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.     

Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease *☆

BACKGROUND： Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson＇s disease; however, the survival rate of transplanted cells has been low. Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals.
OBJECTIVE： To observe whether survival of transplanted cells, transplantation efficacy, and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins （PNS） in a rat model of Parkinson＇s disease.
DESIGN, TIME AND SETTING： Cellular and molecular biology experiments with randomized group design. The experiment was performed at the Animal Experimental Center, First Hospital of Sun Yat-sen University from April to October 2007.
MATERIALS： Thirty-two adult, healthy, male Sprague Dawley rats, and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected. The right ventral mesencephalon was injected with 6-hydroxydopamine to establish a model of Parkinson＇s disease. 6-hydroxydopamine and apomorphine were purchased from Sigma, USA.
METHODS： Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium. Lesioned animals were randomly divided into four groups （n = 8）： dopaminergic neuron, dopaminergic neuron ＋ PNS, PNS, and control. The dopaminergic neuron group was injected with 3 μL cell suspension containing dopaminergic neurons differentiated from neural stem cells. The dopaminergic neurons ＋ PNS group received 3 μ L dopaminergic cell suspension combined with PNS （250 mg/L）. The PNS group received 3 μL PNS （250 mg/L）, and the control group received 3 μL DMEM/F12 culture medium.
MAIN OUTCOME MEASURES： The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry. The rats were intraperitoneally injected with apomorphine （0.5 mg/kg） to induce rotational behavior. RESU     

碱性成纤维细胞因子在局灶性脑缺血后内源性神经干细胞增殖过程中对β-catenin的影响

BACKGROUND： The Wnt/β-catenin signaling pathway plays an important role in neural development. ,β-catenin is an important component of the Wnt/β-catenin signaling pathway. The Wnt signaling pathway has been shown to regulate the interaction of neural stem cells with the extracellular matrix.
OBJECTIVE： To investigate the effects of basic fibroblast growth factor （bFGF） on β-catenin protein and mRNA expression, and on hippocampal neural stem cell proliferation in a rat model of cerebral ischemia/reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiology experiment was performed in Shenyang Medical College between August 2006 and August 2008. MATERIALS： A total of 72 healthy male Wistar rats, aged 3 months, were used in this study. bFGF was provided by Beijing SL Pharmaceutical Co.,Ltd., China. METHODS： Rats were randomly divided into 3 groups： sham-operated, ischemia/reperfusion, and bFGF-treated （n = 24 per group）. Focal cerebral ischemia/reperfusion was induced in rats from the ischemia/reperfusion group and the bFGF-treated group by 2 hour right middle cerebral artery occlusion and 2 hour restoration of blood flow using the suture method. The ischemia/reperfusion and bFGF-treated groups were intraperitoneally administered 500 IU/mL of bFGF, or the same volume of physiological saline, once a day at postoperative days 1 3, and once every 3 days thereafter. Simultaneously, the sham-operated group underwent experimental procedures identical to the ischemia/reperfusion and bFGF-treated groups, with the exception of ischemia/reperfusion induction and drug administration. At 2 hours, 2, 6, 13, and 20 days after ischemiaJreperfusion induction, 50 mg/kg bromodeoxyuridine （BrdU） was administered to each group, twice daily, to label proliferating neural stem cells. MAIN OUTCOME MEASURES： The effects of bFGF on BrdU labeling, and ,8 -catenin mRNA and protein expression, in neural stem cells were examined by immunohistochemistry, Western blot, RT-PCR, and in situ hybridization techniques. RESULTS： In the sham-operated group, only a few BrdU-immunoreactive neural stem cells were found. In the ischemia/reperfusion group, BrdU-immunoreactive cells began to increase from 3 days after ischemia/reperfusion induction, reached a peak level at 7 days, and gradually reduced from 21 days. At 3, 7, 14, and 21 days after ischemia/reperfusion induction, the numbers of BrdU-immunoreactive cells were significantly greater in the bFGF-treated group than in the ischemia/reperfusion group. The sham-operated group exhibited slight expression of β-catenin and β-catenin mRNA. In the ischemia/reperfusion group, the expression of β-catenin and β-catenin mRNA gradually increased with reperfusion time, peaked at 14 days after reperfusion, and gradually decreased thereafter; by 21 days, the expression was markedly lower. Following bFGF injection, the expression of hippocampal BrdU, β-catenin, and β-catenin mRNA had apparently increased in each group. CONCLUSION： bFGF promotes neural stem cell proliferation, and the expression of β-catenin and β-catenin mRNA in the ischemic brain tissue. These findings indicate that bFGF promotion of neural stem cell proliferation may be mediated by Wnt/β-catenin signaling pathway.     

Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease

BACKGROUND:Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson's disease;however,the survival rate of transplanted cells has been low.Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals.OBJECTIVE:To observe whether survival of transplanted cells,transplantation efficacy.and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins(PNS) in a rat model of Parkinson's disease.DESIGN,TIME AND SETTING:Cellular and molecular biology experiments with randomized group design.The experiment was performed at the Animal Experimental Center,First Hospital of Sun Yat-sen University from April to October 2007.MATERIALS:Thirty-two adult,healthy,male Sprague Dawley rats,and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected.The right ventral mesenceDhalon was injected with 6-hydroxydopamine to establish a model of Parkinson's disease.6-hydroxydopamine and apomorphine were purchased from Sigma.USA.METHODS:Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium.Lesioned animals were randomly divided into four groups(n=8):dopaminergic neuron,dopaminergic neuron PNS,PNS,and control.The dopaminergic neuron group was iniected with 3 μ L cell suspension containing dopaminergic neurons difierentiated from neural stem cells.The dopaminergic neurons PNS group received 3 μ L dopaminergic cell suspension combined with PNS (250 mg/L).The PNS group received 3 μL PNS(250 mg/L),and the control group received 3 μL DMEM/F12 culture medium.MAIN OUTCOME MEASURES:The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry.The rats were intraperitoneally injected with apomorphine (0.5 mg/kg)to induce rotational behavior.RESULTS:Cell counts of tyrosine hydroxylase-positive neurons in the dopaminergic neuron PNS group were(732±82.6)cells/400-fold field.This was significantly greater than the dopaminergic neuron group [(326±34.8)cells/400-fold field,P<0.01].Compared to the control group,the rotational asymmetry of rats that received dopaminergic neuron transplants was significantly decreased,beginning at 20 days after operation(P<0.0 1).Rotational asymmetry was fugher reduced between 10～60 days post-surgery in the dopaminergic neuron PNS group,compared to the dopaminergic neuron group(P<0.01).CONCLUSION:Panax notoginseng saponins can increase survival and effectiveness of dopaminergic neurons differentiated from neural stem cells for transplantation in a rat model of Parkinson's disease.     

静电纺壳聚糖/胶原蛋白复合纳米纤维的细胞相容性

BACKGROUND： Due to the lack of autograft transplant rejection, Schwann cells （SCs） can promote the proliferation of embryonic stem cells and the induction of dopaminergic neurons. Mesencephalic stem cells can be induced to produce dopaminergic neurons. The therapeutic effects of co-grafts of SCs and neural stem cells （NSCs） deserves further study and verification in Parkinsonian animal models. OBJECTIVE： To investigate the effects of Schwann cells and mesencephalic NSC co-grafts in Parkinsonian animal models on animal behavior and histology. DESIGN： Randomized controlled experiment. SETTING： Fudan University; Institute of Neuroscience, Chinese Academy of Sciences. MATERIALS： The following animals were obtained from the Experimental Animal Center, Shanghai Institute for Biological Science, Chinese Academy of Sciences： 5 Sprague-Dawley rats, embryonic day （E） 13-16; 16 neonatal Sprague-Dawley rats, postnatal day 1-3; and 18 adult SD rats of both genders. Animal experimentation met animal ethical approval. METHODS： The experiment was performed at the Department of Anatomy, Histology and Embryology, Shanghai Medical Center, Fudan University from September 2005 to January 2007. The mesencephalic NSCs were obtained from the brains of SD rats at E 13-16, and SCs were harvested from the sciatic nerves of neonatal rats at day 1-3. Hemiparkinsonian rats （n =18） were selected for transplantation after estimating rotational behavior in response to apomorphine and were randomly assigned to three groups： control group, NSC group, and co-graft group. There were 6 rats in each group. Either phosphate buffered saline （PBS）, NSCs, or SCs plus NSCs were transplanted into the right neostriatum of Parkinsonian rats, respectively. MAIN OUTCOME MEASURES： （1） Rotational behavior was induced by apomorphine （0.05 mg/kg, i.p.） 2, 4, 6, 8, and 10 weeks after transplantation, and the number of rotations were counted. （2） Differentiation and survival of dopaminergic neurons in the right neostr     

