Foundation Item: Supported by national science foundation of China(30470598)Neuroprotective Effects of Edaravone on Neonatal Mice with Hypoxia-Ischemia Brain Damage / 实用儿科临床杂志

ObjectiveTo evaluate the neuroprotective effect and possible mechanisms of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) in neonatal Harlequin (Hq) mutant mice brain after hypoxia-ischemia brain injury(HIBD) insult.MethodsThe nine-day-old male Hq mutant mouse pups were assigned randomly either edaravone (n=16) and vehicle (n=17) treatment group. The Hq mice were subjected to left common carotid artery occlusion combined with inhalation 100 mL/L oxygen for 45 minutes. The mice were injected intraperitoneally either with edaravone (10 mg/kg) or equivalent volume of saline immediately after artery occlusion and after hypoxia. Nitrotyrosine and lipid peroxidation formation were evaluated at 3 h and 24 h after hypoxia-ischemia(HI) by using immunohistochemistry staining. Nitrotyrosine formation and caspases activation were evaluated either by immunoblotting or fluorogenic activity measurement at 24 h after HI. Brain injury was evaluated at 72 h by neuropathological score and calculating the infarct volume.ResultsBrain injury encompassed cortex, hippocampus, striatum and thalamus. Edaravone treatment reduced brain injury significantly in all the brain regions. The total infarct volume was reduced 52.8% in edaravone treatment group compared with vehicle group (P<0.001). The edaravone treatment reduced nitrotyrosine formation as well as lipid peroxidation formation significantly, but without obviously effect on caspases activation.ConclusionEdaravone affords neuroprotection after neonatal HI insult, which correlated with the reduction of free radical formation.

Changes of cell proliferation and differentiation in the developing brain of mouse / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To investigate the cell proliferation and differentiation in the developing brain of mouse.</p><p><b>METHODS</b>C57/BL6 mice were divided into 3 groups at random. Bromodeoxyuridine (BrdU) was injected into the brains in different development periods once a day for 7 d. The brains were retrieved 4 weeks after the last BrdU injection. Immunohistochemical and immunofluorescent studies were carried out for detecting cell proliferation (BrdU) and cell differentiation (NeuN, APC, Iba1, and S100beta), respectively.</p><p><b>RESULTS</b>The number of BrdU labeled cells decreased significantly with the development of the brain. Cell proliferation was prominent in the cortex and striatum. A small portion of BrdU and NeuN double labeled cells could be detected in the cortex at the early stage of development, and in the striatum and CA of the hippocampus in all groups. The majority of BrdU labeled cells were neuroglia, and the number of neuroglia cells decreased dramatically with brain maturation. Neurogenesis is the major cytogenesis in the dentate gyrus.</p><p><b>CONCLUSION</b>These results demonstrated that cell proliferation, differentiation and survival were age and brain region related.</p>

Therapy Effect of N-Acetylcysteine on Lipopolysaccharide-Sensitized Neonatal Rat with Hypoxic-Ischemic Brain Damage / 实用儿科临床杂志

Objective To evaluate the effect of N- acetylcysteine(NAC) on lipopolysaccharide (LPS) - sensitized neonatal rats with hypoxic- ischemic brain damage(HIBD) and possible mechanism except the antioxidant. Methods With the total number of 98 Wistar pups at postnatal day 8 of either sex was used in this study. There were 86 pups which were divided into three groups to evaluate the brain injury:vehicle group ( n = 29) ,low dose (25 mg/kg) ( n = 31 ) and high dose NAC (200 mg/kg) ( n - 26) treatment group. The pups were injected with LPS(0.1 mg/kg)intraperitoneally 3 days before hypoxic- ischemic(HI) insult. Multiple dose of NAC (25 mg/kg or 200 mg/kg) or vehicle was injected intraperitoneally before and after HI. Brain injury was evaluated 7 days after HI. For the Caspase - 3 activity and immunoblotting analysis, the samples were collected at 24 h after HI treated either with vehicle or high dose NAC ( n = 6 per group). Results The brain injury volume was significantly reduced by high dose NAC (200 mg/kg) treatment compared with that of vehicle (77% reduction, P ＜ 0.001 ). The tissue loss was reduced 67 % ( P ＜ 0.001 ) in high dose NAC treated group compared with that of vehicle. However,there was no significant reduction of brain injury in the low dose NAC treatment group compared with vehicle group. Caspase - 3 like activity measurement showed that the activity decreased 53 % after high dose NAC treatment ( P ＜ 0. 001 ) compared with that of vehicle treatment. The immunoblots showed that the active form of Caspase - 3, 17 kDa band, was abolished by the high dose NAC treatment. Conclusions NAC treatment attenuate LPS - sensitized neonatal HI brain injury is dose dependent. The neuroprotective effect involves Caspase - 3 inhibition.

