Effect of epidermal growth factor on the migration of neural stem cells

INTRODUCTION Since neural stem cells (NSCs) are separated from corpus striatum of adult rats in 1992 [1], researches on NSCs become a hot topic all over the world because they are characterized by excellent superiority and complete cure on treating geneti…     

Differentiation of endogenous neural precursors following spinal cord injury in adult rats☆

BACKGROUND： Studies have shown that cell death can activate proliferation of endogenous neural stem cells and promote newly generated cells to migrate to a lesion site.
OBJECTIVE： To observe regeneration and differentiation of neural cells following spinal cord injury in adult rats and to quantitatively analyze the newly differentiated cells.
DESIGN, TIME AND SETTING： A cell biology experiment was performed at the Institute of Orthopedics and Medical Experimental Center, Lanzhou University, between August 2005 and October 2007.
MATERIALS： Fifty adult, Wistar rats of both sexes; 5-bromodeoxyuridine （BrdU, Sigma, USA）; antibodies against neuron-specific enolase, glial fibrillary acidic protein, and myelin basic protein （Chemicon, USA）.
METHODS： Twenty-five rats were assigned to the spinal cord injury group and received a spinal cord contusion injury. Materials were obtained at day 1, 3, 7, 15, and 29 after injury, with 5 rats for each time point. Twenty-five rats were sham-treated by removing the lamina of the vertebral arch without performing a contusion.
MAIN OUTCOME MEASURES： The phenotype of BrdU-labeled cells, i.e., expression and distribution of surface markers for neurons （neuron-specific enolase）, astrocytes （glial fibrillary acidic protein）, and oligodendrocytes （myelin basic protein）, were identified with immunofluorescence double-labeling. Confocal microscopy was used to detect double-labeled cells by immunofluorescence. Quantitative analysis of newly generated cells was performed with stereological counting methods.
RESULTS： There was significant cell production and differentiation after adult rat spinal cord injury. The quantity of newly-generated BrdU-labeled cells in the spinal cord lesion was 75-fold greater than in the corresponding area of control animals. Endogenous neural precursor cells differentiated into astrocytes and oligodendrocytes, however spontaneous neuronal differentiation was not detected. Between 7 and 29 d after spinal cord injury, newl     

Zhichan decoction induces differentiation of dopaminergic neurons in Parkinson’s disease rats after neural stem cell transplantation

The goal of this study was to increase the dopamine content and reduce dopaminergic metabolites in the brain of Parkinson’s disease rats. Using high-performance liquid chromatography, we found that dopamine and dopaminergic metabolite（dihydroxyphenylacetic acid and homovanillic acid） content in the midbrain of Parkinson’s disease rats was increased after neural stem cell transplantation ＋ Zhichan decoction, compared with neural stem cell transplantation alone. Our genetic algorithm results show that dihydroxyphenylacetic acid and homovanillic acid levels achieve global optimization. Neural stem cell transplantation ＋ Zhichan decoction increased dihydroxyphenylacetic acid levels up to 10-fold, while transplantation alone resulted in a 3-fold increment. Homovanillic acid levels showed no apparent change. Our experimental findings show that after neural stem cell transplantation in Parkinson’s disease rats, Zhichan decoction can promote differentiation of neural stem cells into dopaminergic neurons.     

Electroacupuncture at Ren channel point combined with injection of basic fibroblast growth factors for neural stem cell proliferation in subventricular zone of cerebral ischemic side

INTRODUCTION Basic fibroblast growth factor (bFGF) significantly promotes the prolif- eration of mesoderm and neuroectoderm-derived cells. It is also the mitogenic factor and angiogenesis factor of endothelial cells, and plays an important role in the pro…     

Cerebral expression of DNA repair protein,Ku70, and its association with cell proliferation following cerebral hypoxia–ischemia in neonatal rats

