Study of subependymal ventricular zone and white matter as an endogenous source of cells for white matter repair in neonatal rats with periventricular leukomalacia in vitro / 中华实用儿科临床杂志

Objective To explore an endogenous self-repair potentiality for injured cerebral white matter from both of subependymal ventricular zone and white matter cell cultures in neonatal rats with oxygen glucose deprivation (OGD) in vitro.Methods The white matter and subependymal ventricular zone tissues from the neonatal rats within 5 days old were separately used to prepare primary glia-derived cell cultures,and these cell cultures were randomly divided into the control group and the OGD group.The double-label fluorescent immunoanalysis was used to observe the proliferation and differentiation of the glia-derived cells came from both of subependymal ventricular zone and white matter activated by OGD.The Hoechst33342/propidium iodide (PI) staining and the flow cytometry technology were used to assess the apoptotic rates of the newborn cells.Results More apoptotic and necrotic cells appeared in the OGD group than those in the control group both in subependymal ventricular zone and white matter cell cultures in the flow cytometry test and Hoechst33342/PI staining at 24 h,48 h,72 h,7 d and 14 d after OGD (all P ＜ 0.01).Furthermore,fluorescence microscope showed that the number of the NG2 + progenitor cells,the O4 + oligodendrocyte precursor cells in the OGD group were all significantly more than those in the control group during 72 h after OGD (all P ＜ 0.05,0.01),while the number of the immature and mature oligodendrocytes in the OGD group decreased significantly compared with those in the control group on 7 d and 14 d after OGD (all P ＜ 0.05,0.01).Conclusions OGD may activate 2 endogenous self-repair pathways from subependymal ventricular zone and white matter in vitro.The activated subependymal ventricular zone and white matter-glial progenitor cells appear to proliferate markedly,and differentiate along an oligodendroglial pathway.However,only a few newly generated precursor cells can be differentiated into the immature or mature oligodendrocytes and OGD may induce the newborn cells to appear apoptotic and necrotic.

Endogenous self-repair in immature white matter induced by ischemia in neonatal rats / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study in vivo the endogenous self-repair mechanism in immature white matter induced by ischemia in neonatal rats with periventricular leukomalacia (PVL).</p><p><b>METHODS</b>Five-day-old neonatal Sprague-Dawley (SD) rats were randomly divided into sham and PVL groups. Rat model of PVL was prepared by ligation of the right common carotid artery following 2 hours of exposure to 8% oxygen. Pathological changes and myelination in the white matter were assessed under light and electron microscopy at 7 and 21 days after PVL. O4-positive OL precursor cells in the white matter were determined with immunofluorescence staining. Activation, proliferation, migration and differentiation of glial progenitor cells in SVZ were observed using immunofluorescent double labeling of either NG2 (marker of progenitor cells) and 5-bromodeoxyuridine (BrdU), or O4 (marker of OL precursor cells) and BrdU.</p><p><b>RESULTS</b>All rats in the PVL group manifested either mild or severe white matter injury under light microscopy, and had higher pathological scores of white matter compared with the sham group at 7 and 21 days after PVL (P<0.05). Electron microscopy showed that the number and thickness of myelin sheath in the PVL group were significantly reduced compared with the sham group (P<0.01). O4-positive OL precursor cells in the white matter observed under fluorescence microscopy were significantly reduced in the PVL group compared with the sham group (P<0.05). BrdU/NG2-positive cells in the SVZ increased significantly in the PVL group 48 hours after PVL and migrated into the periventricular area, reaching a peak on day 7 after PVL. BrdU/O4-positive newborn cells began to appear in the periventricular area 72 hours after PVL, and the number of BrdU/O4-positive cells in the PVL group was statistically more than in the sham group on day 21 after PVL (P<0.05).</p><p><b>CONCLUSIONS</b>Ischemia may induce brain self-repair in neonatal rats, resulting in activation and proliferation of NG2 glial progenitor cells in the SVZ migration and differentiation into OL precursor cells in periventricular white matter.</p>

