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.
Animals ; Anoxia ; Brain ; Brain Injuries ; Carotid Arteries ; Cell Culture Techniques ; Cell Survival ; Cells, Cultured ; Eosine Yellowish-(YS) ; Erythropoietin ; Hematoxylin ; Humans ; Ischemia ; Ligation ; Models, Animal ; N-Methylaspartate ; Neurons ; Neuroprotective Agents ; Pregnancy ; Rats ; Real-Time Polymerase Chain Reaction ; Receptors, N-Methyl-D-Aspartate ; RNA, Messenger

PURPOSE: Recently, Geneticin (G418) were known to exert neuroprotective effects in the hypoxic-ischemic (H-I) brain injury, but the mechanism is still unclear. The roles of fibroblast growth factor (FGF) and FGF receptor (FGFR) ware not well known in the H-I brain injury. We investigated the neuroprotective effects of systemically administrated Geneticin through the regulation of FGFR following the H-I brain injury METHODS: The cortical neuron cell culture of Spague-Dawley (SD) rat embryo brain (E18) was done in a hypoxic incubator. The cultured cells were divided three groups: a normoxia group, a hypoxia group, and an Geneticin-treated group. After verifying the desired amount of cellular injury in the hypoxia group, the Geneticin-treated group (after an H-I insult) was further divided into two groups. This produced four final groups: normoxia, hypoxia, and Geneticin-treated groups before H-I insult and a Geneticin-treated group after HI insult. The expression of FGFR-2 and FGFR-3 mRNA was measured using Northern blotting. RESULTS: The expression of FGFR-2 and FGFR-3 mRNA was notably increased in the hypoxic group compared to the normoxic group. In both Geneticin-treated groups before and after a hypoxic insult, the expression of FGFR-2 and FGFR-3 mRNA was decreased. CONCLUSION: It suggests that FGFR has an important role in hypoxic brain injury. Geneticin appears to exert a protective effect through down regulation of the expression of FGFR mRNA. However, more experiments are needed in order to demonstrate the usefulness of Geneticin as a preventative and rescue treatment for H-I brain injuries of neonatal brain.
Animals ; Anoxia ; Blotting, Northern ; Brain ; Brain Injuries ; Cell Culture Techniques* ; Cells, Cultured ; Down-Regulation ; Embryonic Structures ; Fibroblast Growth Factors* ; Fibroblasts* ; Incubators ; Neurons* ; Neuroprotective Agents ; Rats ; Receptors, Fibroblast Growth Factor* ; RNA, Messenger

PURPOSE: Transforming growth factor (TGF)-beta1 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-beta1 on hypoxic-ischemic (HI) brain injury in neonatal rats is not clear. The aim of this study was to determine whether TGF-beta1 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-beta1 (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% O2). TGF-beta1 (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-beta1-treated group. In the in vivo model, the expression of inducible NOS (iNOS) decreased in the hypoxia group and increased in the TGF-beta1-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-beta1-treated group except NR2C. CONCLUSION: The administration of TGF-beta1 could significantly protect against perinatal HI brain injury via some parts of the NO-mediated or excitotoxic mechanism.
Animals ; Anoxia ; Astrocytes ; Brain ; Brain Injuries ; Incubators ; Ischemia ; N-Methylaspartate ; Neurons ; Neuroprotective Agents ; Nitric Oxide ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type II ; Rats ; Receptors, N-Methyl-D-Aspartate ; Transforming Growth Factor beta1 ; Transforming Growth Factors

PURPOSE: Transforming growth factor (TGF)-beta1 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-beta1 on hypoxic-ischemic (HI) brain injury in neonatal rats is not clear. The aim of this study was to determine whether TGF-beta1 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-beta1 (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% O2). TGF-beta1 (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-beta1-treated group. In the in vivo model, the expression of inducible NOS (iNOS) decreased in the hypoxia group and increased in the TGF-beta1-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-beta1-treated group except NR2C. CONCLUSION: The administration of TGF-beta1 could significantly protect against perinatal HI brain injury via some parts of the NO-mediated or excitotoxic mechanism.
Animals ; Anoxia ; Astrocytes ; Brain ; Brain Injuries ; Incubators ; Ischemia ; N-Methylaspartate ; Neurons ; Neuroprotective Agents ; Nitric Oxide ; Nitric Oxide Synthase ; Nitric Oxide Synthase Type II ; Rats ; Receptors, N-Methyl-D-Aspartate ; Transforming Growth Factor beta1 ; Transforming Growth Factors

