determined. RESULTS: We did not detect any significant difference in the allele frequencies between the healthy newborns and the newborns whose serum total bilirubin levels were >12.9 mg/dL. However, the patients with short (<24 GT) dinucleotide repeat in the HO-1 gene promoter on either allele had significantly higher prolonged unconjugated hyperbilirubinaemia than the healthy newborns. There was no significant difference between the groups 2 and 3. CONCLUSION: The results indicate that polymorphism of HO-1 gene promoter region can be an underlying cause of the prolonged unconjugated hyperbilirubinaemia associated with breast milk. In this patient population, short repeat alleles of the HO-1 gene promoter polymorphism were associated with prolonged jaundice.

Protective effects of topiramate against hyperoxic brain injury in the developing brain.

Recent studies have shown that exposure to hyperoxia in infant rats leads to extensive apoptotic degeneration in the cortex and white matter of the developing brain. Besides its antiepileptic effects, topiramate exerts neuroprotective effects in animal models of stroke, hypoxia ischemia, excitotoxic insults, and status epilepticus. In the present study, we investigated the effects of topiramate against hyperoxia-induced neurodegeneration in the developing brain. Eighteen Wistar rat pups were divided into three groups: control group, hyperoxia+phosphate buffered saline treated group and hyperoxia+topiramate treated group. Hyperoxia groups were exposed to 80% oxygen (n=12) in plexiglas chambers in which the oxygen concentration was monitored twice daily from birth until postnatal day five. The hyperoxia+topiramate group received an intraperitoneal injection of topiramate at a dose of 80 mg/kg/day. At postnatal day 5, all animals were killed. Neuronal cell death and apoptosis were evaluated. Histopathological examination showed that topiramate significantly diminished apoptosis in the CA1 region and dentate gyrus of hippocampus. Topiramate may offer a therapeutic potential for neuroprotection under conditions of hyperoxic brain injury.

Inhibition of vascular endothelial growth factor-induced retinal neovascularization by retinoic acid in experimental retinopathy of prematurity.

Vascular endothelial growth factor (VEGF) has an important role in the pathogenesis of retinopathy of prematurity (ROP) and inhibition of VEGF expression in the neovascular phase might prevent destructive neovascularization in ROP. It is suggested that retinoids exert a highly potent antiangiogenic activity by inhibiting VEGF expression. The aim of this study was to demonstrate the preventive effect of retinoic acid (RA) on the VEGF-induced retinal neovascularization in a rat model of ROP. Wistar albino rats were placed into incubators at birth and exposed to an atmosphere alternating between 50 % and 10 % O(2) every 24 hours. After 14 days, the animals were removed to room air and received either an intraperitoneal injection of RA (5 mg/kg/day) (n=9) or saline (n=4) daily for six days, and sacrificed at 21 days. Other rats (n=4) were raised in room air and served as age-matched controls. The globe of each eye was cut through the cornea and embedded in paraffin. Serial sections were stained with hematoxylin-eosin for quantification of neovascular nuclei. The avidin-biotin peroxidase method was performed for evaluation of VEGF expression. The average number of neovascular nuclei was significantly lower in the control group compared to that in the ROP groups. In addition, it significantly decreased in the RA-treated ROP group compared to that of the saline-administrated ROP group. VEGF immunostaining was overall negative in room air-exposed rats. The VEGF immunostaining score significantly decreased in the RA-treated ROP group compared to that in the saline-administered ROP group. RA treatment might be beneficial in preventing neovascularization resulting from oxygen-induced retinopathy by downregulation of VEGF expression.

Erythropoietin attenuates lipopolysaccharide-induced splenic and thymic apoptosis in rats.

Apoptosis of lymphoid tissues during sepsis is well documented and linked to the pathobiology of organ failure and death. In this study, we evaluated the effect of a single dose of recombinant erythropoietin (EPO) on thymic and splenic apoptosis in an endotoxic sepsis model. Young male Wistar rats were divided into 3 groups and administered intraperitoneally (IP) either normal saline; lipopolysaccharide (LPS) 10 mg/kg; or EPO (5000 U/kg) 30 min before lipopolysaccharide. Six hours following LPS administration animals were sacrificed. Apoptosis was assessed by hematoxylin-eosin staining, terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick end labeling (TUNEL), and caspase-3 immunostaining. When compared with animals given LPS, animals pretreated with EPO displayed reduced splenic and thymic TUNEL positivity of 44+/-3 (p<0.05) and 143+/-4 (p<0.05) nuclei per high power field (hpf), respectively. Caspase-3 positivity was also significantly reduced in the spleen and thymus, with 31+/-4 (p<0.05) and 93+/-3 (p<0.05) positive stained nuclei per hpf, respectively. Serum nitrite levels were elevated in animals given lipopolysaccharide. Pretreatment with EPO attenuated the increase in nitrite levels; however, this did not reach statistical significance. We conclude that a single dose of recombinant erythropoietin can reduce thymic and splenic apoptosis associated with lipopolysaccharide administration.