丙戊酸对脊髓损伤大鼠内源性神经干细胞增殖的影响

BACKGROUND： Valproic acid has been reported to decrease apoptosis, promote neuronal differentiation of brain-derived neural stem cells, and inhibit glial differentiation of brain-derived neural stem cells.
OBJECTIVE： To investigate the effects of valproic acid on proliferation of endogenous neural stem cells in a rat model of spinal cord injury.
DESIGN, TIME AND SETTING： A randomized, controlled, neuropathological study was performed at Key Laboratory of Trauma, Buming, and Combined Injury, Research Institute of Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA between November 2005 and February 2007.
MATERIALS： A total of 45 adult, Wistar rats were randomly divided into sham surgery （n = 5）, injury （n = 20）, and valproic acid （n = 20） groups. Valproic acid was provided by Sigma, USA. METHODS： Injury was induced to the T10 segment in the injury and valproic acid groups using the metal weight-dropping method. The spinal cord was exposed without contusion in the sham surgery group. Rats in the valproic acid group were intraperitoneally injected with 150 mg/kg valproic acid every 12 hours （twice in total）.
MAIN OUTCOME MEASURES： Nestin expression （5 mm from injured center） was detected using immunohistochemistry at 1,3 days, 1, 4, and 8 weeks post-injury.
RESULTS： Low expression of nestin was observed in the cytoplasm, but rarely in the white matter of the spinal cord in the sham surgery group. In the injury group, nestin expression was observed in the ependyma and pia mater one day after injury, and expression reached a peak at 1 week （P 〈 0.05）. Expression was primarily observed in the ependymal cells, which expanded towards the white and gray matter of the spinal cord. Nestin expression rapidly decreased by 4 weeks post-injury, and had almost completely disappeared by 8 weeks. At 24 hours after spinal cord injury, there was no significant difference in nestin expression between the valproic acid and injury groups. At 1 week, there was a significant increase in the number of nestin-positive cells surrounding the central canal in valproic acid group compared with the injury group （P 〈 0.05）. Expression reached a peak by 4 weeks, and it was still present at 8 weeks.
CONCLUSION： Valproic acid promoted endogenous neural stem cell proliferation following spinal cord injury in rats.     

