Objective: Neonatal asphyxia is a leading cause of long-term neurologic disabilities varying from mild behavioural dysfunctions to severe seizure, mental retardation, and/or cerebral palsy amongst the newborn. At present, adjunctive therapeutic option and ongoing research into the pathophysiology of neuronal injury after neonatal asphyxia in the developing brain are still limited.  Here, we report that granulocyte colony-stimulating factor (G-CSF) exerts multidimensional beneficial effects involving recovery of synaptic efficacy, neurogenesis, and long-term functional outcome in the developing brain after neonatal asphyxia.

Methods: Ten-day-old (P10) rat pups were subjected to experimental hypoxia insult. G-CSF (10-50 g/kg, single injection/day, P14-19) was subcutaneously administered to test animals and immunoflourecin imaging with confocal laser microscopy, co-immunoprecipitation with immunobloting, whole-cell patch-clamping technique, and chromatin immunoprecipitation were used to analyse synaptic alterations within the hippocampal CA1 subregion (an essential integration area for mammalian learning and memory) (P20). The performance of long-term learning and memory was assessed at later ages (P37-58).

Results: Delayed treatment of G-CSF reversed neonatal asphyxia-induced decreases in the expression of phosphorylated cAMP-responsive element-binding protein-mediated signalling, synaptic plasticity, and neurogenesis, as well as brain-derived neurotrophic factor (BDNF) expression through epigenetic modification with trimethylation of lysine 4 on histone H3 in bdnf promoter gene, corresponding to long-term functional outcome without introducing apparent adverse effects.

Interpretation: Using clinically relevant therapeutic scheme of peripherally delayed treatment with G-CSF is able to promote a combined beneficial mechanism involving neurogenesis with synaptic plasticity recovery, and improve long-term functional outcome in neonatal asphyxia animals.

Hypoxic-ischemic encephalopathy is a major cause of neurological disabilities in childhood. A new approach to studying treatment for neonatal hypoxic-ischemic encephalopathy is investigating states of tolerance. Tolerance is attained by preconditioning tissue to sublethal stress, which causes the tissue to become more tolerant of subsequent lethal insult. Although neurons are the cellular target of preconditioning, ischemic tolerance occurring on vascular cells may also contribute greatly to neuroprotection. Elucidating the protective mechanisms of preconditioning may offer potential therapies against hypoxic-ischemic brain injury.

Our study showed that in 7-day-old rat pups, ligating the carotid artery 1 h before hypoxia damaged the ipsilateral cerebral hemisphere; in contrast, ligating the artery 24 h before hypoxia provided complete neuroprotection. The protective effect of the 24-h artery ligation preconditioning model requires the activation of cAMP response element-binding protein (CREB), a transcription factor implicated in synaptic plasticity, learning and memory, and survival of the nervous system. Vessels and nerves guide each other to their target during development, and they use common signals, such as VEGF-A, to determine the fate of neurons and endothelial cells. Because VEGF-A is highly expressed in neurons and vessels throughout the development, VEGF-A may be a major protector through its dual neuronal and vascular effects in the developing brain. We tested the hypothesis that VEGF-A/VEGFR-2 signaling that leads to CREB activation is the shared pathway underlying the protective effect of preconditioning in neurons and endothelial cells. VEGF-A, VEGFR-1, or VEGFR-2 was inhibited by antisense oligodeoxynucleotides (ODN) in vivo, and by a VEGF-A neutralizing antibody or VEGFR-2 inhibitor in vitro. CREB phosphorylation (pCREB), and VEGF-A and VEGFR-2 expression were increased and co-localized in vascular endothelial cells and neurons in the ipsilateral cerebral cortex 24 h post-ligation. The antisense ODN blockades of VEGF-A and VEGFR-2 decreased pCREB and reduced the protection of 24-h ligation preconditioning. Furthermore, oxygen-glucose deprivation (OGD) preconditioning upregulated VEGF-A, VEGFR-2, and pCREB levels, and protected immortalized H19-7 neurons and b.End3 endothelial cells against 24-h OGD cell death. Blocking VEGF-A or VEGFR-2 reduced CREB activation and the effects of OGD preconditioning in neurons and endothelial cells. Transfecting a serine-133 phosphorylation mutant CREB also inhibited the protective effect of OGD preconditioning. We conclude that VEGF-A/VEGFR-2 signaling leading to CREB phosphorylation is the shared pathway underlying the preconditioning-induced protective effect in neurons and vascular endothelial cells in the developing brain.

We then tested the hypotheses that selectively activating VEGFR-2 with VEGF-E, instead of activating VEGFR-1 with placenta growth factor (PlGF), phosphorylated CREB and provided neuroprotection. We found that VEGFR-2 instead of VEGFR-1 was expressed in vessels and neurons of postpartum day 7 rat brain. VEGF-A and VEGF-E treatment before hypoxic-ischemia selectively activated VEGFR-2 and CREB, and provided protection against hypoxic-ischemic brain injury. Furthermore, selectively activating VEGFR-2 with VEGF-E instead of VEGFR-1 with PlGF after hypoxic-ischemia also provided significant neuroprotection.