We hypothesized that increased Ku70 expression could be involved in recovery following cerebral hypoxia–ischemia. We investigated the progression of cerebral alterations in Ku70 expression at different time points (24 h, 72 h, 1 week, 4 weeks and 8 weeks) after hypoxia–ischemia (right carotid artery occlusion plus 1.5 h of hypoxia) in neonatal rats. To determine whether in addition to its known role of DNA repair, Ku70 was associated with cell death or cell proliferation we performed double staining for Ku70 and DNA fragmentation or bromodeoxyuridine, respectively. The results show that Ku70 expression was increased in the infarct core and peri-infarct regions at 24 h following hypoxia–ischemia. The increased Ku70 expression was transient in the infarct core with a loss of Ku70 positive cells over days. In contrast, in the peri-infarct region the expression of Ku70 remained increased at chronic times 8 weeks following the insult. Cells positive for DNA fragmentation were not co-localized with cells positive for Ku70 after an insult. However, most of the cells positive for bromodeoxyuridine indicative of cell proliferation were positive for Ku70 in the peri-infarct region at 8 weeks after the insult. Considering the roles of Ku70 in DNA repair or inhibiting apoptosis and its co-localization within cells that had undergone proliferation, Ku70 may be considered a potential novel target to enhance recovery following hypoxia–ischemia.     

Mild hypothermia combined with neural stem cell transplantation for hypoxic-ischemic encephalopathy： neuroprotective effects of combined therapy

Neural stem cell transplantation is a useful treatment for ischemic stroke, but apoptosis often occurs in the hypoxic-ischemic environment of the brain after cell transplantation. In this study, we determined if mild hypothermia(27–28°C) can increase the survival rate of neural stem cells(1.0 × 105 /μL) transplanted into neonatal mice with hypoxic-ischemic encephalopathy. Long-term effects on neurological functioning of the mice were also examined. After mild hypothermia combined with neural stem cell transplantation, we observed decreased expression levels of inflammatory factor nuclear factor-kappa B and apoptotic factor caspase-3, reduced cerebral infarct volumes, increased survival rate of transplanted cells, and marked improvements in neurological function. Thus, the neuroprotective effects of mild hypothermia combined with neural stem cell transplantation are superior to those of monotherapy. Moreover, our findings suggest that the neuroprotective effects of mild hypothermia combined with neural stem cell transplantation on hypoxic-ischemic encephalopathy are achieved by anti-inflammatory and anti-apoptotic mechanisms.     

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson’s disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.     

Differentiation of embryonic versus adult rat neural stem cells into dopaminergic neurons in vitro*☆

BACKGROUND：It has been reported that the conversion of neural stem cells into dopaminergic neurons in vitro can be increased through specific cytokine combinations. Such neural stem cell-derived dopaminergic neurons could be used for the treatment of Parkinson’s disease. However, little is known about the differences in dopaminergic differentiation between neural stem cells derived from adult and embryonic rats. OBJECTIVE： To study the ability of rat adult and embryonic-derived neural stem cells to differentiate into dopaminergic neurons in vitro. DESIGN： Randomized grouping design. SETTING： Department of Neurosurgery in the First Affiliated Hospital of Sun Yat-sen University. MATERIALS： This experiment was performed at the Surgical Laboratory in the First Affiliated Hospital of Sun Yat-sen University （Guangzhou, Guangdong, China） from June to December 2007. Eight, adult, male, Sprague Dawley rats and eight, pregnant, Sprague Dawley rats （embryonic day 14 or 15） were provided by the Experimental Animal Center of Sun Yat-sen University. METHODS： Neural stem cells derived from adult and embryonic rats were respectively cultivated in serum-free culture medium containing epidermal growth factor and basic fibroblast growth factor. After passaging, neural stem cells were differentiated in medium containing interleukin-1α, interleukin-11, human leukemia inhibition factor, and glial cell line-derived neurotrophic factor. Six days later, cells were analyzed by immunocytochemistry and flow cytometry. MAIN OUTCOME MEASURES： Alterations in cellular morphology after differentiation of neural stem cells derived from adult and embryonic rats; and percentage of tyrosine hydroxylase-positive neurons in the differentiated cells. RESULTS： Neural stem cells derived from adult and embryonic rats were cultivated in differentiation medium. Six days later, differentiated cells were immunoreactive for tyrosine hydroxylase. The percentage of tyrosine hydroxylase positive neurons was （5.6 ± 2.8）% and （17.8     