Developmental changes of glutamate acid decarboxylase-67 in mouse brain after hypoxia ischemia / 神经科学通报·英文版

Objective To study the developmental changes of glutamic acid decarboxylase-67 (GAD-67, a GABA synthetic enzyme) in normal and hypoxic ischemic (HI) brain. Methods C57/BL6 mice on postnatal day (P) 5, 9, 21and 60, corresponding developmentally to premature, term, juvenile and adult human brain were investigated by using both Western blot and immunohistochemistry methods either in normal condition or after hypoxic ischemic insult. Results The immunoreactivity of GAD-67 was up regulated with brain development and significant difference was seen between mature (P21, P60) and immature (P5, P9) brain. GAD-67 immunoreactivity decreased in the ipsilateral hemisphere in all the ages after hypoxia ischemia (HI) insult, but, significant decrease was only seen in the immature brain. Double labeling of GAD-67 and cell death marker, TUNEL, in the cortex at 8h post-HI in the P9 mice showed that (15.6 +/- 7.0)%TUNEL positive cells were GAD-67 positive which was higher than that of P60 mice. Conclusion These data suggest that GABAergic neurons in immature brain were more vulnerable to HI insult than that of mature brain.

Expression of p53 in neonatal mice following hypoxia-ischemia and effects of its inhibitor on neonatal brain injury / 中国当代儿科杂志

<p><b>OBJECTIVE</b>p53-induced apoptosis is crucial in the development of hypoxic-ischemia (HI) brain damage and neurodegenerative disorders. Some experimental research has shown that a synthetic inhibitor of p53 can protect neurons against apoptosis. This study aimed to explore the expression of p53 in neonatal mice following HI brain damage and the effect of p53 inhibitor (pifithrin-alpha, PFT-alpha) on brain damage.</p><p><b>METHODS</b>HI was induced in 9-day-old mice pups by ligation of left carotid artery and 10% oxygen exposure for 55 minutes. The pups were sacrificed and the brains were taken out at 3, 8, 24, and 72 hrs post-HI. The brains were sectioned and stained with antibody against p53 and microtubule-associated protein 2 (MAP-2). PFT-alpha was injected intraperitoneally: in experiment 1, immediately after HI with different dosages (1, 2 and 8 mg/kg); in experiment 2, 2 mg/kg at different HI times (1 hr before HI, and immediately and 1 hr after HI). Control animals without HI received injections of 0.5% dimethyl sulfoxide. Brain damage was evaluated by gross morphology scoring at 72 hrs after HI.</p><p><b>RESULTS</b>The number of p53 positive cells in the cortex, hippocampus and striatum of the ipsilateral hemisphere increased significantly and peaked at 3-8 hrs post-HI when compared with those of contralateral hemisphere as well as normal controls. The positive cells distributed mainly in the MAP-2 negative area. Both different dosages and different injection time PFT-alpha treatment did not reduce the extent of brain damage.</p><p><b>CONCLUSIONS</b>The immunoactivity of p53 increased significantly as early as 3 hrs post-HI. The distribution area of p53 expression was consistent with that of brain damage. The p53 inhibitor PFT-alpha has no protective effects against HI brain damage in neonatal mice.</p>

Neuroprotective effect of hypothermia on hypoxic-ischemic brain injury in mice / 中国当代儿科杂志