Spontaneous intestinal perforation after oral ibuprofen treatment of patent ductus arteriosus in two very-low-birthweight infants.

AIM: To discuss intestinal side effects of ibuprofen in the treatment of patent ductus arteriosus, after having observed two cases of spontaneous intestinal perforation following ibuprofen treatment. METHODS: Clinical and laboratory records of two preterm infants, who developed intestinal perforation after ibuprofen administration, were evaluated. RESULTS: Gestational ages of infants were 29 wk (male) and 30 wk (female). Both infants developed intestinal perforations without signs of necrotizing enterocolitis. The perforations cured with Penrose drainage alone. CONCLUSION: Although ibuprofen is a reasonable treatment alternative to indomethacin, randomized controlled trials, which address potential adverse effects including spontaneous intestinal perforation, are needed.

Synchronized ventilation of very-low-birth-weight infants; report of 6 years' experience.

OBJECTIVES: To evaluate the effects of long-term patient triggered ventilation (PTV) using assist/control or synchronized intermittent mandatory ventilation (SIMV) in very-low-birth-weight infants with respiratory distress. METHODS: Ninety-seven very-low-birth-weight infants who had undergone synchronized ventilation for respiratory distress or insufficiency were assessed from January 1995 to December 2000. Death, oxygen support, pneumothorax development while ventilated, intracranial hemorrhage, necrotizing enterocolitis, periventricular leukomalacia, retinopathy of prematurity and duration of ventilation were noted as the mean outcome measures. RESULTS: The mean birth weight was 1139 +/- 268 g (range 450-1500 g) and the mean gestational age was 29.0 +/- 2.8 weeks (range 23-36 weeks). Eighty-four per cent of 97 infants survived. Antenatal steroids were administered to only 20% of mothers. Surfactant was administered to all of the 67% of infants with respiratory distress syndrome. The mean duration of ventilator support was 4.7 +/- 7.3 days (1-43 days) for survivors and 8.9 +/- 11 days (1-45 days) for infants who died. No respiratory paralysis was necessary in any case during ventilation and pneumothorax was diagnosed in only eight infants. Severe intracranial hemorrhage (grade > or = III) and periventricular leukomalacia developed in 15% and 12% of infants, respectively. Necrotizing enterocolitis (Bell's classification stage > or = 2) and retinopathy of prematurity were noted in two infants. Four infants had evidence of chronic lung disease. The rate of survival without major morbidity was 83.5%. CONCLUSION: Patient-triggered ventilation, initially PTV with Asist/Control and subsequently with SIMV in very-low-birth-weight infants with respiratory distress is feasible, but optimization of trigger and ventilator performance with respect to respiratory diagnosis is essential.

Long-term follow-up of otherwise healthy term infants with marked hyperbilirubinaemia: should the limits of exchange transfusion be changed in Turkey?

AIM: To evaluate prospectively non-haemolytic term infants with marked hyperbilirubinaemia treated by phototherapy only for evidence of bilirubin toxicity at 2-6 y of age, and to determine the suitability for Turkish children of the exchange transfusion limits recently reported by the American Academy of Pediatrics. METHODS: The study group included a total of 30 children, aged 2-6 y, who had developed marked hyperbilirubinaemia (20-24 mg dl(-1), 342-410 micromol l(-1)) during the newborn period (gestational age >37 wk, birthweight >2500 g) and were treated without exchange transfusion because intensive phototherapy, instituted during the preparations for exchange transfusion, was successful in decreasing their serum bilirubin levels. The control group consisted of 30 children of the same age group without clinical jaundice in the newborn period. Physical and neurological examinations, brainstem auditory-evoked potentials (BAEPs) and developmental tests for Turkish children were performed in both the study and control children. RESULTS: There was no difference between the groups with regard to mean BAEP latencies and developmental scores. None of the infants had hearing loss, developmental delay or abnormal neurological findings. CONCLUSION: The results suggest that successful intensive phototherapy without exchange transfusion in otherwise healthy term newborn infants with marked hyperbilirubinaemia (20-24 mg dl(-1), 342-410 micromol l(-1)) might not increase the risk of bilirubin brain injury and that the conventional limit of 20 mg dl(-1) (342 micromol l(-1)) could be changed to 22-24 mg dl(-1) (376-410 micromol l(-1)) for healthy term infants in Turkey. These limits, however, address only infants who do not have haemolytic disease, and the data are not sufficient to draw conclusions on the safety of even higher bilirubin levels (i.e. >24 mg dl(-1), 410 micromol l(-1)) in this population.

Transient tachypnea of the newborn (TTN): a role for polymorphisms of surfactant protein B (SP-B) encoding gene?