Effect of single or combined application of UDP-glucose, GDNF and memantine on improvement of white matter injury in neonatal rats assessed with light and electron microscopy pathologically / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate pathologically the effect of the single or combined application of UDP-glucose, GDNF and memantine on the improvement of white matter injury in neonatal rats with periventricular leukomalacia (PVL) under light and electron microscopy.</p><p><b>METHODS</b>A five-day-old neonatal rat model for PVL was established by ligation of the lateral common carotid artery following 120-minute hypoxia. Rats were randomly divided into six groups (30 rats in each group): sham-operated, PVL, UDP-glucose (UDP-glucose 2000 mg/kg intraperitoneally after PVL), GDNF (GDNF 100 μg/kg intracerebrally after PVL), tmemantine (memantine 20 mg/kg intraperitoneally after PVL), and a combination administration of three drugs (UDP-glucose, GDNF and memantine). The rats were sacrificed 7 or 21 days after PVL for assessment of pathological changes in the white matter under both light and electron microscopy. The number and thickness of the myelin sheath in the white matter were measured under electron microscopy, and both of pathological grading and scoring were undertaken under light microscopy.</p><p><b>RESULTS</b>There was rare and sparse myelinogenesis with a loose arrangement of nerve fibers in the white matter under electron microscopy in the PVL group at 7 and 21 days after PVL. The number and thickness of the myelin sheath in the PVL group were significantly less than in the sham-operated, UDP-glucose, GDNF, memantine and combination administration groups (P<0.01). The results of pathological grading of white matter under light microscopy showed that all rats in the PVL group manifested either mild injury (38%-50%) or severe injury (50%-62%) at 7 and 21 days after PVL. The majority of rats (50%-88%) in the four drug administration groups had normal white matter at 7 and 21 days after PVL. The pathological scores at 7 and 21 days after PVL in the PVL group were the highest, and they were significantly higher than in the other five groups (P<0.05).</p><p><b>CONCLUSIONS</b>The single or combined application of UDP-glucose, GDNF and memantine may significantly improve pathological changes in the white matter of rats with PVL. The favorable effect is inferred to be closely correlated with the improvement of brain microenvironment and the enhancement of nerve regeneration promoted by the three drugs.</p>

Effects of glial cell line-derived neurotrophic factor and memantine on long-term prognosis in neonatal rats with periventricular leukomalacia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the effects of glial cell line-derived neurotrophic factor (GDNF) and memantine on the long-term prognosis in neonatal rats with ischemia-induced periventricular leukomalacia (PVL).</p><p><b>METHODS</b>Thirty-two 5-day-old neonatal rats were randomly divided into 4 groups: sham-operated, PVL, GDNF-treated and memantine-treated. PVL was induced by right carotid artery ligation and hypoxia in the PVL, GDNF-treated and memantine-treated groups. GDNF (100 μg/kg) or memantine (20 mg/kg) was injected in the two treatment groups immediately after PVL inducement. The weight of the rats was measured immediately before and after hypoxia ischemia (HI). Both of Morris water maze test and Rivlin inclined plane test were performed at 26 days old (21 days after HI). The values of the escape latency (EL) and swimming distance, and the maximum inclined plane degree which the rats could stand at least 5 seconds were compared among the four groups.</p><p><b>RESULTS</b>The lower weight, the prolonged mean values of EL and swimming distance and the reduced maximum inclined plane degree were observed in the PVL group compared to those in the sham-operated, GDNF-treated and memantine-treated groups. There were no significant differences in the weight, the values of EI and swimming distance and the maximum inclined plane degree between the two treatment groups and the sham-operated group.</p><p><b>CONCLUSIONS</b>The administration of either GDNF or memantine can markedly increase the abilities of spatial discrimination,learning and memory, and motor coordination, promote weight gain, and improve long-term prognosis in rats with PVL.</p>