<p><b>OBJECTIVE</b>The study was to investigate the effect of different temperatures during hypoxia on brain injury in mice of different ages.</p><p><b>METHODS</b>Newborn C57/BL6 mice at 7 days or 21 days of life were subjected to left carotid artery ligation followed by exposure with 10% oxygen. The mice were kept in a incubator with a predetermined, constant temperature, either 34 degrees centigrade (Hypothermia group) or 36 degrees centigrade (Normothermia group). Brain injury was evaluated 7 days after hypoxia-ischemia (HI). Active caspase-3 and apoptosis-inducing factor (AIF) expressions in the brain tissue were detected by immunohistochemistry and Western Blot was used to evaluate the phosphor-Akt (P-Akt) expression in the brain tissue at 24 hrs post-HI.</p><p><b>RESULTS</b>Brain injuries, including the cortex, hippocampus, striatum and thalamus injuries, occurred in the Normothermia group at 7 days post-HI. The brain cortex showed cystic cavitation in the postnatal day (P)7 pups mice and laminar infarct of the brain cortex was observed in P21 mice. In the Hypothermia group, the P7 mice did not present with laminar infarct of the cortex and had lower scores of neuropathological lesions in cortex, hippocampus, striatum and thalamus than P7 mice from the Normothermia group (P < 0.01); the cortex injuries were significantly relieved but the injuries of hippocampus, striatum and thalamus in P21 mice were similar to those from the Normothermia group. Active caspase-3 (7.0 +/- 5.6) and AIF positive cells (3.7 +/- 6.2) in the cortex of P7 mice from the Hypothermia group were significantly lower than those of the Normothermia group (51.5 +/- 23.2 and 31.8 +/- 22.4) at 24 hrs post-HI (P < 0.01). Wetstern Blot showed the P-Akt expression was obviously decreased in the ipsilateral hemisphere to the occlusion compared with that of the contralateral hemisphere after HI in the Normothermia group (P < 0.05), while in the Hypothermia group the P-Akt expression was not significantly different between the two hemispheres.</p><p><b>CONCLUSIONS</b>Hypothermia has protective effects against HI insults. The protection was more pronounced for the immature brain than the mature brain.</p>

Effect of Intrauterine Infection and Interuterine Asphyxia to Fetal Rats′ Brain Damage,Cell Apoptosis and Expression of Glial Fibrillary Acidic Protein / 实用儿科临床杂志

Objective To find out the associated effect of intrauterine infection and interuterine asphyxia to fetal rat′s brain damage,cell apoptosis,and expression of glial fibrillary acidic protein(GFAP).Methods Pregnant rats of gestation 18 days were randomly divided into four groups:1.NS plus sham operation,2.intrauterine infection,3.intrauterine asphyxia,4.intrauterine infection plus intrauterine asphyxia.The fetal rats′ brains were taken out 72 h after different disposal and given HE coloration,immunohistochemistry of TUNEL and GFAP,respectively.Results The level of brain cell edema and tissue disorganization of group intrauterine infection plus intrauterine asphyxia were more serious than those of group intrauterine infection or group intrauterine asphyxia.TUNEL and GFAP had the same results:The number of positive cells in group intrauterine infection plus intrauterine asphyxia more than that in group intrauterine infection,and which in group intrauterine asphyxia more than that in group NS plus sham operation.There was significant difference between the first three groups and the group NS plus sham operation(P=0).There was also significant difference between group intrauterine infection plus intrauterine asphyxia and group intrauterine infection or group intrauterine asphyxia(P=0).Conclusions Both intrauterine infection and intrauterine asphyxia may induce premature rat brain damage,the association of intrauterine infection and intrauterine asphyxia may aggravate the degree of fetal rat brain damage,also increase the number of apoptosis cell and the expression of GFAP.