Dose-dependent effects of procyanidin on nerve growth factor expression following cerebral ischemia/ reperfusion injury in rats★

BACKGROUND： Recently, grape seed procyanidin （GSP） has been shown to be exhibit antioxidant effects, effectively reducing ischemia/reperfusion injury and inhibiting brain cell apoptosis. OBJECTIVE： To study the effects of GSP on nerve growth factor （NGF） expression and neurological function following cerebral ischemia/reperfusion injury in rats. DESIGN： Randomized controlled study based on SD rats. SETTING： Weifang Municipal People＇s Hospital. MATERIALS： Forty-eight healthy adult SD rats weighing 280-330 g and irrespective of gender were provided by the Experimental Animal Center of Shandong University. GSP derived from grape seed was a new high-effective antioxidant provided by Tianjin Jianfeng Natural Product Researching Company （batch number： 20060107）. Rabbit-anti-rat NGF monoclonal antibody was provided by Beijing Zhongshan Biotechnology Co., Ltd., and SABC immunohistochemical staining kit by Wuhan Boster Bioengineering Co., Ltd. METHODS： The present study was performed in the Functional Laboratory of Weifang Medical College from April 2006 to January 2007. Forty-eight SD rats were randomly divided into the sham operation group, ischemia/reperfusion group, high-dose GSP （40 mg/kg） group, or low-dose GSP （10 mg/kg） group （n = 12 per group）. Ischemia/reperfusion injury was established using the threading embolism method of the middle cerebral artery. Rats in the ischemia/reperfusion model group were given saline injection （2 mL/kg i.p.） once daily for seven days pre-ischemia/reperfusion, and once more at 15 minutes before reperfusion. Rats in the high-dose and low-dose GSP groups were injected with GSP （20 or 5 mg/mL i.p., respectively, 2 mL/kg） with the same regime as the ischemia/reperfusion model group. The surgical procedures in the sham operation group were as the same as those in the ischemia/reperfusion model group, but the thread was approximately 10 mm long, thus, the middle cerebral artery was not blocked. MAIN OUTCOME MEASURES： NGF expression in the     

Protective effects and time course of Huangqi on early-stage free radical injury following brain trauma in rats★

BACKGROUND： Huangqi （Astragalus mongholicus）, a Chinese herb, has already been included in the ＂Chinese Pharmacopoeia＂ for the treatment of ischemic cerebrovascular disease. Secondary injury following brain injury is associated with free radical production, and Huangqi possesses the ability to ameliorate free radical-mediated injury. OBJECTIVE： This study was designed to observe the correlation between anti-free-radical properties of Huangqi and early histological changes of brain tissues following traumatic brain injury. DESIGN, TIME AND SETTING： This study, a randomized, controlled, animal experiment, was performed from May 2006 to June 2007 at the Experimental Center of Science and Technology, School of Basic Science, Liaoning Medical University, Jinzhou City, Liaoning Province, China. MATERIALS： Healthy, adult, Sprague Dawley rats of either gender were included. Huangqi injection was purchased from Heilongjiang Provincial Zhenbaodao Pharmaceutical Co., Ltd., China （National License Medical Number： Z23020781）. Na^＋-K^＋-adenosine triphosphatase （ATPase）, Ca^2＋-ATPase, and Mg^2＋-ATPase, as well as kits to measure superoxide dismutase （SOD） activity and malondialdehyde （MDA） content, were purchased from Nanjing Jiancheng Biological Reagent Company, China. METHODS： Seventy-two rats were randomly divided into three groups, with 24 rats in each group： （1） sham-operated group： rats were only exposed, but not injured; （2） model group： brain focal laceration rat models were established by free-falling. These groups were intraperitoneally injected with saline, once every 10 hours; （3） Huangqi group： rats were intraperitoneally injected with 4 mL/kg Huangqi （2 g/mL）, once every 10 hours, following brain focal laceration by free-falling. MAIN OUTCOME MEASURES： Ultrastructural changes in brain tissue were observed under an electron microscope 24 hours after injury. The water content of brain tissue was measured using the dry-wet weight method. In additio     

