Oligodendrocyte transcription factor 1 mRNA and protein expression in organotypic rat brain slices

Numerous studies have confirmed that oligodendrocyte transcription factor 1 （Olig-1） is vital for myelin repair. However, the effects of hypoxia and ischemia on Olig-1 expression remain unknown. In this study, Olig-1 mRNA and protein expressions were analyzed by in situ hybridization and immunohistochemistry, to determine the expression profile of Olig-1 in rat brain slices exposed to hypoxia and ischemia. Brains were obtained from 2-day-old Sprague-Dawley rats, and sections were randomly assigned to control and hypoxia/ischemia groups. Hematoxylin-eosin staining revealed karyorrhexis and karyopyknosis in cells from the hypoxia/ischemia group. Under electron microscopy, mitochondria swelling and neuropil edema were observed in the hypoxiaJischemia group. Olig-1 mRNA and protein expressions were increased at 1 day after hypoxia and ischemia treatment. These results suggest that in situ hybridization and immunohistochemistry could be used simultaneously to detect mRNA and protein expression in brain slices.

Expression of hypoxia-inducible factor 1 alpha and oligodendrocyte lineage gene-1 in cultured brain slices after oxygen-glucose deprivation

Oligodendrocyte lineage gene-1 expressed in oligodendrocytes may trigger the repair of neuronal myelin impairment, and play a crucial role in myelin repair. Hypoxia-inducible factor la, a transcription factor, is of great significance in premature infants with hypoxic-ischemic brain damage There is little evidence of direct regulatory effects of hypoxia-inducible factor le on oligodendrocyte lineage gene-l. In this study, brain slices of Sprague-Dawley rats were cultured and subjected to oxygen-glucose deprivation. Then, slices were transfected with hypoxia-inducible factor la or oligodendrocyte lineage gene-1. The expression levels of hypoxia-inducible factor la and oligodendrocyte lineage gene-1 were significantly up-regulated in rat brains prior to transfection, as detected by immunohistochemical staining. Eight hours after transfection of slices with hypoxia-inducible factor la, oligodendrocyte lineage gene-1 expression was upregulated, and reached a peak 24 hours after transfection. Oligodendrocyte lineage gene-1 transfection induced no significant differences in hypoxia-inducible factor la levels in rat brain tissues with oxygen-glucose deprivation. These experimental findings indicate that hypoxia-inducible factor la can regulate oligodendrocyte lineage gene-1 expression in hypoxic brain tissue, thus repairing the neural impairment.

Protective effect of bone marrow-derived mesenchymal stem cells on dopaminergic neurons against 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in rat brain slices

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.

Protective effects of costunolide on mouse brain slice injury induced by oxygen glucose deprivation/reoxygenation

OBJECTIVE To investigate the protective effects and mechanisms of costunolide against mousebrain slice injury induced by oxygen-glucose deprivation/reoxygenation(OGD/R).METHODS Mouse brain slice injury was induced by OGD/R in vitro,and the degree ofinjury was evaluated by measuring the release of lactate dehydrogenase(LDH)and 2,3,5-triphenyltetrazolium chloride(TTC)staining.Western blotting was used to analyze the expression of Bax,Bcl-2,Cyt-c,caspase-9,caspase-7 and caspase-3.RESULTS Compared with OGD/R,1,5,and 10μmol·L^-1 costu⁃nolide decreased the LDH levels,increased the TTC staining intensity,inhibited Bax,Cyt-c,caspase-9,caspase-7,caspase-3 expression levels,and enhanced Bcl-2 expression level.CONCLUSION Costunolide has latent neuroprotective activi⁃ties by the regulation of apoptosis via the mitochondrial apoptosis pathway.

Inhibitory effect of morphine on excitatory synaptic transmission via presynaptic mechanism in rat SON neurons in brain slices

Objective: To observe the effects of morphine on the excitatory postsynaptic currents (EPSCs) and miniature EPSCs (mEPSCs) in rat supraoptic nucleus (SON) neurons and to explore its synaptic mechanism. Methods: Using whole-cell voltage-clamp recording technique in the brain slices, the EPSCS and mEPSCs of rat SON neurons were recorded, respectively. Results: Morphine (20μmol/L) decreased the frequency of EPSCs and mEPSCs (by 65% for EPSCS and by 45% for mEPSCs), and reduced the amplitude of EPSCs by 44% in all SON neurons, but the amplitude distribution of mEPSCs was not affected. Conclusion: Morphine inhibits the excitatory transmissions via presynaptic mechanisms in SON neurons from rat brain slices.