Cytochrome C release and apoptosis in neonatal rat cerebral hypoxia-ischemia / 中华儿科杂志

<p><b>OBJECTIVE</b>To study the relation of cytochrome C release from mitochondria to cytosol and neuronal apoptosis after cerebral hypoxia-ischemia (HI) in neonatal rats.</p><p><b>METHODS</b>Hypoxia-ischemia was induced in 7-day-old rat pups by ligation of left carotid artery and 7.7% oxygen was inhaled for 55 min. The pups were sacrificed and the brains were taken out at different recovery time. Some of the brains were homogenized and cellular fraction of mitochondria and cytosol was isolated with different speed centrifugation. The cellular fraction was used for Western blotting. Some of the brains were sectioned and stained with antibody against cytochrome C and TUNEL as well as double labeling with different combinations.</p><p><b>RESULTS</b>Western blots showed that cytochrome C in mitochondria was not reduced significantly at 1 h, but reduced markedly at 14 h in ipsilateral hemisphere post-HI. However, the immunoreactivity of cytochrome C in cytosol was increased markedly at 1 h post-HI and reached peak at 14 h post-HI. The number of cytochrome C positive cells in the cortex was increased significantly at 1 h (8.4 +/- 1.8/visual field) compared to normal control (1.5 +/- 0.8/visual field) (P < 0.01) and reached peak at 14 h (29.0 +/- 5.2/visual field) post-HI. The number of TUNEL positive cells increased significantly at 1 h post-HI (14 +/- 3/visual field) compared to normal control (1.5 +/- 0.8/visual field) (P < 0.01) and reached peak at 24 h (286 +/- 86/visual field). The double labeling of cytochrome C and active caspase-3 showed that they colocalized well at 3 h after HI. Furthermore, the positive cells showed nuclei condensation. There were more active caspase-3 positive cells at late recovery (24 h and on) after HI. The double labeling of cytochrome C and TUNEL showed only part of Positive cells colocalized. The cells with cytochrome C strong staining showed TUNEL negative or weakly positive. The cells with TUNEL strong staining showed weakly cytochrome C staining.</p><p><b>CONCLUSION</b>Cytochrome C release is one of the early biochemical changes of neuronal apoptosis after hypoxia-ischemia in neonatal rat brain.</p>

Effect of Intrauterine Asphyxia on Expression of Tissue-Type Plasminogen Activator in Fetal Rat′s Brain / 实用儿科临床杂志

0.05),but there were significant difference between the two groups from 12 hours to 48 hours after operation(all P

Changes of Cytokines Measurement in Serum and Cerebrospinal Fluid in Asphyxia Ne onates and Its Relationship with Prognostic / 实用儿科临床杂志

Objective To study the dynamic changes of cytokines including tumor necrosis factor-alpha (TNF-?),interleukin-6 (IL-6),IL-8 in serum and cerebrospinal fluid (CSF) of asphyxia neonates,and to analyze the relationship between cytokines levels and severity of brain damage and neurological outcome. Methods The concentrations of TNF-?,IL-6,IL-8 in serum and CSF were measured by radioimmunoassay in 63 asphyxia neonates. Neurological development was evaluated at 12 months by children′s developmental scale of china.Results The serum concentrations of TNF-?, IL-6,IL-8 were significantly higher in asphyxiated neonates than those in the controls,and they were correlated with the degree of encephalopathy. The level of serum TNF-? was hig-(hest) at the first day and IL-6 was highest at the third day. There was no marked dynamic changes within 5 days in serum IL-8 level. The concentrations of TNF-?,IL-8 in CSF were higher at the first and the third day.The dynamic changes of IL-6 in CSF were similar in serum and they were positively correlated. The serum concentration of IL-6 in severe brain injury group was much higher than those of normal and mild group.The CSF concentration of IL-6 in severe brain injury group was much higher than that of normal group. The CSF concentration of IL-8 in severe brain injury group was much higher than those of the normal and mild group. Conclusions The concentrations of TNF-?,IL-6 and IL-8 are increased both in serum and CSF in asphyxiated neonates which are correlated with severity of hypoxic-ischemic encephalopathy. Cytokine-mediated inflammatory reactions may participate in the mechanism of hypoxic-ischemic brain injury after asphyxiaion.The concentration of IL-6 in serum and IL-6, IL-8 in CSF are correlated with the neurological outcome.

