An enriched environment increases noradrenaline concentration in the mouse brain

Exposure to an enriched environment has been shown to have many positive effects on brain structure and function. In the present study, we examined the effects of environmental enrichment on monoaminergic neurons in the mouse brain. After being exposed to an enriched environment for 40 days, noradrenaline content was increased significantly in the parieto-temporo-occipital cortex, the cerebellum and the pons/medulla oblongata. In contrast, no changes were observed in serotonin or dopamine levels in these same regions.     

高效制备昆明鼠胚胎成纤维细胞饲养层

背景：小鼠胚胎成纤维细胞作为饲养层是胚胎干细胞培养最常用的方法,能有效抑制胚胎干细胞分化并促进其增殖,但其制备过程繁琐,工作量大,准备周期长。 目的：探索建立小鼠胚胎成纤维细胞饲养层简单、高效的培养体系。 方法：取13.5 d胎龄胎鼠用改良组织块法及简化酶消化法分离培养原代成纤维细胞,倒置显微镜下观察不同方法培养的原代和传代鼠胚胎成纤维细胞的生长形态、结构及细胞数量变化。收集鼠胚胎成纤维细胞进行冻存,复苏后细胞经不同浓度作用时间的丝裂霉素C处理,制备饲养层。 结果与结论：两种简化方法培养的原代鼠胚胎成纤维细胞生长状态良好,得到高效优质足量细胞,操作过程简单,省去了多次消化、离心、细胞计数等繁琐操作,均适宜于鼠胚胎成纤维细胞的原代培养。简化复苏法复苏后的细胞,按其生长汇合情况,以丝裂霉素C 10 mg/L作用1.5~2.0 h或1 mg/L培养过夜,省时并可获得细胞生长状态最佳的饲养层。     

Phospholipase A2 activity in rat embryonic brain and in primary cultures of cortical neurons

Summary. We investigated, with the aid of a radio-enzymatic method, the developmental changes in the activity of 85 kDa calcium-dependent cytosolic phospholipase A₂ (cPLA₂) in pre-term embryonic rat brain and in primary cultures of cortical neurons. PLA₂ activity was highest in the brains of 18 day embryos (E18) and gradually decreased toward birth (E18 through E21). No significant differences were found regarding cPLA₂ activity in different topographies, namely hippocampus, cortex, and total brain. In primary cultures of E18/E19 cortical neurons, cPLA₂ activity was higher than the respective embryonic tissue activity, straight from early development. In vitro cPLA₂ activity peaked on the 4^th day in culture, and decreased after the six days of incubation, when the cultures became mature. This result reinforces previous evidence that cPLA₂ plays an important role in the early development of the nervous system, and further suggests that it may be implicated in neurodevelopmental processes and in in vitro neuronal survival. We believe that we have produced a useful model for the tissue culture study of PLA₂ metabolism and biochemistry, which can be further addressed to the investigation of the biology of neurodegenerative and neuropsychiatric disorders. Received December 26, 2001; accepted January 23, 2002     