L-Serine Decreases Taurine Concentration in the Extracellular Fluid of Brain Slices

L-Serine is considered a functional amino acid in the central nervous system, and induces sedation and hypnotic effects in some animal models of acute and chronic stress. Accordingly, while L-serine is a candidate anti-stress factor, the central mechanism of L-serine is not clear. The present study clarifies the action of L-serine using acute chick brain slices. We investigated the changes in some extracellular fluid amino acid concentrations in response to L-serine perfusion. Taurine concentration decreased while L-alanine concentration increased following L-serine perfusion. To examine the involvement of the taurine transporter, the effect of L-serine on the taurine concentration in the presence and absence of Na+ was also investigated. Na+ had no effect on taurine concentration induced by L-serine perfusion. These results suggest that L-serine has an ability to promote L-alanine synthesis facilitating the catabolism of taurine. In conclusion, L-serine modifies the metabolism of taurine and L-alanine in the extracellular space in chick brain.

Human Brain Slice Culture: A Useful Tool to Study Brain Disorders and Potential Therapeutic Compounds

Investigating the pathophysiological mechanisms underlying brain disorders is a priority if novel therapeutic strategies are to be developed. In vivo studies of animal models and in vitro studies of cell lines/primary cell cultures may provide useful tools to study certain aspects of brain disorders. However, discrepancies among these studies or unsuccessful translation from animal/cell studies to human/clinical studies often occur, because these models generally represent only some symptoms of a neuropsychiatric disorder rather than the complete disorder. Human brain slice cultures from postmortem tissue or resected tissue from operations have shown that, in vitro, neurons and glia can stay alive for long periods of time, while their morphological and physiological characteristics, and their ability to respond to experimental manipulations are maintained. Human brain slices can thus provide a close representation of neuronal networks in vivo, be a valuable tool for investigation of the basis of neuropsychiatric disorders, and provide a platform for the evaluation of novel pharmacological treatments of human brain diseases.A brain bank needs to provide the necessary infrastructure to bring together donors, hospitals, and researchers who want to investigate human brain slices in cultures of clinically and neuropathologically well-documented material.

Negative regulation of miRNA-9 on oligodendrocyte lineage gene 1 during hypoxic-ischemic brain damage

Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair, miRNA-9 is involved in the occurrence of many related neurological disorders. Bioin- formatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups： control group; oxygen-glucose deprivation group （treatment with 8% O2 ＋ 92% N2 and sugar-free medium for 60 minutes）; transfection control group （after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid） and miRNA-9 transfection group （after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid）. From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligoden- drocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.

Preferential Transport and Metabolism of Glucose in Bergmann Glia over Purkinje Cells:A Multiphoton Study of Cerebellar Slices

了解不同类型的细胞如何处理葡萄糖有助于解释能量供应是如何是如何根据大脑能量需求来进行调整的。荧光追踪结合共聚焦显微镜技术已用于研究培养的脑细胞摄取葡萄糖的实时动态过程。本文采用这种技术利用多光子显微镜观察急性制备的大鼠小脑脑片。带荧光的葡萄糖类似物2NBDG和6NBDG在小脑皮质的分子层中的转运速度比其在蒲肯野细胞胞体和颗粒细胞中快若干倍。洗脱游离示踪剂后,可见大部分磷酸化示踪剂都位于Bergmann胶质细胞,用胶质细胞标记物sulforhodamine 101免疫染色后进一步确认这一结果。有效回收荧光光漂白后显示,2NBDG-P可通过Bergmann胶质细胞之间的缝隙连接沿着分子层水平扩散。本文的结果表明在急性小脑切片中,Bergmann胶质细胞对葡萄糖的转运能力和糖酵解率高于蒲肯野细胞若干倍。由于小脑主要由葡萄糖提供能量,蒲肯野神经元被认为比Bergmann胶质细胞更耗能量,这些结果表明,在胶质细胞和神经元之间存在类似乳酸的能量代谢物介导的环路。