1400W blocks death pathway of LPS-induced activated-microglia to preOLs / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the efficacy of inductible nitric oxide synthase (iNOS) inhibitor 1400W in vivo in blocking the death pathway of lipopolysaccharide (LPS)-induced activated-microglia to preoligodendrocytes (preOLs) in neonatal rats with infective-type periventricular leukomalacia (PVL) induced by LPS.</p><p><b>METHODS</b>Two-day-old neonatal rats were randomly divided into: a sham-operated group, an untreated PVL group, and four 1400W-treated PVL groups that were subcutaneously administrated with 20 mg/kg of 1400W at 0 h, 8 hrs, 16 hrs, and 24 hrs after LPS induction, respectively. The brain specimens were obtained 5 days after LPS induction. The pathological assessment of cerebral white matter was performed under a light microscope. Concentrations of nitric oxide (NO) were measured by nitric acid-deoxidize colorimetry. Synthesis of iNOS was determined by Western blot analysis. Peroxynitrite (ONOO(-)) level and the amount of preOLs were determined by immunocytochemistry. RETHODS: The obvious injuries of periventricular white matter, massive loss of positive O4-labelled preOLs, and increased levels of NO, ONOO(-) and iNOS were observed in neonatal rats with PVL. Compared to the untreated PVL group, the use of 1400W at 0 h, 8 hrs and 16 hrs after LPS induction significantly improved white matter injuries, reduced the levels of NO, ONOO(-) and iNOS, and increased the amount of O4-labelled preOLs. However, the use of 1400W at 24 hrs after LPS induction did not result in the improvements.</p><p><b>CONCLUSIONS</b>iNOS inhibitor 1400W can effectively block the toxicity of LPS-activated microglia to preOLs and protect cerebral white matter through inhibiting iNOS and reducing the production of NO and ONOO(-). The use of 1400W within 16 hrs after LPS induction may provide cerebral protections in neonatal rats with PVL.</p>

Effect of 1400W, an inhibitor of inducible nitric oxide synthetase, on blocking the toxicity of lipopolysaccharide-induced activated microglia to preoligodendrocytes / 中华儿科杂志

<p><b>OBJECTIVE</b>To explore the toxicity of LPS-induced activated microglia to preoligodendrocytes (preOLs) and the effect of 1400W, a selective inhibitor of inducible nitric oxide synthetase (iNOS), on the blockage of the toxicity.</p><p><b>METHODS</b>Co-cultured microglia and preOLs obtained from two-day-old Sprague-Dawley (SD) rats were divided into three groups: co-culture control group, co-culture LPS group and co-culture LPS plus 1400W group. After cultured cells were induced by LPS (100 ng/ml) for 48 hours, the concentration of nitric oxide (NO) was measured by nitric acid-oeoxidize-colorimetry, the level of peroxynitrite (ONOO(-)) was determined by immunocytochemistry, and the synthetic level of iNOS was detected by Western blotting, respectively. The morphologic observation of apoptotic preOLs stained with Hoechst 33342/PI and the apoptotic rate of preOLs detected by flow cytometry were processed simultaneously. Data were analyzed with SPSS 11.0 software.</p><p><b>RESULTS</b>Compared to co-culture control group, there was significant increase in levels of NO [(82.27+/-3.41) micromol/L vs. (167.86+/-9.87) micromol/L, t=8.593, P<0.01], ONOO(-)[(6.14+/-1.27) x 10(7)/L vs. (34.38+/-7.75) x 10(7)/L, t=5.892, P<0.01], and iNOS [(0.18+/-0.027) vs. (0.79+/-0.068), t=9.26, P<0.01] induced by LPS in co-culture LPS group, and with a higher apoptotic rate of preOLs [(6.73+/-1.39)% vs. (24.77+/-2.05)%, t=12.619, P<0.01]. However, all levels of NO [(69.55+/-5.07) micromol/L, t=8.896, P<0.01], ONOO(-) [(10.33+/-3.47) x 10(7)/L, t=14.96, P<0.01] and iNOS (0.35+/-0.042, t=5.506, P<0.01) decreased significantly with the use of 1400W at a dose of 10 micromol/L in co-culture LPS plus 1400W group, and the apoptotic rate of preOLs [(11.8+/-2.06)%, t=7.715, P<0.01] was also reduced evidently.</p><p><b>CONCLUSIONS</b>NO, ONOO(-) and iNOS, etc. play important roles in the death pathway of preOLs induced by LPS. 1400W can block effectively the toxicity of LPS-activated microglia toxicity to preOLs through inhibiting iNOS selectively and reducing the production of NO and ONOO(-), and improve the survival rate of preOLs.</p>