Effect of Angelica sinensis on neural stem cell proliferation in neonatal rats following intrauterine hypoxia

BACKGROUND:Angelica sinensis is a widely used herb in Chinese traditional medicine.It has been shown to improve hypoxia in embryonic rats and reduce nestin expression in neural stem cells,resulting in proliferation of neural stem cells.OBJECTIVE:To study the protective effect of Angelica on neural stem cell proliferation in neonatal rats after intrauterine hypoxia.DESIGN,TIME AND SETTING:The randomized,controlled,experiment was performed at the Department of Histology and Embryology,Luzhou Medical College,China from July 2007 to January 2008.MATERIALS:Because gestational days 14-15 are a key stage in rat nervous system development,21 healthy,pregnant Sprague Dawley rats(14 days after conception)were used for this study.Nestin monoclonal primary antibody was obtained from Chemicon,USA.Angelica parenteral solution(250 g/L)was obtained from Pharmaceutical Preparation Section,Second Affiliated Hospital of Wuhan University,China.METHODS:Rats were randomly divided into a control group(n=5),a hypoxia group(n=8),and an Angelica group(n=8).Saline(8 mL/kg)was injected into the caudal vein of rats in the hypoxia group once a day for seven consecutive days.Intrauterine hypotonic hypoxia was induced using 13% O2 for two hours per day on three consecutive days.Rats in the Angelica group received injections of Angelica parenteral solution(250 g/L);all other protocols were the same as the hypoxia group.The control group procedures were identical to the hypoxia group,but under normal,non-hypoxic conditions.After birth,brain tissues were immediately obtained from neonatal rats and prepared for nestin immunohistochemistry.MAIN OUTCOME MEASURES:Nestin-positive cells in hippocampal CA3 area of neonatal rats in each group were quantified using image analysis to detect signal absorbance.RESULTS:The number of nestin-positive cells increased in the hippocampal CA3 area of neonatal rats in the hypoxia group.The number of nestin-positive cells was less in the Angelica group than in the hypoxia group.Integral absorbance of nestin-positive cells in the hippocampal CA3 area of neonatal rats was significantly higher in the hypoxia group,compared with the control group(P<0.05).The integral absorbance of nestin positive cells was lower in the Angelica group,compared with the hypoxia group(P<0.05).CONCLUSION:Intrauterine hypoxia,induced for 2 hours daily for three consecutive days,with an oxygen concentration of 13%,stimulated the proliferation of neural stem cells.Angelica injection has a protective effect on neural stem cells from neonatal rats following intrauterine hypoxia by decreasing proliferation of neural stem cells.     