睾丸支持细胞对体外培养鼠胎脑皮质细胞的影响

背景：目前绝大多数研究者将目标指向功能细胞，为改善细胞的生存环境而给予一些生长因子。睾丸支持细胞能分泌大量营养物质及生长因子，且其所具有的Fas配体可降低移植入体内后宿主的免疫排斥。 目的：探讨睾丸支持细胞对体外培养鼠胚胎大脑皮质细胞的影响。 设计、时间及地点：细胞学体外观察实验，于2005-10/2007-09在首都医科大学生殖医学研究中心实验室完成。 材料：健康SPF级10~15 d龄雄性ICR小鼠40只，用于提取睾丸支持细胞；15 d龄胎鼠40只，用于提取大脑皮质细胞。 方法：将分离的大脑皮质细胞密度调整为2×109 L-1，分为2组：实验组接种于预铺有70%汇合传代的睾丸支持细胞培养瓶中，加入含体积分数为0.1的FBS、1×105 U/L青霉素、0.01 g/L链霉素的DMEM/F12完全培养基，置于37 ℃、体积分数为0.05的CO2饱和湿度环境下培养17 d；对照组接种于预铺有0.05%多聚右旋赖氨酸的培养瓶中单独培养。 主要观察指标：采用免疫组织化学法、免疫细胞化学法、免疫细胞荧光法进行细胞鉴定，检测神经元、神经球数量及酪氨酸羟化酶阳性率。 结果：两组细胞神经元特异性烯醇化酶、巢蛋白、酪氨酸羟化酶均呈阳性表达。与培养2 h比较，随培养时间的延长两组贴壁神经元数均明显减少（P < 0.01），但实验组各时间点贴壁神经元数均明显多于对照组（P < 0.01）。实验组于培养3 d出现神经球，数量多且变化较快，7 d后开始呈现明显的下降趋势，至17 d神经球接近消失；对照组仅可见散在巢蛋白阳性表达细胞，未出现神经球。实验组酪氨酸羟化酶阳性细胞率随培养时间的延长而逐渐升高，至11 d达高峰，且各培养时间点酪氨酸羟化酶阳性细胞率均高于对照组（P < 0.01）。 结论：睾丸支持细胞可有效促进鼠胎脑皮质细胞的贴壁及生长，在促进神经干细胞增殖的同时诱导其向多巴胺能神经元分化。     

A method for the collection of defined areas from the embryonic rat brain for cell and tissue culture

Tissue for the culture of cerebral neurons has frequently been taken from brains of the embryonic rats. In many cases it was impossible to obtain regularly and reproducibly small, defined pieces of tissue, e.g. diencephalic nuclei, of the extremely soft embryonic cerebral tissue. On the other hand, as a basis for tissue cultures, well defined samples are more and more considered essential. Therefore a method for collecting samples of defined small regions from the di- and mesencephalic rat brain, 17 days of gestation, was developed. It is applicable to cell and tissue culture. Embryonic brains are prepared aseptically and embedded in congealing Agarose. Stabilized in this gel they are cut into 225 micron slices using a tissue chopper. Tissue samples desired for culture are then punched out from the respective brain slice, which previously had been compared with corresponding reference micrographs. The correct localization of the tissue punch within the fresh brain slice is controlled histologically. The embedding procedure for performing the histology of the brain slices of 225 micron width is described. For application of the introduced method, coordinates for slicing and histological reference micrographs are given for di- and mesencephalic areas.     

Subtle sex differences in vasopressin mRNA expression in the embryonic mouse brain

Arginine vasopressin (AVP) is a neuropeptide which acts centrally to modulate numerous social behaviors. One receptor subtype through which these effects occur is the AVP 1a receptor (AVPR1A). The modulatory effects of Avp via the AVPR1A varies by species as well as sex, since both AVP and the AVPR1A tend to be expressed more prominently in males. Beyond these neuromodulatory effects there are also indications that the AVP system may play a role in early development to, in part, organize sex‐specific neural circuitry that is important to sexually dimorphic social behaviors in adulthood. However, to date, AVP's role in early development is poorly understood, particularly with respect to its differential effect on males and females. In order to determine the timing and distribution of the AVP system in early brain development, we examined the brains of male and female C57BL/6J mice between embryonic day (E) 12.5 and postnatal day (P) 2 and quantified Avp and Avpr1a mRNA using qPCR and AVPR1A protein using receptor autoradiography. The mRNA for Avp was measurable in males and females starting at E14.5, with males producing more than females, while Avpr1a mRNA was found as early as E12.5, with no difference in expression between sexes. AVPR1A binding was observed in both sexes starting at E16.5, and while there were no observed sex differences, binding density and the number of neuroanatomical areas did increase over time. These data are significant as they provide the first whole‐brain characterization of the vasopressin system in the embryonic mouse. Further, these findings are consistent with data from other species, that have documented a sex difference in the vasopressin system during early brain formation.     