These findings suggest that VEGFR-2, but not VEGFR-1, is pivotal for neuroprotection in the developing brain, and that pharmacological activation of VEGFR-2/CREB signaling may be an important strategy for treating neonatal hypoxic-ischemic brain injury.

BACKGROUND
We reported at 2006 ASPR the prophylactic indomethacin (IND) to reduce the incidence of severe intraventricular hemorrhage (IVH) in Japanese extremely low birthweight (ELBW) infants in a large randomized controlled tria. Our hypothesis is that the reduced incidence of severe IVH may result in improved neurological sequela, namely prevalence of cerebral palsy at three years of age.

OBJECTIVE
To compare the prevalence of cerebral palsy around 3 years between the two groups (IND or placebo) in a follow-up study.

DESIGN / METHODS
We studied 469 infants between November 1999 and August 2003. The trial was conducted in 21 Level III neonatal intensive care units in Japan. Newborn infants with birthweights between 400-1000 g were randomly assigned to either IND group (235 infants) or placebo groups (234 infants). Three doses of IND (0.1 mg/kg/dose) starting within 6 hours of birth or placebo were given with 6-hour continuous infusion every 24 hours. Neurological assessment of each infant was conducted by qualified physicians around three years of age.

RESULTS
Of the 418 survived infants, 378 (90.4 %), 191 IND infants and 187 placebo infants, were assessed around three years of age.

Cerebral palsy or suspected cerebral palsy was present in 63 infants (16.3 %), 31 in IND group, 32 in placebo group. There is no significant difference (p=0.87) in the prevalence of cerebral palsy or suspected cerebral palsy or death between the IND group (53 infants, 22.6%) and the placebo group (61 infants, 26.1%).

Subgroup analysis was made for IVH grade three and four, and CP or death for each birth weight groups. In 400-599g group the number of CP or death in the IND group was 7 in 22, whereas it was 18 in 27 in the placebo group. The odds ratio is 0.23 and the IND has been shown to be effective to reduced CP or death.

CONCLUSION

1. For the whole study subjects, there was no difference in the number of infants with “CP or death” by the prophylactic use of IND compared with placebo
2. The prevalence of CP in each grade of IVH showed no evidence of difference between IND and placebo groups
3. The prevalence of PVL showed no evidence of difference in IND and placebo groups
4. In a subgroup analysis, the prevalence of “CP or death” was significantly lower in infants with birthweight 400-599g in IND group than placebo group

Application of prophylactic IND to the smallest group of ELBW 400-599g is shown as effective in preventing the death or development of CP at 3-5 years

Background
Results of previous systematic reviews of randomised controlled trials (RCT) have shown that probiotic supplementation reduces the risk of definite necrotising enterocolitis (NEC: RR 0.36, 95% CI 0.20-0.65, p <0.0008) and all cause mortality (RR 0.47, 95% CI 0.30-0.73, p < 0.0007) significantly while reducing the time to full feeds (WMD -2.74 days, 95% CI -4.98 to -0.51) in preterm (Gestation <34 weeks) very low birth weight (VLBW: birth weight <1500 grams) neonates. Subsequent to these reviews few more RCTs have been published in this area. Trial sequential analysis (TSA) is useful to assess if results of cumulative meta analyses are conclusive.
Aim
Update our 2007 systematic review and conduct a TSA to assess if its results are conclusive.

Methods
Standard search strategy recommended by the Cochrane Neonatal Review Group was followed. Cochrane CENTRAL, MEDLINE, CINHAL, EMBASE data bases and proceedings of Pediatric Academic Society meetings and Gastroenterology conferences were searched. RCTs of any probiotic supplement started within first 10 days, and continued for ≥ 7 days in preterm VLBW neonate, and reporting on ≥ Stage II NEC (Modified bell Staging) were included in the analysis. Trial quality (Jadad score), and possibility of publication bias (Funnel plot) were assessed.

Results
11 good quality RCTs including 4 new trials were included in analysis by fixed effects model. There was no statistical heterogeneity. The probiotic products, protocols, types of milk, and enteral feeding regime varied between trials. The previous results, that risk of definite NEC and all cause mortality (but not of late onset blood culture positive sepsis) are reduced significantly, were confirmed. The confidence intervals were narrow, and the p values were extremely low (< 0.00001). No significant adverse effects were reported in any of the trials. TSA indicated that the results were conclusive for at least 30% reduction in the incidence of NEC.

Conclusion
Probiotics significantly reduce the risk of definite NEC and all cause mortality in preterm VLBW neonates. These conclusive results along with those from observational studies on routine use, use in extremely low birth weight neonates, and safety in terms of long term neurodevelopmental impairment, justify change in practice if a safe and suitable product is available. Further placebo controlled trials are not necessary in this area.