宫内缺氧及当归注射液干预对幼年大鼠海马齿状回神经元与学习记忆能力的影响

BACKGROUND： The present study analyzed the effect of 3 days （2 h/d） intrauterine hypoxia on learning and memory in juvenile rats, as well as the therapeutic effects of Angelica sinensis on dentate gyrus neurons, as well as learning and memory. OBJECTIVE： To explore the effects of intrauterine hypoxia on hippocampal dentate gyrus neurons, as well as learning and memory, in juvenile rats; to explore N-methyI-D-aspartate receptor-1 （NMDAR1） expression in the dentate gyrus of neonatal rats following intrauterine hypoxia, as well as prolonged hypoxia; to investigate the regulatory mechanisms of Angelica sinensis. DESIGN, TIME AND SETTING： A randomized and controlled experiment based on developmental neurobiology was performed at the Department of Histology and Embryology in Luzhou Medical College from October 2007 to October 2008. MATERIALS： Angelica sinensis solution （250 g/L） was obtained from Central South Hospital of Wuhan University, China. Neuron-specific enolase and NMDAR1 mRNA in situ hybridization reagents were provided by Wuhan Boster Biological Technology, China. Image-Pro Plus 6.0 analysis system was purchased from Media Cybernetics, USA. METHODS： Healthy pregnant Sprague Dawley rats （n = 30） were randomly divided into control （n = 10）, hypoxia （n = 10）, and Angelica （n = 10） groups. The Angelica and hypoxia pregnant rats were placed in a three-gas incubator （oxygen concentration： 13%） starting with day 14 of pregnancy for 2 hours/day for 5 consecutive days to establish a fetal rat intrauterine hypoxia model. One hour prior to modeling, the pregnant rats from the Angelica and hypoxia groups received Angelica sinensis and normal saline （8 mL/kg） injections, respectively, through the caudal vein. The control group procedures were identical to the hypoxia group, but lacked the hypoxic conditions. MAIN OUTCOME MEASURES： Brain tissues of neonatal rats were used to detect expression of NMDAR1 mRNA, and brain tissues of juvenile rats aged 30 days were used to determine neuron-specific enolase mRNA expression by in situ hybridization. Microscopic images （400x） of the hippocampal dentate gyrus were collected. The integral optical density （IOD） value of positive NMDAR1 mRNA cells in the dentate gyrus of neonatal rats, as well as the quantity and the IOD value of positive neuron-specific enolase mRNA cells in the dentate gyrus of juvenile rats, were analyzed with Image-Pro IPP6.0 software. At 30 days after birth, learning and memory parameters were measured in the juvenile rats using Morris water maze. RESULTS： The quantity and the IOD value of positive neuron-specific enolase mRNA cells in the dentate gyrus of the hypoxia group juvenile rats were significantly less than the control group （P 〈 0.05）, and also less than the Angelica group （P 〈 0.05）. The IOD value of positive NMDAR1 mRNA cells in the dentate gyrus of the hypoxia group neonatal rats was significantly greater than the control group, and also greater than the Angelica group （P 〈 0.05）. In the Morris water maze, the searching time during the probe trial and reversal probe trial was shorter in the hypoxia group juvenile rats compared with the control group, and the Angelica group was prolonged compared with the hypoxia group （P 〈 0.05）. CONCLUSION： Intrauterine hypoxia increased expression of NMDAR1 mRNA in the dentate gyrus of neonatal rats, reduced the number of dentate gyrus neurons, and negatively affected learning and memory in juvenile rats. In contrast, Angelica sinensis injection improved the intrauterine hypoxic condition, increased the number of dentate gyrus neurons, and improved the learning and memory deficits of the juvenile rats.     

稳定增殖神经干细胞的实验研究

目的探讨稳定、可靠建立神经干细胞体外增殖的方法,并对增殖培养的细胞进行鉴定。方法获取胚胎大鼠的脑组织,通过加入神经生长因子和采用保留细胞联系技术操作,使脑组织中的神经干细胞在体外克隆增殖并稳定传代。以免疫荧光方法对增殖克隆的神经干细胞球进行鉴定。结果神经干细胞不断增殖形成神经干细胞球且神经干细胞能快速稳定传代增殖。培养的细胞为神经干细胞特异性巢蛋白(nestin)染色阳性细胞。结论在神经生长因子的作用下,利用保留细胞联系技术操作,神经干细胞可以在体外快速、稳定克隆增殖并传代。     

快速老化与正常老化小鼠脑内神经干细胞增殖的差异**

背景：在老化过程中,脑内环境改变可引起脑内神经干细胞增殖能力改变。脑内神经干细胞与衰老和退行性神经病变疾病密切相关,增殖能力与年龄存在负相关,但以快速老化小鼠为衰老模型的相关研究未见报道。目的：比较快速老化与正常老化小鼠嗅球、海马、皮质神经干细胞增殖的差异。方法：分别取6只快速老化小鼠(SAMP8)和6只正常老化小鼠(SAMR1)的嗅球、海马、皮质组织,在固定、冰冻切片后,运用Ki-67/Nestin免疫荧光双标检测3个脑区的神经干细胞增殖情况。免疫荧光双标在荧光显微镜下通过Leica Qwin v3采图,在40倍物镜和10倍目镜下采图,每一张切片随机选取5 个相邻视野,通过Image-pro-Plus软件完成图像分析。结果与结论：正常老化小鼠和快速老化小鼠均有神经干细胞增殖现象,但二者存在差异,其差异主要表现在海马和嗅球两个脑区(P < 0.05)。提示快速老化可能会导致海马、嗅球神经干细胞增殖能力降低。     