OBJECTIVE: TGF-beta1 is an important neuronal survival factor to neurons from damage induced by cerebral ischemia. We examined whether treatment with the TGF-beta1 has neuroprotective effects on HI brain injury in neonatal rats using Rice-Vannucci model (in vivo) and in rat brain cortical cell culture induced by hypoxia (in vitro). METHODS: Seven-day-old Sprague-Dawley (SD) rat pups were subjected to left carotid occlusion followed by 2 hour of hypoxic exposure. At the before and after 30 minutes of HI, the animals were injected intracerebrally with TGF-beta1 0.5 ng/kg. In addition, brain cortical cell culture model using pregnant SD rats for 19 days were experimented and induced for hypoxia cell injury. The cell were treated with TGF-beta1 1 ng/mL, 5 ng/mL and 10 ng/mL separately. and incubated in 1% O2 incubator. Apoptosis was measured in the injured hemispheres 7 days after the HI insults using western blot for pro-apoptotic marker-bax, caspase-3 and anti-apoptotic marker-Bcl-2. RESULTS: In western blot and real-time PCR showed Caspase-3, Bax and Bax/Bcl-2 levels was reduced and Bcl-2 level was increased in vivo. In brain cortical cell culture, Bcl-2 expression was greater in the group with low dose of TGF-beta1 (1 ng/mL) in western blot. CONCLUSION: This study thus suggests that the neuroprotective role of TGF-beta1 against HI brain injury is mediated through an anti-apoptotic effect, which offers the possibility of TGF-beta1 application for the treatment of neonatal HI encephalopathy.
Animals ; Anoxia ; Apoptosis ; Blotting, Western ; Brain ; Brain Injuries ; Brain Ischemia ; Caspase 3 ; Cell Culture Techniques ; Incubators ; Neurons ; Neuroprotective Agents ; Rats ; Real-Time Polymerase Chain Reaction ; Transforming Growth Factor beta1

PURPOSE: Resveratrol, extracted from red wine and grapes, has an anti-cancer effect, an antiinflammatory effect, and an antioxidative effect mainly in heart disease and also has neuroprotective effects in the adult animal model. No studies for neuroprotective effects during the neonatal periods have been reported. Therefore, we studied the neuroprotective effect of resveratrol on hypoxic-ischemic brain damage in neonatal rats via anti-apoptosis. METHODS: Embryonic cortical neuronal cell culture of rat brain was performed using pregnant Sprague-Dawley (SD) rats at 18 days of gestation (E1 8) for the in vitro approach. We injured the cells with hypoxia and administered resveratrol (1, 10, and 30 microg/mL) to the cells at 30 minutes before hypoxic insults. In addition, unilateral carotid artery ligation with hypoxia was induced in 7 -day-old neonatal rats for the in vivo approach. We injected resveratrol (30 mg/kg) intraperitoneally into animal models. Real-time PCR and Western blotting were performed to identify the neuroprotective effects of resveratrol through anti-apoptosis. RESULTS: In the in vitro approach of hypoxia, the expression of Bax, caspase-3, and the ratio of Bax/Bcl-2, indicators of the level of apoptosis, were significantly increased in the hypoxia group compared to the normoxia group. In the case of the resveratrol-treated group, expression was significantly decreased compared to the hypoxia group. And the results in the in vivo approach were the same as in the in vitro approach. CONCLUSION: The present study demonstrates that resveratrol plays neuroprotective role in hypoxic-ischemic brain damage during neonatal periods through the mechanism of anti-apoptosis.
Adult ; Animals ; Anoxia ; Apoptosis ; Blotting, Western ; Brain ; Brain Injuries ; Carotid Arteries ; Caspase 3 ; Cell Culture Techniques ; Heart Diseases ; Humans ; Infant, Newborn ; Ligation ; Models, Animal ; Neurons ; Neuroprotective Agents ; Pregnancy ; Rats ; Real-Time Polymerase Chain Reaction ; Stilbenes ; Vitis ; Wine