Cerebral pathological evaluation following neural stem cells intraventricular transplantation in neonatal rats with periventricular leukomalacia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To evaluate the brain pathological changes following exdogenous neural stem cells (NSCs) intraventricular transplantation in neonatal rats with periventricular leukomalacia (PVL), and to explore the feasibility of NSCs transplantation for the treatment of PVL in premature infants.</p><p><b>METHODS</b>NSCs were prepared from E14 embryonic rat brain. Two-day-old neonatal rats were randomly divided into six groups: PVL, PVL+culture medium, PVL+NSCs, sham operation, sham operation+culture medium, and sham operation+NSCs (18-21 rats each group). Intraventricular transplantation of exdogenous NSCs was performed 72 hrs after PVL induction or sham operation. The cerebral pathological evaluation was undertaken by light microscopy 7, 14 and 21 days after transplantation.</p><p><b>RESULTS</b>The pathological changes in the cerebral white matter were gradually improved with the prolonged time after transplantation. After 21 days of transplantation, 50% of the cerebral white matter showed mild pathological changes and 50% of that showed severe pathological changes, with neuronal pathological scores of 1.28+/-0.86, in the untreated PVL group. In the PVL+NSCs group, 30% of normal white matter, 40% of mild and 30% of severe pathological changes in the white matter were observed, with neuronal pathological scores of 0.32+/-0.16, 21 days after transplantation. There were very significant differences in both of pathological changes in the cerebral white matter and neuronal pathological scores between the PVL and PVL+NSCs groups (x2=10.7, P<0.01; F=29.664, P<0.01).</p><p><b>CONCLUSIONS</b>Intraventricular transplantation of exdogenous NSCs can apparently improve cerebral white matter damage. It is suggested that intraventricular transplantation of NSCs is of a great potential feasibility for the treatment of PVL in premature infants.</p>

Establishment of a neonatal rat model of periventricular leukomalacia and its concomitant cataract / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To establish a reliable neonatal rat model of periventricular leukomalacia (PVL) which is expected to be similar to PVL of human preterm infants pathologically, and to explore the concomitant eye lesions in the PVL model.</p><p><b>METHODS</b>Two-old-day neonatal rats were randomly divided into a PVL group and a sham-operated group (n=19 each). The PVL model was established by the ligation of bilateral common carotid arteries, followed by a 30-min exposure to 8% oxygen. The cerebral infarction area was assessed with TTC staining 1 day after operation. Cerebral pathology was examined under a light micsrocope 2 and 21 days after operation. The examinations of eyes under a slip lamp and the pathology of eyeballs under a light microscope were performed 21 days after operation.</p><p><b>RESULTS</b>The TTC staining cerebral slices showed there were extensive white areas of infarction in the brain of the PVL group, with an infarction area of 53.45 +/- 33.90 mm3 and a percentage of infarction of (24.98 +/- 15.44)% . Significant cystic necrosis and apoptosis around the periventricular and subcortical white matter and mild damage in cortical neurons were observed in the PVL group 2 days after operation. The more obvious cystic necrosis around the periventricular area was found in the PVL group 21 days after operation. There were no pathological changes in the brain of the sham-operated group. All of rats in the PVL group had bilateral cataracts, however, no pathological changes were observed in their postbulbar tissues. The sham-operated group did not show eye abnormal.</p><p><b>CONCLUSIONS</b>The PVL animal model that was similar to PVL of human preterm infants pathologically was successfully established by the ligation of bilateral common carotid arteries, followed by 30-min hypoxia exposure, with a positive effect and a good repeatability. Cataract can also be induced by the method.</p>