多层螺旋CT冠脉造影在心脏血管狭窄中的应用价值

目的探讨多层螺旋CT冠脉造影(CTA)在心脏血管狭窄中的诊断情况。方法收集2019年3月至2022年5月在本院进行CTA、数字减影全脑血管造影术(DSA)检查的患者86例,以DSA检查为金标准,统计CTA检查技术的诊断效能。结果经CTA技术检查后,灵敏度为92%、特异度为81.82%、阳性预测值为97.18%、阴性预测值为60.00%、准确度为90.70%。结论CTA在心脏血管狭窄具有较好的诊断效能,值得在临床上推广应用。

Training stem cells for treatment of malignant brain tumors

The treatment of malignant brain tumors remains a challenge. Stem cell technology has been applied in the treatment of brain tumors largely because of the ability of some stem cells to infiltrate into regions within the brain where tumor cells migrate as shown in preclinical studies. However, not all of these efforts can translate in the effective treatment that improves the quality of life for pa-tients. Here, we perform a literature review to identify the problems in the field. Given the lack of efficacy of most stem cell-based agents used in the treatment of malignant brain tumors, we found that stem cell distribution(i.e., only a fraction of stem cells applied capable of targeting tumors) are among the limiting factors. We provide guidelines for potential improvements in stem cell distribution. Specifically, we use an engineered tissue graft platform that replicates the in vivo microenvironment, and provide our data to validate that this culture platform is viable for producing stem cells that have better stem cell distribution than with the Petri dish culture system.

氯化胆碱和蔗糖切片液制作的小鼠脑片下丘脑视前区神经元电生理特性的比较

目的比较不同切片液对小鼠下丘脑视前区(mPOA)脑片神经元电生理特性的影响。方法选取6~8周龄C57雌性小鼠,异氟烷麻醉后心脏灌注(通混合气950 mL/L O_(2),50 mL/L CO_(2))4℃氯化胆碱切片液或蔗糖切片液后取脑,振动切片机制备含mPOA脑区的冠状脑片,全细胞膜片钳记录mPOA电生理特性。结果采用蔗糖切片液制作的脑片mPOA神经元的内在兴奋性要优于氯化胆碱切片液,具体表现为细胞静息膜电位更超极化,动作电位幅度、不同电流刺激平均放电幅度、放电个数均高,半宽、基强度较小。而氯化胆碱切片液制作的脑片突触活性要更好,表现为兴奋性微小突触后电流频率高于蔗糖切片液制作脑片的细胞。结论蔗糖切片液和氯化胆碱切片液在保证细胞活性的情况下,蔗糖切片液更适合记录mPOA神经元的内在兴奋性,而氯化胆碱切片液更适合突触特性的记录。

不同病理分级脑胶质瘤患者多层螺旋CT灌注成像参数的差异及临床意义

目的 探讨不同病理分级脑胶质瘤患者多层螺旋CT灌注成像(MSCTPI)参数[血流量(BF)、血容量(BV)、表面渗透性(PS)]的差异及临床意义。方法 将103例脑胶质瘤患者作为脑胶质瘤组,82例行去骨瓣减压手术的颅脑外伤患者作为对照组,两组患者均行MSCTPI检查,比较两组患者及不同病理分级脑胶质瘤患者的BF、BV、PS值,并分析BF、BV、PS值与脑胶质瘤病理分级的相关性。结果 脑胶质瘤组患者BF、BV、PS值均明显高于对照组,差异均有统计学意义(P﹤0.01)。随着脑胶质瘤患者病理分级的升高,BF、BV、PS值均逐渐升高,差异均有统计学意义(P﹤0.05)。经Pearson相关分析发现,BF、BV、PS值与脑胶质瘤病理分级均呈正相关(P﹤0.01)。结论 不同病理分级脑胶质瘤患者的MSCTPI参数存在较大差异,且BF、BV、PS值与脑胶质瘤病理分级均呈正相关,对该疾病的诊疗有较高的指导意义。