BACKGROUND: Transient tachypnea of the newborn (TTN) is usually a benign self-limiting respiratory disorder in the immediate neonatal period. The lipophilic surfactant-associated protein B (SP-B) was demonstrated to be the most relevant structural component of the surfactant system for immediate postnatal pulmonary adaptation. We hypothesized genetic variations of surfactant protein B (heterozygous 121 ins 2 mutation er intron 4 polymorphisms) to be related to TTN. PATIENTS AND METHOD: We screened genomic DNA of 83 healthy term neonates (gestational age: 39 (37 - 41) completed weeks [median and range]; birth weight: 3325 +/- 541 grams [mean +/- SD]) and 75 infants presenting with TTN (gestational age: 38 (37 - 41) completed wecks [median and range]; birth weight: 3091 +/- 435 grams [mean +/- SD]) by means of PCR-amplification, fragment length and sequence analysis. TTN was diagnosed an the basis of the clinical signs with respiratory rate > 60 breaths/minute, fraction of inspired oxygen > 0.21, and characteristic radiographic findings within less than 24 hours after birth. Newborns with any infection, pulmonary or cardiac congenital malformations, postnatal asphyxia and infants born to diabetic mothers were excluded. RESULTS: In TTN-group the frequency of male infants (68.4 % versus 44.6 %, p < 0.05) and caeserian section were significantly higher (68.4 % versus 30.1 %, p < 0.05). We did not find any statistical difference in frequency of intron 4 variations between controls and TTN-group (8.4 % versus 10.7 %). None of the infants were heterozygous for the 121ins2 SP-B mutation. CONCLUSIONS: WC conclude polymorphisms of intron 4 and heterozygous 121 ins 2 mutation not to associated with TTN.

Analyses of polymorphism for UGT1*1 exon 1 promoter in neonates with pathologic and prolonged jaundice.

In this study, we investigated whether a TATA box polymorphism in the promoter of the UGT1*1 exon I, the most common detected DNA polymorphism in Gilbert's syndrome, is a contributory factor in unexplained pathologic or prolonged jaundice. 38 neonates who had unexplained pathologic jaundice, 37 neonates who had unexplained prolonged jaundice, and 35 healthy, nonjaundiced neonates were enrolled in the study. Genotypes were assigned as follows: 6/6 (homozygous for a normal allele bearing the sequence [TA](6)TAA), 7/7 (homozygous for an abnormal allele with the sequence [TA](7)TAA), and 6/7 (heterozygous with one of each allele). Of the 110 infants, 10 (9%) had 7/7, 51 (46%) had 6/7, and 49 (45%) had 6/6 genotype; the differences between the three groups were not statistically significant. Also no differences were observed among different genotypes and mean serum total bilirubin concentrations. In conclusion, we showed that TA 7/7 and TA 6/7 genotypes are not rare in our population and that the presence of these polymorphisms alone does not play a significant role in the etiology of unexplained pathologic or prolonged neonatal hyperbilirubinemia.

Outcome of very-low-birth-weight infants in a developing country: a prospective study from the western region of Turkey.

OBJECTIVE: To illustrate neonatal outcomes, including morbidity, birth weight and gestational age-specific mortality, and care practices for very-low-birth-weight infants admitted to our tertiary neonatal intensive care unit in Turkey and compare these with the corresponding data from recent reports from developed countries. METHODS: Perinatal data were collected prospectively from January 1996 to December 2000. Perinatal events and the neonatal course to 120 days of life, discharge, or death were evaluated. RESULTS: Of 173 infants, 82% survived until discharge to home or to 120 days of life. Survival was 13% for infants of 501-750 g at birth, 74% for those of 751-1000 g, 92% for those of 1001-1250 g and 87% for those of 1251-1500 g. Mortality rates were greater for male than for female infants (25% vs. 12%). The mean birth weight was 1218 (450-1500) g and the mean gestational age was 29.8 (23-36) weeks. The birth weight and gestational age distributions showed that the majority of infants (48%) weighed 1251-1500 g and were between 28 and 31 weeks' gestation (57%). Antenatal steroids were administered to only 19% of mothers. The overall Cesarean section rate was 77%. Respiratory distress syndrome was diagnosed in 36% and surfactant was administered to 98% of these infants. The rate of ventilator support was 54% for a mean duration of 9 days. Air leak syndromes were diagnosed in only nine infants (5%). Severe intracranial hemorrhage (grade > II) and periventricular leukomalacia developed in 9% of infants. Four infants had evidence of chronic lung disease. Retinopathy of prematurity (stage > II) was noted in only one infant, and proven necrotizing enterocolitis (Bell's classification stage > 2) was not observed. The rate of survival without major morbidity was 91%. The mean hospital stay was 40 days for survivors and 19 days for infants who died. CONCLUSION: Despite marked differences in socioeconomic conditions and tertiary care facilities, the mortality (except in the smallest babies) and morbidity rates were comparable with those of recent studies from developed countries.