Protective effect of Angelica sinensis on cerebral neurons from rat embryos under hypoxia

BackgroundThe enhanced expression of c-Fos protein in nerve cells after hypoxia is the marker for converting extracellular hypoxia information to intracellular changes at hypoxia, and it is suspected that the increase of c-Fos protein can lead to the synthesis and excretion of related neurotrophic factor and nerve growth factor. However, it is still unclear what functional changes of nerve cells are induced by the increase of c-Fos protein at hypoxia, and whether it is good for the survival of damaged neurons.ObjectiveTo observe the expression of c-Fos in the cerebral neurons from embryos of rats with hypoxia in uterus, and investigate the pathway for the protective effect of Angelica sinensis injection on the cerebral neurons from rat embryos under hypoxia.DesignA completely randomized controlled study.SettingDepartment of Histology and Embryology, Luzhou Medical College.MaterialsTwelve female Wistar rats in oestrum and 1 male adult Wistar rat with body mass of 220 to 250 g were selected. Rabbit-anti-rat neuro-specific enolase (NSE) and rabbit-anti-rat c-Fos were purchased from Wuhan Boster Biological Technology Co., Ltd.; Double-staining kit was bought from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. Angelica sinensis injection was produced by the Department of Pharmacy, the Second Affiliated Hospital of Hubei Medical University.MethodsThe experiments were completed in the experimental animal center and the Department of Histology and Embryology of Luzhou Medical College from December 2004 to December 2005.

Twelve adult female Wistar rats in oestrum and 1 male Wistar rat were housed in one rearing cage. The appearance of vaginal embolus at 8:00 in the next morning was recorded as 0 day of pregnancy and the rats were recorded for 15 days, and they were divided randomly into three groups, control group (n =4), hypoxia group (n =4) and Angelica group (n =4). The pregnant rats in the hypoxia group were firstly injected with saline (8 mL/kg), then put into 2 L wide-mouthed bottle containing 100 g sodalime, and then the lid of the bottle was closed tightly to induce hypotonic hypoxia for 1 hour followed by 1-hour re-oxygenation. The pregnant rats were killed under anesthesia, and then fetuses were taken out by rapid cesarean. Part of the brain tissues were exposed and then fixed in formaldehyde (40 g/L). The pregnant rats in the Angelica group were treated the same as those in the hypoxia group except that saline was replaced by 250 g/L Angelica sinensis injection which was injected via caudal vein (8 mL/kg). The rats in the control group were injected with saline (8 mL/kg) slowly via caudal vein, but not put into the wide-mouthed bottle for hypoxia, and then the brain tissues were removed and fixed as those in the hypoxia group after 1 hour.

Twenty embryos from rats were chosen randomly in each group and then routinely embedded in paraffin. Paraffin sections of 4 μm thick were prepared through the anterior fontanelle of head of the fetal rats. The sections were immunohistologically stained with c-Fos/NSE.

The one-way analysis of variance (ANOVA) was used to compare the differences of measurement data among the groups, and the q test was applied in the two-two comparison.Main outcome measuresThe numbers of c-Fos and c-Fos/NSE positive neurons in cerebrum from rat embryos were observed.Results

Numbers of NSE positive neurons in cerebrum of rat embryos in the control group, hypoxia group and Angelica group were (84.3±9.0), (90.2±12.5) and (86.7±9.7) cells/high power field (P > 0.05).

The number of c-Fos/NSE positive neurons was more in the hypoxia group than in the control group and Angelica group [(38.4±5.28), (11.35±2.67), (20.65±4.07) cells/high power field, q =29.17, 19.14, P < 0.05].ConclusionHypoxia can stimulate the expression of c-Fos in cerebral neurons from rat embryos. Angelica sinensis injection could reducing the damage of hypoxia to neurons and play a neuroprotective role by decreasing the expression of c-Fos protein in hypoxic neurons.     