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     

骨髓间充质干细胞对1-甲基-4-苯基吡啶离子损伤的离体纹状体-黑质脑片多巴胺能神经元的保护作用

BACKGROUND： To date, the use of bone marrow-derived mesenchymal stem cells （MSCs） for the treatment of Parkinson’s disease have solely focused on in vivo animal models. Because of the number of influencing factors, it has been difficult to determine a consistent outcome.
OBJECTIVE： To establish an injury model in brain slices of substantia nigra and striatum using 1-methyl-4-phenylpytidinium ion （MPP＋）, and to investigate the effect of MSCs on dopaminergic neurons following MPP＋ induced damage.
DESIGN, TIME AND SETTING： An in vitro, randomized, controlled, animal experiment using I mmunohistochemistry was performed at the Laboratory of the Department of Anatomy, Fudan University between January 2004 and December 2006.
MATERIALS： Primary MSC cultures were obtained from femurs and tibias of adult Sprague Dawley rats. Organotypic brain slices were isolated from substantia nigra and striatum of 1-day-old Sprague Dawley rat pups. Monoclonal antibodies for tyrosine hydroxylase （TH, 1：5 000） were from Santa Cruz （USA）; goat anti-rabbit IgG antibodies labeled with FITC were from Boster Company （China）.
METHODS： Organotypic brain slices were cultured for 5 days in whole culture medium supplemented with 50% DMEM, 25% equine serum, and 25% Tyrode’s balanced salt solution. The medium was supplemented with 5 μg/mL Ara-C, and the culture was continued for an additional 5 days. The undergrowth of brain slices was discarded at day 10. Eugonic brain slices were cultured with basal media for an additional 7 days. The brain slices were divided into three groups： control, MPP＋ exposure, and co-culture. For the MPP＋ group, MPP＋ （30 μmol/L） was added to the media at day 17 and brain slices were cultured for 4 days, followed by control media. For the co-culture group, the MPP＋ injured brain slices were placed over MSCs in the well and were further cultured for 7 days.
MAIN OUTCOME MEASURES： After 28 days in culture, neurite outgrowth was examined in the brain slices under phase-contrast microscopy. The percent of area containing dead cells in each brain slice was calculated with the help of propidium iodide fluorescence. Brain slices were stained with antibodies for TH to indicate the presence of dopaminergic neurons. Transmission electron microscopy was applied to determine the effect of MSCs on neuronal ultrastructure.
RESULTS： Massive cell death and neurite breakage was observed in the MPP＋ group. In addition, TH expression was significantly reduced, compared to the control group （P 〈 0.01）. After 7 days in culture with MSCs, the co-culture group presented with less cell damage and reduced neurite breakage, and TH expression was increased. However, these changes were not significantly different from the MPP＋ group （P 〈 0.01）. Electron microscopy revealed reduced ultrastructural injury to cells in the brain slices. However, vacuoles were present in cells, with some autophagic vacuoles.
CONCLUSION： Bone marrow-derived MSCs can promote survival of dopaminergic neurons following MPP＋-induced neurotoxicity in co-cultures with substantia nigra and striatum brain slices.     

Response of the sensorimotor cortex of cerebral palsy rats receiving transplantation of vascular endothelial growth factor 165-transfected neural stem cells

Neural stem cells are characterized by the ability to differentiate and stably express exogenous ge- nes. Vascular endothelial growth factor plays a role in protecting local blood vessels and neurons of newborn rats with hypoxic-ischemic encephalopathy. Transplantation of vascular endothelial growth factor-transfected neural stem cells may be neuroprotective in rats with cerebral palsy. In this study, 7-day-old Sprague-Dawley rats were divided into five groups： （1） sham operation （control）, （2） cerebral palsy model alone or with （3） phosphate-buffered saline, （4） vascular en- dothelial growth factor 165 ＋ neural stem cells, or （5） neural stem cells alone. The cerebral palsy model was established by ligating the left common carotid artery followed by exposure to hypox- ia. Phosphate-buffered saline, vascular endothelial growth factor ＋ neural stem cells, and neural stem cells alone were administered into the sensorimotor cortex using the stereotaxic instrument and microsyringe. After transplantation, the radial-arm water maze test and holding test were performed. Immunohistochemistry for vascular endothelial growth factor and histology using hematoxylin-eosin were performed on cerebral cortex. Results revealed that the number of vas- cular endothelial growth factor-positive cells in cerebral palsy rats transplanted with vascular endothelial growth factor-transfected neural stem cells was increased, the time for finding water and the finding repetitions were reduced, the holding time was prolonged, and the degree of cell degeneration or necrosis was reduced. These findings indicate that the transplantation of vascu- lar endothelial growth factor-transfected neural stem cells alleviates brain damage and cognitive deficits, and is neuroprotective in neonatal rats with hypoxia ischemic-mediated cerebral palsy.     