Neuroprotective effect and mechanisms of hypothermia in neonatal rat cerebral hypoxic-ischemic damages / 中华儿科杂志

<p><b>OBJECTIVE</b>Recent studies suggest that hypothermia may be a potential treatment for perinatal hypoxic-ischemic (HI) brain damage. But the mechanisms of this effect are not well known. In the present study, the protective effect of systemic hypothermia as well as effect on apoptosis and associated biochemical events were investigated on neonatal rats with HI brain damage.</p><p><b>METHODS</b>Seven-day-old Wistar rats were subjected to left carotid artery ligation and hypoxia was persisted for 60 min. Immediately at the end of hypoxia, the animals were maintained either at 36 degrees C or 30 degrees C for 10 h at random. Caspase-2, 3 activity in brain homogenate was detected with Western blotting at 24 h post-HI (n = 8 for each group). Immunoactivity of microtubule-associated protein-2 (MAP-2), active caspase-3, apoptosis inducing factor (AIF) and oligonucleotide hairpin probe staining were detected at 72 h post-HI. The infarct volume, neuronal loss in CA(1) sector of hippocampus as well as brain injury scoring were calculated according to MAP-2 staining and hematoxylin and eosin staining.</p><p><b>RESULTS</b>Caspase-2, 3 activities were much higher in the normothermia group [(27.7 +/- 14.7), (94.9 +/- 53.1) pmol/(min.mg protein)] at 24 h post-HI than those of hypothermia [(7.9 +/- 3.4), (21.1 +/- 18.7) pmol/(min.mg protein)] and normal control groups [(7.6 +/- 0.7), (12.9 +/- 0.5) pmol/(min x mg protein)] (P < 0.01). The activities were not significantly different between hypothermia group and normal control group. Western blotting showed that caspase-3 activation process was blocked by hypothermia. The number of active caspase-3 and AIF positive cells in the cortex of ipsilateral hemisphere was much higher in the normothermia group (median: 148.5; 22/field) than that of hypothermia group (median: 48.5; 9/field) (P < 0.05). The number of apoptotic cells as judged by oligonucleotide hairpin probe labeling was much higher in normothermia group (median: 144/field) than that of hypothermia group (median: 133/field) (P < 0.05). The brain injury scoring, infarct volume and neuronal loss in CA(1) area of hippocampus were much less in the hypothermia group [10.4 +/- 2.9; 40.5 +/- 34.8)mm(3); 25.7 +/- 11.5] than that of normothermia group [14.2 +/- 3.5; (73.9 +/- 22.4) mm(3); 37.4 +/- 10.6, P < 0.05].</p><p><b>CONCLUSIONS</b>Systemic hypothermia for 10 h after hypoxia-ischemia seemed to be effective in reducing brain damage and the mechanism is associated with alteration of apoptotic pathway.</p>

Observation of Erythropoietin Acrossing Blood - Brain Barrier of Premature Infants / 实用儿科临床杂志

Objective To observe whether recombinant human erythropoietin (rhu- EPO) could cross blood- brain barrier (BBB) of premature infants. Methods Thirty - six premature infants, with gestational age 28 - 35 weeks, birth weight0.05).Conclusion Rhu- EPO can cross the BBB of premature infants.

Neuroprotective Effects of Edaravone on Neonatal Mice with Hypoxia-Ischemia Brain Damage / 实用儿科临床杂志

Objective To evaluate the neuroprotective effect and possible mechanisms of edaravone(3-methyl-1-phenyl-2-pyrazolin-5-one) in neonatal Harlequin(Hq) mutant mice brain after hypoxia-ischemia brain injury(HIBD) insult.Methods The nine-day-old male Hq mutant mouse pups were assigned randomly either edaravone(n=16) and vehicle(n=17) treatment group.The Hq mice were subjected to left common carotid artery occlusion combined with inhalation 100 mL/L oxygen for 45 minutes.The mice were injected intraperitoneally either with edaravone(10 mg/kg) or equivalent volume of saline immediately after artery occlusion and after hypoxia.Nitrotyrosine and lipid peroxidation formation were evaluated at 3 h and 24 h after hypoxia-ischemia(HI) by using immunohistochemistry staining.Nitrotyrosine formation and caspases activation were evaluated either by immunoblotting or fluorogenic activity measurement at 24 h after HI.Brain injury was evaluated at 72 h by neuropathological score and calculating the infarct volume.Results Brain injury encompassed cortex,hippocampus,striatum and thalamus.Edaravone treatment reduced brain injury significantly in all the brain regions.The total infarct volume was reduced 52.8% in edaravone treatment group compared with vehicle group(P