OBJECTIVE: Resveratrol, a polyphenolic phytoalexin, is extracted abundantly from the red wine and grapes and biosynthesized as a defense agent to infection, ultraviolet and ozon etc. Recently, The cancer-preventive, anti-inflammatory and anti-oxidative effects of resveratrol have been reported. The aim of this study was to investigate the effect of resveratrol on the expression of nitric oxide synthases in hypoxic-ischemic brain injury in the neonatal rat model. METHODS: Embryonic cortical neuronal cell culture of rat brain was performed with pregnant Sprague-Dawley (SD) rats at 18 days of gestation (E18) for in vitro approaches. In addition, unilateral carotid artery ligation was induced in seven-days old neonatal rats for in vivo approaches. The real-time PCR using iNOS, eNOS and nNOS primer, and the western blotting using the same antibodies were done to identify the effects of resveratrol. RESULTS: The expression of iNOS, eNOS and nNOS in both cell culture and animal model of neonatal HI brain injury revealed that, as indicated by western blotting and real-time PCR, the expression of iNOS was decreased in the hypoxia group while those of eNOS and nNOS were increased in the hypoxia group compared with the normoxia group. The expression of iNOS was increased in the resveratrol-treated group while those of eNOS and nNOS decreased in the resveratrol-treated group compared with a hypoxic group. CONCLUSION: The present study demonstrates resveratrol might affect nitric oxide synthases expression in HI injury of the perinatal period
Animals ; Anoxia ; Antibodies ; Blotting, Western ; Brain ; Brain Injuries ; Carotid Arteries ; Cell Culture Techniques ; Ligation ; Models, Animal ; Neurons ; Nitric Oxide ; Pregnancy ; Rats ; Real-Time Polymerase Chain Reaction ; Sesquiterpenes ; Stilbenes ; Vitis ; Wine

PURPOSE: Resveratrol (trans-3,4',5-trihydroxystilbene), abundant in skin of grapes and red wines, has been known to protect heart cells from hypoxia/ischemia injury through its anti-oxidant properties and may also exert its cardioprotective action. There, to date, are no reports about the relationship with nitric oxide (NO)-mediated mechanism. Therefore, we investigated whether resveratrol can regulate the expression of NO synthase (NOS) in an in vitro hypoxic model of cultured H9c2 cardiomyoblasts. METHODS: The cultured H9c2 cardiomyoblasts were divided into four groups: a normal control group, a hypoxic group, two groups each treated with resveratrol before and after hypoxic insult. The control cells were placed in 5% CO2 incubator, and the hypoxic and resveratrol-treated groups were placed in 1% O2 incubator. NO activity was determined for all three isoforms of NOS; induced NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) using real-time PCR. RESULTS: The expressions of iNOS and eNOS were decreased in the hypoxic group compared to the control group, whereas the expression of nNOS was greater in the hypoxic group than in the control group. In contrast, the group treated with resveratrol before hypoxic insult showed increased expressions of iNOS and eNOS as compared to the hypoxic group. CONCLUSION: The results of the present study demonstrate that activation of iNOS and eNOS, but not nNOS, may be one of the mechanisms involved in the protective effect on resveratrol against hypoxic myocardial injury.
Anoxia ; Heart ; Incubators ; Neurons ; Nitric Oxide Synthase* ; Nitric Oxide* ; Protein Isoforms* ; Real-Time Polymerase Chain Reaction ; Skin ; Vitis ; Wine