Differentiation of endogenous neural stem cells in adult versus neonatal rats after brachial plexus root avulsion injury

An experimental model of brachial plexus root avulsion injury of cervical dorsal C5-6 was established in adult and neonatal rats.Real-time PCR showed that the levels of brain-derived neurotrophic factor,nerve growth factor and neurotrophin-3 in adult rats increased rapidly 1 day after brachial plexus root avulsion injury,and then gradually decreased to normal levels by 21 days.In neonatal rats,levels of the three neurotrophic factors were decreased on the first day after injury,and then gradually increased from the seventh day and remained at high levels for an extended period of time.We observed that greater neural plasticity contributed to better functional recovery in neonatal rats after brachial plexus root avulsion injury compared with adult rats.Moreover, immunohistochemical staining showed that the number of bromodeoxyuridine/nestin-positive cells increased significantly in the spinal cords of the adult rats compared with neonatal rats after brachial plexus root avulsion injury.In addition,the number of bromodeoxyuridine/glial fibrillary acidic protein-positive cells in adult rats was significantly higher than in neonatal rats 14 and 35 days after brachial plexus injury.Bromodeoxyuridine/β-tubulin-positive cells were not found in either adult or neonatal rats.These results indicate that neural stem cells differentiate mainly into astrocytes after brachial plexus root avulsion injury.Furthermore,the degree of neural stem cell differentiation in neonatal rats was lower than in adult rats.     

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.     

促红细胞生成素干预缺氧缺血性脑损伤新生鼠神经干细胞巢蛋白的表达☆

背景: 巢蛋白是一种存在于神经干细胞的特异性抗原,在神经系统发生病变或损伤引起再生时广泛表达,因此巢蛋白表达常用作判定神经系统发生病变或损伤后能否促进神经再生的一种手段。 目的：从神经再生和神经干细胞激活的角度,探讨外源性促红细胞生成素对新生鼠缺氧缺血性脑损伤后神经干细胞巢蛋白表达的影响。 方法：结扎大鼠右侧颈总动脉和8%低氧暴露2 h制备新生大鼠缺氧缺血性脑损伤模型。对照组仅游离右侧颈总动脉,不予结扎和缺氧处理。干预组大鼠缺氧缺血后立即腹腔注射重组人促红细胞生成素5 000 IU/kg,1次/d,连用3 d。缺氧缺血性脑损伤组大鼠缺氧缺血后连续腹腔注射等量生理盐水溶液3 d。每组随机取8只分别于术后4,7,14 d处死。应用免疫组化方法和计算机图像分析技术检测不同时点海马齿状回巢蛋白标记阳性细胞的变化。 结果与结论：各时点缺氧缺血性脑损伤组巢蛋白阳性细胞数较对照组增加(P < 0.05);各时点干预组巢蛋白阳性细胞较对照组和缺氧缺血性脑损伤组均增加(P < 0.05)。3组大鼠海马齿状回区巢蛋白阳性细胞数均于术后 7 d 达高峰。结果提示早期给予重组人促红细胞生成素可促使新生鼠缺氧缺血性脑损伤后海马齿状回区巢蛋白表达增加,促进神经干细胞的增殖再生,在缺氧缺血性脑损伤后神经再生、修复中发挥一定的保护作用。     

Differential expression of Musashi1 and nestin in the adult rat hippocampus after ischemia

Both nestin and the neural RNA-binding protein Musashi1 (Msi1) are expressed in neural stem cells in the subventricular zone. Neurogenesis in the hippocampus has received much attention, so we evaluated the expression of Msi1 and nestin in the adult rat hippocampus after transient forebrain ischemia. Both Msi1 and nestin were induced in the reactive astrocytes after ischemia, especially in the CA1 region, until 35 days after ischemia. Induction of both molecules suggested that reactive astrocytes might have immature characteristics. In the subgranular zone (SGZ) of the hippocampal dentate gyrus, Msi1-positive cells formed clusters after ischemia. These cells were labeled by bromodeoxyuridine (BrdU) but did not express glial fibrillary acidic protein. In contrast, very few nestin-positive cells were labeled by BrdU. Our results suggest that neuronal progenitor cells in the SGZ expressed Msi1 but not nestin.     