Variance of Perinatal Asphyxia in Latest 10 Years and Analysis of Mortality and Risk Factors of Perinatal Asphyxia / 实用儿科临床杂志

Objective To analyze the incidence rate and mortality of perinatal asphyxia and effects of new resuscitation technique on asphyxia as well as the risk factors of asphyxia in latest 10 years.Methods A retrospective evaluation was done for all the newborns who were born in the provincial women and children′s health care hospital from 1995 to 2004.The morbidity,mortality and fatality rate were calculated for each observed year and different seasons.The influence of gender,body weight,gestational age as well as polyembryony and mode of delivery on the asphyxia was analyzed.Results The morbidity of mild birth asphyxia was decreased dramatically and maintained at about 1.5% after using new resuscitation technique,however,there were no obvious effects on the sever asphyxia.In the same time,no big influence on fatality rate of birth asphyxia was observed.The incidence rate was highest in April,but the mortality and fatality of asphyxia was highest in July.The incidence of asphyxia was also related with gender,polyembryony,birth weight,prematurity babies and aids to delivery from voginal.Conclusions The incidence of perinatal asphyxia is related with the gender,polyembryony,birth weight and gestation age as well as seasons.New resuscitation technique can reduce the morbidity of mild birth asphyxia,and no effect on the severe asphyxia as well as fatality rate.

Effect of Subhypothermia and Erythropoietin Combined Treatment on Neonatal Hypoxic-Ischemic Brain Damage / 实用儿科临床杂志

Objective To explore the effect of systemic subhypothermia and erythropoietin combined treatment on neonatal hypoxic-ischemic brain damage(HIBD).Methods Sixty-five asphyxiated newborns enrolled in this study were divided randomly into combination group(n=16),hypothermia group(n=24)and conventional group(n=25).Subhypothermia was adepted in hypotheria group as well as conventional therapy.Subhypotheria and erythropoietin were both applied in combination group excepy for conventional therapy.The short-term effect was evaluated by neonatal neurological score as well as neonatal behavioral neurological assessment(NBNA)on postnatal days of 7,14 and 28,and the long-term effect was evaluated by using intellectual development table made by Children's Development Center of China(CDCC)at 3 and 6 month of age.Results The neonatal neurological score in combination group at 24,48,72 and 80 h were lower than that of 0 and 12 h.NBNA evaluation was much higher on 14 d and 28 d in combination group and hypothermia group than that in conventional group(Pa0.05).Conclusions Combination therapy with systemic subhypothermia and erythropoietin exerts obvious short and longer-time neuroprotective effect on HIBD,but there are no differences compared with hypothermia alone.

Neuroprotective Effect of Recombinant Human Erythyropoietin Theraphy for Neonatal Hypoxic-Ischemic Encephalopathy / 实用儿科临床杂志

Objective To evaluate the neuroprotective effect and safety of neonatal hypoxic-ischemic encephalopathy(HIE)treated with recombinant human erythropoietin(rhEPO).Methods Fifty-three neonates with HIE were randomly divided into rhEPO treated group(n=29) with the dosage of 300 U/(kg?time),three times a week for 2 weeks and control group(n=24)without rhEPO.All supportive measures were same between 2 groups.Neurological scoring was evaluated at d3,d5 and d7 Neonatal behavioral neurological assessment(NBNA) was evaluated at d7,d14 and d28.The neurodevelopment quote was evaluated at age of 3 and 6 months.Blood pressure,liver and renal function,blood electrolytes and blood hemoglobin,platelet and reticular red blood cell count were monitored before and after treatment in all infants.Results The neurological scoring between two groups had no difference at d3.The significant difference was found at d7(P0.05).Conclusions Teraphy with rhEPO on neonatal HIE infants can promote neurological recovery,and there is no serious side effect with rhEPO treatment.