Effect of different blood glucose levels on the expression of cerebral GLUT3 mRNA in neonatal rats with hypoxia-ischemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Concerns of the effect of glucose on perinatal hypoxic-ischemic brain damage are increasing. It was previously considered that the glucose transporter (GLUT) genes and their productions played an important role in the regulation of cerebral energy metabolism. The present study aimed to explore the effect of different blood glucose levels on the expression of cerebral GLUT3 mRNA in neonatal rats with hypoxia-ischemia (HI), and to evaluate the neuroprotective effect of glucose against HI insults.</p><p><b>METHODS</b>A total of 250 7-day-old neonatal SD rats were randomly divided into 10 groups (n=25 each): Normal control, Sham-operated, HI, Hypoglycemia, Hypoglycemia pre- and post-HI, Mild hyperglycemia pre- and post-HI, Severe hyperglycemia pre- and post-HI. Blood glucose levels of normal, hypoglycemia, mild hyperglycemia and severe hyperglycemia were defined as 5-7 mmol/L, 3-4 mmol/L, 10-15 mmol/L and 16-25 mmol/L, respectively. The expression of GLUT3 mRNA was detected with RT-PCT technique at 2, 24, 48 and 72 hrs and at 7 days after HI.</p><p><b>RESULTS</b>There was a correlation between increases in GLUT3 mRNA expression and postnatal age in the Normal control group. HI significantly enhanced the expression of GLUT3 mRNA from 2 hrs, peaking at 24 hrs after HI, and then significantly decreased at 72 hrs and 7 days after HI when compared with the Normal Control group (P < 0.01). GLUT3 mRNA expression in the Hypoglycemia pre-HI group was the lowest among all groups with HI at each time point after HI, and a statistically significant difference was found at 72 hrs after HI when compared with the HI group (P < 0.05). The expressional levels of GLUT3 mRNA in the Severe hyperglycemia pre-HI group were strikingly higher than those in any other groups with HI (P < 0.05 or 0.01). The GLUT3 mRNA expression patterns in the Mild and Severe hyperglycemia post-HI and the Hypoglycemia post-HI groups were similar to the Hypoglycemia pre-HI group.</p><p><b>CONCLUSIONS</b>GLUT3 mRNA expression and the synthesis of GLUT3 can be down-regulated by hypoglycemia pre-HI, coupled with aggravation of cerebral pathology, but up-regulated by higher hyperglycemia pre-HI, coupled with improvement of cerebral pathology. This suggested that adequate glucose supplement before HI can improve the cerebral function against HI insults in neonatal rats.</p>

Long-term effects of memantine therapy on neonatal rats with hypoxic-ischemic brain damage / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Animal trials have demonstrated that memantine has neuroprotective effects on hypoxic-ischemic (HI) brain damage. Whether memantine can improve the long-term prognosis of rats with HI brain damage has not been reported. This study was designed to investigate the long-term effect of memantine therapy on neonatal rats with HI brain damage.</p><p><b>METHODS</b>Sixty postnatal 7-day-old newborn rats were randomly assigned into Normal control, HI and Memantine treated groups. Memantine (20 mg/kg) was administered immediately after HI in the Memantine-treated group. All subjects received a 5-day training of Morris water maze test from 23 days old. The escape latency (EL) was recorded at 28 and 35 days old.</p><p><b>RESULTS</b>The EL values of the Normal control, HI and Memantine-treated groups at 28 days old were 23.1 +/- 21.8, 35.1 +/- 5.3, and 20.6 +/- 3.4 seconds, respectively. There was a significant difference in the EL value between the HI and the Normal control groups (P < 0.05). The EL value of the Normal control, HI and Memantine-treated groups at 35 days old were 19.7 +/- 16.7, 35.6 +/- 32.3, and 16.3 +/- 13.2 seconds, respectively. A prolonged EL induced by HI still existed (P < 0.05 vs Normal controls) but memantine treatment shortened the EL (P < 0.01 vs HI group) at 35 days old.</p><p><b>CONCLUSIONS</b>Administering memantine immediately after HI can markedly increase the abilities of spatial discrimination, learning and memory and improve the long-term prognosis in rats with HI brain damage.</p>