MRI自动定位技术在脑转移瘤中的应用

目的:评估MRI自动定位技术用于检测脑转移瘤的诊断效能,并与传统的手动定位方法进行比较.方法:连续性纳入32例确诊的脑转移瘤患者,他们均在2种不同的MRI扫描仪上进行颅脑增强MRI检查,即配备颅脑自动定位技术的Siemens Aera(1.5 T)扫描仪和传统手工定位的GE 750 W扫描仪(3.0 T).将Siemens Aera和GE 750 W扫描仪获得的颅脑增强MRI图像分别设为A组和B组.计算2组的诊断效能和诊断信心.结果:对于A组,脑转移瘤阳性数目的诊断灵敏度为87.4%(153/175),对照既往的MRI增强检查后,A组的诊断灵敏度提高到98.3%(172/175),且差异具有统计学意义(P<0.05).对于B组,相应的诊断灵敏度分别为88.9%(112/126)和99.2%(125/126),同样差异具有统计学意义(P<0.05).A组和B组之间的诊断灵敏度差异无统计学意义(P>0.05).A组和B组的平均诊断置信度分别为3.9和3.8(P>0.05).结论:本研究初步表明参照既往影像检查可以提高检测脑转移瘤的诊断灵敏度.MRI自动定位技术有助于快速发现病变,并且与传统手动定位方法相比,这种技术不会牺牲诊断灵敏度和诊断信心.

多排螺旋CT诊断新生儿低血糖脑损伤的效果研究

目的:研究观察多排螺旋CT诊断新生儿低血糖脑损伤的临床效果。方法:选取郑州儿童医院于2021年1月至2022年2月期间收治的40例新生儿低血糖脑损伤患儿进行研究。患儿均接受多排螺旋CT检查以及脑电图检查,观察并记录成像情况后分析多排螺旋CT检查对新生儿低血糖脑损伤的诊断效果(以脑电图检查作为诊断金标准)。结果:用多排螺旋CT诊断这些患儿病情的准确率为95.00%(38/40)。进行多排螺旋CT检查的结果显示,这些患儿中脑内损伤位置为颞叶的患儿有7例,为顶枕叶的患儿有15例,为额叶的患儿有11例,为额颞叶的患儿有4例。多排螺旋CT检查与脑电图检查检出脑内损伤位置的分布情况相比,差异无统计学意义(P>0.05)。结论:多排螺旋CT在新生儿低血糖脑损伤诊断中的应用价值较高,具有诊断效率高、应用安全性高、操作简单方便、扫描速度快、成像结果清晰等优点,能够为临床干预新生儿低血糖脑损伤提供依据,进而可减少患儿因治疗不及时产生的脑瘫、智力低下等后遗症,改善其预后。

大鼠脑片损伤模型和新型定量评价方法的建立

目的 建立不同性质脑损伤实验模型和一种方便、快速、灵敏的定量评价方法。方法 制备大鼠皮层和海马脑片 ,检测活性后 ,分别接受缺氧缺糖 (OGD)、谷氨酸、过氧化氢 (H2 O2 )损伤 ,氯胺酮、D AP5、异丙酚预处理 ,随后与TTC溶液共同孵育 ,有机溶剂抽提 ,酶标仪测定OD4 90 值 ,同时测定孵育上清液乳酸脱氢酶 (LDH)释放率。结果 室温下恢复孵育 90min后 ,海马脑片CA1区锥体细胞层可记录到群体峰电位。随着OGD时间延长 ,皮层和海马脑片TTC染色明显降低 ,孵育上清液LDH释放率逐渐增加 ,组织损伤百分率与LDH释放率明显正相关 :皮层r =0 960 9,P <0 0 1 ;海马r =0 892 1 ,P <0 0 5。和对照组相比 ,谷氨酸 1mmol·L- 1 和H2 O2 2mmol·L- 1 明显降低脑片TTC染色。氯胺酮5μmol·L- 1 、D AP550 μmol·L- 1 、异丙酚 5μmol·L- 1 分别抑制OGD 1 0min、谷氨酸、H2 O2 损伤所致脑片TTC染色降低。结论 利用大鼠脑片建立的缺氧缺糖、谷氨酸和过氧化氢损伤实验模型 ,以及新鲜脑片与TTC溶液共同孵育 ,有机溶剂抽提、比色的定量评价方法具有方便、快速、灵敏的特点 。

