• Neonatal Medicine
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• v.18 no.1
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• pp.59-69
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• 2011

Purpose: Current studies have demonstrated the neuroprotective effects of 6-cyano-7-nitroquinoxalin-2,3-dione (CNQX) in many animal models of brain injury, including hypoxic-ischemic (HI) encephlopathy, trauma and excitotoxicity, but limited data are available for those during the neonatal periods. Here we investigated whether CNQX can protect the developing rat brain from HI injury via mediation of nitric oxide synthase. Methods: In an in vivo model, left carotid artery ligation was done in 7-day-old Sprague-Dawley (SD) rat pups. The animals were divided into six groups; normoxia (N), hypoxia (H), hypoxia with sham-operation (HS), hypoxia with operation (HO), HO treated with vehicle (HV), and HO treated with CNQX at a dose of 10 mg/kg (HC). Hypoxia was made by exposure to a 2 hr period in the hypoxic chamber (92% $N_2$, 8% $O_2$). In an in vitro model, embryonic cortical neuronal cell culture of SD rats at 18-day gestation was done. The cultured cells were divided into three groups: normoxia (N), hypoxia (H), and hypoxia treated with CNQX (HC). The N group was prepared in 5% $CO_2$ incubators and the other groups were placed in 1% $O_2$) incubators (94% $N_2$, 5% $CO_2$) for 16 hr. Results: In the in vitvo and in vivo models, the expressions of iNOS and eNOS were reduced in the hypoxia group when compared to the normoxia group, whereas they were increased in the CNQX-treated group compared to the hypoxia group. In contrast, the expression of nNOS was showed reversely. Conclusion: CNQX has neuroprotective property over perinatal HI brain injury via mediation of nitric oxide synthase.

• Clinical and Experimental Pediatrics
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• v.52 no.5
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• pp.594-602
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• 2009

Purpose : Transforming growth factor (TGF)-${\beta}1$ reportedly increases neuronal survival by inhibiting the induction of inducible nitric oxide synthase (NOS) in astrocytes and protecting neurons after excitotoxic injury. However, the neuroprotective mechanism of $TGF-{\beta}1$ on hypoxic-ischemic (HI) brain injury in neonatal rats is not clear. The aim of this study was to determine whether $TGF-{\beta}1$ has neuroprotective effects via a NO-mediated mechanism and N-methyl-D-aspartate (NMDA) receptor modulation on perinatal HI brain injury. Methods : Cortical cells were cultured using 19-day-pregnant Sprague-Dawley (SD) rats treated with $TGF-{\beta}1$ (1, 5, or 10 ng/mL) and incubated in a 1% O2 incubator for hypoxia. Seven-day-old SD rat pups were subjected to left carotid occlusion followed by 2 h of hypoxic exposure (7.5% $O_2$). $TGF-{\beta}1$ (0.5 ng/kg) was administered intracerebrally to the rats 30 min before HI brain injury. The expressions of NOS and NMDA receptors were measured. Results : In the in vitro model, the expressions of endothelial NOS (eNOS) and neuronal NOS (nNOS) increased in the hypoxic group and decreased in the 1 ng/mL $TGF-{\beta}1-treated$ group. In the in vivo model, the expression of inducible NOS (iNOS) decreased in the hypoxia group and increased in the $TGF-{\beta}1$-treated group. The expressions of eNOS and nNOS were reversed compared with the expression of iNOS. The expressions of all NMDA receptor subunits decreased in hypoxia group and increased in the $TGF-{\beta}1$-treated group except NR2C. Conclusion : The administration of $TGF-{\beta}1$ could significantly protect against perinatal HI brain injury via some parts of the NO-mediated or excitotoxic mechanism.

• Neonatal Medicine
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• v.16 no.2
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• pp.221-233
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• 2009

Purpose: Erythropoietin (EPO) has neuroprotective effects in many animal models of brain injury, including hypoxic-ischemic (HI) encephalopathy, trauma, and excitotoxicity. Current studies have demonstrated the neuroprotective effects of EPO, but limited data are available for the neonatal periods. Here in we investigated whether recombinant human EPO (rHuEPO) can protect the developing rat brain from HI injury via modulation of NMDA receptors. Methods: In an in vitro model, embryonic cortical neuronal cell cultures from Sprague-Dawley (SD) rats at 19-days gestation were established. The cultured cells were divided into five groups: normoxia (N), hypoxia (H), and 1, 10, and 100 IU/mL rHuEPO-treated (H+E1, H+ E10, and H+E100) groups. To estimate cell viability and growth, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was done. In an in vivo model, left carotid artery ligation was performed on 7-day-old SD rat pups. The animals were divided into six groups; normoxia control (NC), normoxia Sham-operated (NS), hypoxia-ischemia only (H), hypoxia-ischemia+vehicle (HV), hypoxia-ischemia+rHuEPO before a HI injury (HE-B), and hypoxia-ischemia+rHuEPO after a HI injury (HE-A). The morphologic changes following brain injuries were noted using hematoxylin and eosin (H/E) staining. Real-time PCR using primers of subunits of NMDA receptors (NR1, NR2A, NR2B, NR2C and NR2D) mRNA were performed. Results: Cell viability in the H group was decreased to less than 60% of that in the N group. In the H+E1 and H+E10 groups, cell viability was increased to >80% of the N group, but cell viability in the H+E100 group did not recover. The percentage of the left hemisphere area compared the to the right hemisphere area were 98.9% in the NC group, 99.1% in the NS group, 57.1% in the H group, 57.0% in the HV group, 87.6% in the HE-B group, and 91.6% in the HE-A group. Real-time PCR analysis of the expressions of subunits of NMDA receptors mRNAs in the in vitro and in vivo neonatal HI brain injuries generally revealed that the expression in the H group was decreased compared to the N group and the expressions in the rHuEPO-treated groups was increased compared to the H group. Conclusion: rHuEPO has neuroprotective property in perinatal HI brain injury via modulation of N-methyl-D-aspartate receptors.