激光调控内源性神经干细胞的增殖分化及脑功能重建**☆

背景: 大量研究表明增强脑内源性神经细胞的增殖能力和自我修复将成为治愈缺血缺氧性脑损伤有价值的方法之一。 目的：观察氦氖激光对新生大鼠缺血缺氧性脑损伤内源性神经干细胞增殖分化及脑功能重建的影响。 方法：7 d龄健康Wistar新生大鼠,建立缺血缺氧性脑损伤模型后第2天开始,激光穴位照射组给予氦氖激光照射。穴位选取顶骨正中的“百会”穴,以及第7颈椎与第1胸椎间、背部正中的“大椎”穴。假手术组和模型组不给予激光照射。于第2疗程结束后,用Y-型迷宫检测各组大鼠的学习记忆能力。随后制备脑海马切片,分别进行巢蛋白和微管关联蛋白2免疫组织化学染色。 结果与结论：①激光穴位照射组大鼠的学习和记忆能力明显高于模型组(P < 0.05),但与假手术组相比,无明显差异(P > 0.05)。②大鼠内源性神经干细胞的表达：与假手术组比较,模型组、激光治疗组齿状回内巢蛋白免疫阳性细胞均明显增多(P < 0.05),且激光治疗组增多幅度大于模型组(P < 0.05)。③神经元特有结构蛋白的表达：激光治疗组大脑皮质微管关联蛋白2表达相当广泛,强阳性染成棕褐色的树突呈条索样、流星样放射状分布,海马各区锥体神经元和齿状回颗粒细胞层神经元排列比较整齐,树突连续阳性染色呈树枝状交叉分布于分子层。假手术组与激光治疗组染色所见无明显差别。模型组微管关联蛋白2表达明显减弱。结果提示激光治疗能够促进缺血缺氧性脑损伤新生大鼠脑内源性神经干细胞增殖,诱导其向神经元方向分化,并达到学习记忆功能的重建。     

Change of neural stem cells in the choroid plexuses of developing rat

Neural stem cells reside in various brain regions. However, neural stem cells in the choroid plexuses are poorly understood. This study was conducted to corroborate the hypotheses that there are neural stem cells in the choroid plexuses, and the change of neural stem cells is age dependent. We examined neural stem cells from rats at postnatal 1, 3, 7 days, 2, 4, 6, and 8 weeks to investigate the distribution and change of the cells in the choroid plexuses. We found nestin-positive cells in the choroid plexuses and these nestin-expressing cells were located principally at the boundary between the choroid plexus epithelium and the underlying connective tissue stroma. Some choroid plexuses of the postnatal 1-, 3-, 7-day, and 2-week rats were stained with line-like markers of nestin. We also observed nestin-positive cells in 4-week rats, but no such cells were detected in the 6- and 8-week rats. These findings indicate that neural stem cells exist in the rat choroid plexuses, and the change of neural stem cells is age-dependent.     

缺血缺氧新生大鼠脑内原位神经干细胞的增殖与分化：可通过针刺任脉、督脉及膀胱经实现？

[摘要] 目的 检测针刺任脉、督脉及膀胱经对新生儿缺血缺氧性脑病模型鼠脑内神经干细胞的影响,分析针刺诱导神经干细胞增殖、分化的机制,为临床针刺治疗新生儿缺血缺氧性脑病提供新的细胞学理论依据。方法 新生7天SD大鼠结扎左侧颈总动脉并缺氧2小时制作新生鼠缺血缺氧性脑病模型。实验动物共分三组：针刺组、对照组和正常组。针刺组每天针刺任脉、督脉及膀胱经治疗一次。对照组及正常组不作针刺处理。各组动物每天两次腹腔注射Brdu用于标记脑内神经干细胞增殖情况。分别于模型建立后3d、7d、14d和28d取脑组织行抗Brdu的免疫组化染色,并于模型建立后40d行免疫荧光双标,分别观察各组动物海马及皮层Brdu阳性细胞数目、形态、分布以及分化情况; 并比较他们之间的差异。结果 抗Brdu的免疫组化染色显示针刺任脉、督脉及膀胱经治疗第3天及第7天时针刺组动物皮层及海马的Brdu阳性细胞数目和对照组相比,差别无统计学意义;针刺治疗第14天及第28天时针刺组动物皮层及海马的Brdu阳性细胞数目明显比对照组多,差别有统计学意义。针刺后第40天免疫荧光双标显示大部分Brdu阳性细胞和神经元标记物NSE共存,少部分和星形胶质细胞标记物GFAP共存。结论 针刺任脉、督脉及膀胱经能促进HIE模型鼠皮层及海马神经干细胞的增殖潜能;针刺任脉、督脉及膀胱经治疗后新生的神经干细胞大部分分化为神经元,提示针刺后新生的神经细胞有可能有效地补充在缺血缺氧中丧失的神经元,并能促进HIE动物功能的恢复。     