不同培养条件对小鼠诱导性多能干细胞分化为神经元样细胞的影响

目的研究小鼠诱导性多能干细胞(i PSC)在不同培养条件诱导下向神经元样细胞分化的能力。方法将小鼠i PSC悬浮培养形成拟胚体,然后随机分为全反式维甲酸(ATRA)组、脑片共培养组和脑组织匀浆上清组,将其诱导分化成神经元样细胞。倒置显微镜下观察细胞形态变化;免疫荧光染色技术对其进行鉴定分析;Western blot法检测巢蛋白(nestin)、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)的表达。结果小鼠i PSC在不同培养条件下都能够向神经元样细胞分化,这些神经元样细胞可以被神经细胞标志物nestin、MAP2标记;ATRA组的nestin、MAP2、GFAP蛋白相对表达量明显高于脑片共培养组和脑匀浆上清诱导组,但脑片共培养组和脑匀浆上清诱导组间无显著性差异。结论脑片微环境和脑组织匀浆上清均可诱导小鼠i PS细胞向神经元样细胞分化,但效果不及ATRA组。

图像分析定量测量脑片体积方法的改良

目的 :改良计算机图像分析法测量大鼠和小鼠脑片的方法 ,并验证与经典方法的相关性以及在评价局灶性脑缺血中的应用。方法 :分别采用测重法、密度法精确测出大鼠、小鼠的脑片体积 ;以图像分析测定脑片面积 ,乘以校正后的脑片厚度 ,得到脑片两侧及左右侧体积数据 ;另对局灶性脑缺血小鼠脑片体积变化 ,以图像分析法测定左右侧脑体积。结果 :图像分析法对测量不同大小的面积有良好的线性 (r=1.0 0 0 ) ;脑片精确测量值 (密度法 )与图像分析测量值之间有良好的相关性 ,在大鼠 r=0 .80 9(n=4 5 ,P<0 .0 0 1) ,在小鼠 r=0 .84 4(n=74 ,P<0 .0 0 1) ;小鼠脑缺血 6、2 4 h后缺血侧体积明显增加。结论 :改良的计算机图像分析法可较为准确地反映脑片体积。

大鼠下丘脑离体脑薄片视上核神经元的全细胞记录

在大鼠下丘脑薄片标本上对52例视上核神经元进行了全细胞膜片箝记录。膜被动及主动电生理参数测量如下：静息电位，59±8mV；输入阻抗，535±129MΩ；时间常数，32±9ms；动作电位幅度，99±11mV；超射值，37±13mV（n＝39）。大多数神经元在接受去极化刺激时出现明显的慢后超极化电位或电流。我们发现，在电压箱状态下几乎所有的视上核神经元均接受兴奋性和／或抑制性突触传λ（n=13）。药理学实验表明，兴奋性突触后电流是由non－NMDA亚型谷氨酸受体介导，而抑制性突触后电流由GABAA受体介导。

不同浓度芬太尼对大鼠脑片缺氧缺糖损伤的保护作用

目的探讨高浓度芬太尼对大鼠脑片缺氧缺糖损伤的保护作用及其机制。方法建立大鼠脑片缺氧缺糖损伤模型,设立对照组(Control)、缺氧缺糖损伤组(OGD)、芬太尼50μg.L-1组(F50)、芬太尼500μg.L-1组(F500)。利用2,3,5-三苯基氯化四氮唑(TTC)染色定量比色、乳酸脱氢酶(LDH)、免疫组化、电镜评价高浓度芬太尼对脑损伤的保护作用。同时激光共聚焦显微镜测定脑片胞内钙变化。结果不同浓度芬太尼(50、500μg.L-1)抑制脑片OGD损伤所致的TTC染色降低;减少LDH释放;减轻神经元凋亡,改善神经元超微结构的病理损伤。OGD损伤增加bax和bc l-2蛋白表达,增加胞内钙离子浓度。不同浓度芬太尼(50、500μg.L-1)进一步上调bc l-2蛋白表达,降低bax蛋白表达,同时抑制胞内钙离子浓度。与芬太尼500μg.L-1相比,芬太尼50μg.L-1作用较强。结论高浓度芬太尼具有脑保护作用,能够抑制缺氧缺糖损伤导致的大鼠脑片神经元损伤及其凋亡过程,且芬太尼对于胞内钙离子的调控可能是其发挥保护作用的重要机制之一。随着芬太尼的剂量增加,其保护作用减弱,但在临床应用的剂量范围内未见明显的毒性作用。