PURPOSE: Taurine (2-aminoethanesulfonic acid) is a simple sulfur-containing amino acid. It is abundantly present in tissues such as brain, retina, heart, and skeletal muscles. Current studies have demonstrated the neuroprotective effects of taurine, but limited data are available for such effects during neonatal period. The aim of this study was to determine whether taurine could reduce hypoxic-ischemic (HI) cerebral injury via anti-apoptosis mechanism. METHODS: Embryonic cortical neurons isolated from Sprague-Dawley (SD) rats at 18 days gestation were cultured in vitro. The cells were divided into hypoxia group, taurine-treated group before hypoxic insult, and taurine-treated group after HI insult. In the in vivo model, left carotid artery ligation was performed in 7-day-old SD rat pups. The pups were exposed to hypoxia, administered an injection of 30 mg/kg of taurine, and killed at 1 day, 3 days, 1 week, 2 weeks, and 4 weeks after the hypoxic insult. We compared the expressions of Bcl-2, Bax, and caspase-3 among the 3 groups by using real-time polymerase chain reaction (PCR) and western blotting. RESULTS: The cells in the taurine-treated group before hypoxic insult, although similar in appearance to those in the normoxia group, were lesser in number. In the taurine-treated group, Bcl-2 expression increased, whereas Bax and caspase-3 expressions reduced. CONCLUSION: Taurine exerts neuroprotective effects onperinatal HI brain injury due to its anti-apoptotic effect. The neuroprotective effect was maximal at 1-2 weeks after the hypoxic injury.
Animals ; Anoxia ; Apoptosis ; Blotting, Western ; Brain ; Brain Injuries ; Carotid Arteries ; Caspase 3 ; Heart ; Ligation ; Muscle, Skeletal ; Neurons ; Neuroprotective Agents ; Pregnancy ; Rats ; Real-Time Polymerase Chain Reaction ; Retina ; Taurine

PURPOSE: Mycophenolic acid (MPA), a potent inhibitor of inosine-monophosphate dehydrogenase (IMPDH), was used as a new immunosuppressive drug since 1990s. It was reported that MPA increased neuronal survival after excitotoxic injury, induced apoptosis in microglial cells, inhibited the induction of inducible nitric oxide synthase (iNOS) in astrocytes. and inhibited microglial cell proliferation in N-methyl-D-aspartate (NMDA) induced hippocampal cells. However, the effects of MPA on the perinatal hypoxic-ischemic (HI) brain injury had not been yet evaluated. Therefore, we examined whether MPA could be neuroprotective in the HI brain injury. METHODS: Cortical cells were cultured using a 18-day-pregnant Sprague-Dawley (SD) rats and incubated in 1% O2 incubator for hypoxia. MPA (10 ug/mL) before or after a HI insult were treated. Seven-day-old SD rat pups were subjected to left carotid occlusion followed by 2.5 hours of hypoxic exposure (8% O2). MPA (10 mg/kg) were administrated intraperitoneally before or after a HI insult. Nitric oxide (NO) activity and expression of N-methyl-D-aspartate (NMDA) receptors also measured using Real-time PCR with primer pairs of isoforms of NOS; iNOS, endothelial NOS (eNOS), neuronal NOS (nNOS), and subunits of NMDA receptors; NR1, NR2A, NR2B, NR2C, NR2D. RESULTS: The expression of iNOS was decreased in the hypoxia group but increased in the MPA-treated group. However express or that eNOS and nNOS were inversed. The expression of all NMDA receptor subunits except NR2B was decreased in the hypoxia group but increased in the MPA-treated group. CONCLUSION: This study indicates that the administration of MPA before a HI insult could significantly protect against perinatal HI brain injury via some parts of NO-mediated or excitotoxic mechanisms.
Animals ; Anoxia ; Apoptosis ; Astrocytes ; Brain Injuries* ; Brain* ; Cell Proliferation ; Incubators ; Mycophenolic Acid* ; N-Methylaspartate* ; Neurons ; Nitric Oxide Synthase Type II ; Nitric Oxide Synthase* ; Nitric Oxide* ; Oxidoreductases ; Protein Isoforms* ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Receptors, N-Methyl-D-Aspartate