吡咯喹啉醌与神经干细胞的增殖

背景：目前国内外学者主要应用生长因子样物质来促进神经干细胞的分裂与增殖,所以作者推测吡咯喹啉醌小分子化合物也能促进神经干细胞的分裂增殖。 目的：体外观察氧化还原酶辅酶吡咯喹啉醌促进鼠成体神经干细胞增殖的作用。 方法：体外培养Wistar胎鼠成体神经干细胞成球体后,于培养液中加入1×10-8 mol/L吡咯喹啉醌,对照组加入同等剂量的Hanks溶液。应用流式细胞仪测细胞周期增殖率,应用免疫组化法和Western Blot测周期蛋白激酶(CDK2)和周期蛋白激酶抑制剂(P27)的表达。 结果与结论：与对照组相比,吡咯喹啉醌处理16 h和24 h后,S期和G2期细胞数的比例显著增高,而细胞的凋亡率降低;CDK2蛋白的表达显著增强,而P27的表达显著降低。结果显示低浓度的吡咯喹啉醌即能促进鼠成体神经干细胞的增殖。     

低氧条件下星形胶质细胞对神经干细胞增殖的影响

目的 探讨体外低氧条件下星形胶质细胞对海马神经干细胞( NSCs)增殖的影响.方法 将分离出的新生小鼠海马NSCs分别与低氧条件星形胶质细胞培养液(低氧组)、低氧条件星形胶质细胞培养液+磷脂酰肌醇-3激酶/蛋白激酶B( PI3K/Akt)通路拮抗剂LY294002(拮抗剂组)以及常氧条件星形胶质细胞培养液(对照组)共培养;分别于培养24 h、48 h、72 h应用四甲基偶氮唑盐(MTT)法检测各组NSCs活性;应用Western Blot法检测各组NSCs磷酸化Akt (p-Akt)和磷酸化糖原合成酶激酶(p-GSK)-3β含量.结果 (1)共培养24h、48 h及72 h,低氧组NSCs活性的光密度(OD)值分别为0.292±0.006、0.382 ±0.005、0.649±0.028,拮抗组分别为0.197±0.003、0.255±0.005、0.325±0.012,对照组分别为0.107±0.006、0.198±0.008、0.254±0.006;低氧组各时间点NSCs活性显著高于拮抗剂组和对照组(均P＜0.05);(2)共培养24h、48 h和72 h,低氧组NSCs p-Akt和p-GSK-3β的吸光度(A)值分别为0.52、0.71、0.86及0.49、0.65、0.82;拮抗剂组分别为0.39、0.42、0.61及0.31、0.35、0.40;对照组分别为0.34、0.38、0.39及0.28、0.31、0.35;低氧组各时间点p-Akt及p-GSK-3β含量显著高于拮抗剂组和对照组(均P＜0.05).结论 缺氧刺激可激活星形胶质细胞PI3K/Akt信号通路,促进NSCs增殖.     

19-Nortestosterone influences neural stem cell proliferation and neurogenesis in the rat brain

Abuse of androgenic anabolic steroids can affect brain function leading to behavioural changes. In this study, the effects of the testosterone analogue, 19-nortestosterone, on rat neural stem cells was examined. The androgen receptor is expressed by cultured embryonic and adult neural stem cells, and is also present in the ventricular epithelium during development and in the adult brain in, among others, dentate gyrus. In neural stem cells stimulated with epidermal growth factor, nandrolone reduced cell proliferation, especially in adult ones. The decrease was abolished by flutamide, a receptor antagonist. Nandrolone also decreased the BrdU labelling of neural stem cells in the dentate gyrus, demonstrating an effect of the hormone on cell proliferation in vivo. The effect of nandrolone was observed with both female and male rats but it was more pronounced in pregnant rats, indicating an involvement of oestrogen in nandrolone action. Nandrolone also decreased the number of newly born neuronal cells in the dentate gyrus of male rats. The results show that nandrolone has important effects on the proliferation and differentiation of neural stem cells expressing the cognate androgen receptor. The data show that the use of nandrolone may severely affect the formation of neural stem cells and could therefore have long-term negative consequences in the brain.