新生大鼠全大脑组织分离诱导分化神经干细胞的体外实验

背景：神经干细胞的供体一般以胎鼠和成年鼠为主，利用细胞培养技术分离步骤较繁琐。 目的：以新生大鼠为神经干细胞供体，拟建立一种较为简便、细胞获得率较高的分离培养方法。 设计、时间及地点：以细胞为对象观察性实验，于2006-10/2007-03在重庆医科大学完成。 材料：新生1~3 d 的Wistar大鼠全大脑。 方法：以胰蛋白酶消化、无血清、悬浮培养原代细胞，并加含体积分数为0.10胎牛血清的DMEM/F12培养液诱导其分化。 主要观察指标：应用相差显微镜观察神经干细胞的生长特点及分化后的细胞形态学变化。应用间接免疫细胞化学染色法鉴定神经干细胞及其分化后神经元和胶质细胞标志蛋白的表达。以BrdU标记神经干细胞，观察其增殖情况。 结果：新生大鼠脑组织分离的细胞具有连续传代和增殖的能力，能稳定表达神经干细胞特异性巢蛋白。诱导分化后的细胞能表达神经元细胞、星形胶质细胞、少突胶质细胞的特异性蛋白。 结论：从新生大鼠脑组织分离培养出的神经干细胞获得率高，保持了干细胞的未分化属性，具有自我更新和多项分化潜能。     

Effect of valproic acid on endogenous neural stem cell proliferation in a rat model of spinal cord injury

BACKGROUND: Valproic acid has been reported to decrease apoptosis, promote neuronal differentiation of brain-derived neural stem cells, and inhibit glial differentiation of brain-derived neural stem cells.OBJECTIVE: To investigate the effects of valproic acid on proliferation of endogenous neural sterm cells in a rat model of spinal cord injury.DESIGN, TIME AND SETTING: A randomized, controlled, neuropathological study was performed at Key Laboratory of Trauma, Buming, and Combined Injury, Research Institute of Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA between November 2005 and February 2007.MATERIALS: A total of 45 adult, Wistar rats were randomly divided into sham surgery (n=5), injury(n=20), and valproic acid (n=20) groups. Valproic acid was provided by Sigma, USA.METHODS: Injury was induced to the T10 segment in the injury and valproic acid groups using the metal weight-dropping method. The spinal cord was exposed without contusion in the sham surgery group. Rats in the valproic acid group were intraperitoneally injected with 150 mg/kg valproic acid every 12 hours (twice in total).MAIN OUTCOME MEASURES: Nestin expression (5 mm from injured center) was detected using immunohistochemistry at 1, 3 days, 1, 4, and 8 weeks post-injury.RESULTS: Low expression of nestin was observed in the cytoplasm, but rarely in the white matter of the spinal cord in the sham surgery group. In the injury group, nestin expression was observed in the ependyma and pia mater one day after injury, and expression reached a peak at 1 week (P<0.05).Expression was primarily observed in the ependymal cells, which expanded towards the white and gray matter of the spinal cord. Nestin expression rapidly decreased by 4 weeks post-injury, and had almost completely disappeared by 8 weeks. At 24 hours after spinal cord injury, there was nosignificant difference in nestin expression between the valproic acid and injury groups. At 1 week,there was a significant increase in the number of nestin-positive cells surrounding the central canal in valproic acid group compared with the injury group (P<0.05). Expression reached a peak by 4 weeks, and it was still present at 8 weeks.CONCLUSION: Valproic acid promoted endogenous neural stem cell proliferation following spinal cord injury in rats.