Toxic Reactions of Memantine in Acute Toxicologic Experiment in Neonatal Rats / 实用儿科临床杂志

Objective To explore the acute toxic reactions of memantine in neonatal rats. Methods Based on Completely Lethal dose(LD_(100)) and median lethal dose (LD_(50))of memantine in SD neonatal rats acquired in a preliminary test of death dose, 60 neonatal rats were randomly divided into normal group which were given water injection intraperitoneally and 5 study groups which were given different doses of memantine intraperitoneally.LD_(50) was calculated with Bliss method and the toxic reactions of memantine were observed in all neonatal rats of 6 groups after administration of memantine. Results LD_(50) of memantine in SD neonatal rats was((74.386?2.811)) mg/kg with 95% confidence at the range of 59.334-93.257 mg/kg.Side effects occurred at 1-4 minutes after administration. Excitatory jitteriness,ataxia,decreased respiratory rate and passivity were usually observed in groups with a lower dosage (52.0 mg/kg,61.2 mg/kg,72.0 mg/kg);some of them also manifested side lying, cyanosis and respiratory failure.While neonatal rats with a higher dosage (85 mg/kg,100 mg/kg)mainly manifested visible symptoms of inhibition, respiratory failure,side (lying) and cyanosis.However,no jitteriness and ataxia were observed in them.The neonatal rats usually died around 1 hour after memantine administration;survival rats usually returned to normal 4-5 hours after administration.Conclusion There is a positive correlation between toxic reactions and the mortality with memantine dosage in neonatal rats.

Pathological Changes of Memantine in Neonatal Rats in Acute Toxicologic Experiment / 实用儿科临床杂志

Objective To investigate the pathomorphology effects of memantine on organs in neonatal rats.Methods Sixty-eight neonatal rats were randomly divided into 7 groups:5 groups by different doses memantine intraperitoneally and the controls by water intraperitoneally.The pathomorphology changes of organs were observed in all dead neonatal rats promptly after administration of memantine and in all survived rats after 7 days recover.Results 1.The ratio of organ weight and body weight in dead neonatal rats were higher than those of controls.2.The result of pathomorphology indicated that neurodegeneration and necrosis in the brain,the liver congestion and cell degeneration.The other organs had not distinct changes.3.The pathologic changes and mortality rate of neonatal rats were positively correlated with the dosage of memantine.Conclusion Memantine will affect liver and brain of neonatal rats.

Influence of Brain Hypoxia-Ischemia on Expression of Glucose Transporter 1 Genes and Glucose Transpsorter 3 Genes in Neonatal Rats / 实用儿科临床杂志

Objective To understand the mechanism of cerebral energy failure after hypoxia ischemia at the molecular level and to establish the protocol for the safe and effective treatment of hypoxic-ischemic encephalopathy(HIE).Methods One hundred neonatal rats were divided into normal control group and hypoxic-ischemic(HI) group. SD rats of both groups were decapitated at the time of 2 h,24 h,48 h,72 h and 7 d after HI.These tissues of cerebrum,cortex and hippocampus were taken out to explore the influence of HI on the expression of GLUT1 and GLUT3 genes with the method of RT-PCR.Results There was an enhancement in the expression of GLUT1 and GLUT3 genes with the increasing of day age. The expression was more intense in hippocampus than that in cortex. However, HI could significantly enhance the expression of GLUT genes. The expression was higher in cortex than that in hippocampus. The expression of two genes reached the peak at 24 h after HI, but was significantly lower than that in control group at 7 d after HI.Conclusion The increased expression of GLUT genes can maintain the energy supplement for the brain and delay a cascade reaction of cerebral energy failure.