Insulin dependent neurite outgrowth in cultured embryonic mouse brain cells

Insulin is shown to promote neuronal regeneration in cultures of cells dissociated from embryonic mouse brain. The cells grown in the absence of serum extend neurites upon exposure to insulin. Insulin also induces ornithine decarboxylase activity in these cultures.     

Accelerated differentiation of oligodendrocytes in neuronal-rich embryonic mouse brain cell cultures

The expression of two oligodendroglial markers, galactocerebroside (GC) and myelin basic protein (MBP), was studied in brain cell cultures (BCC) from 14-day-old mouse embryos by immunocytochemical methods. The presence of neurons and astrocytes was also investigated. Results show that oligodendrocytes simultaneously express both GC and MBP already at 7 days in vitro. These cultures are rich in neurons, and the astrocyte layer is also well represented. A comparison is made between these data and those previously obtained by the use of newborn mouse brain cell cultures, which are very poor in neurons. The differentiation of oligodendrocytes, as reflected in the expression of MBP, is accelerated in embryonic mouse BCC when compared to neonatal mouse BCC. We therefore speculate that neurons are involved in the enhancement of the ability of oligodendrocytes to express myelin related components in culture.     

Selection of differentiated neurons in embryonic mouse brain cultures using arabinofuranosylcytosine

Treatment of primary mouse embryonic brain cultures with arabinofuranosylcytosine (AraC) results in selective survival of differentiated neurons. These neurons are nonreplicating and are electrically active. Selection of neurons with AraC is superior to that with fluorodeoxyuridine and provides neuron-enriched cultures suitable for a variety of studies of neuron function in vitro.     

Dendritic cells and dendritic-like microglia in focal cortical ischemia of the mouse brain

Intracerebral dendritic cells (DC) have recently been identified in neuroinflammation initiated peripherally by brain-targeted autoimmunity or infection. The present study detects DC in photochemically induced cortical ischemia of the mouse brain, a brain-intrinsic lesion model characterized by the lack of an overt T cell response. Concomitant to leukocyte infiltration of the infarcted area, cells expressing the pan-DC surface marker CD11c appeared at the lesion and persisted for weeks. These DC were located at the border zone of the infarct and remote from the lesion in degenerating corticothalamic fibre tracts and subcortical nuclei. All CD11c+ brain cells displayed a uniform CD11b+/CD8alpha-/CD205- surface phenotype, indicating a myeloid origin, and were immature DC based on their MHC class II+/CD40-/CD80+/CD86+/- profile. By expressing high levels of CD45, most DC from ischemic brain seemed to be blood-derived while a minority were CD45(low), thus corresponding to resident microglia. Consistently, round-shaped CD11c+ cells were found at the lesion whereas CD11c+ cells at subcortical sites were ramified like parenchymal microglia. These findings evidence a recruitment of myeloid DC to ischemic brain lesions and suggest that reactive microglia in remote areas transform into dendritic-like cells. Brain-infiltrating DC and their microglial counterparts may play a role in the inflammatory response to cerebral ischemia independently of T cells.     

Genetic inactivation of adenosine A2A receptors attenuates acute traumatic brain injury in the mouse cortical impact model

The inactivation of the A_2A receptor (A_2AR) has been shown to neuroprotect against brain injury in several animal models of neurological disorders including stroke and Parkinson's disease. However, despite marked elevation of adenosine level, the role of the A_2A in traumatic brain injury (TBI) remains unclear. In the present study, we investigated the effects of genetic inactivation of A_2ARs in the acute stage. The A_2AR knock-out (KO) mice and their wild-type (WT) littermates were subjected to cortical impact injury by a dropping weight. The control group was only craniotomized without TBI. At 24 h post-TBI, the neurological deficit scores of the KO mice were significantly lower than that of WT littermates. Consistent with the behavioral changes, the brain water contents as well as histological changes and the TUNEL-positive cells of the injured cortex of the KO mice were significantly lower than that of WT littermates. Furthermore, the glutamate level in the cerebral spinal fluid (CSF) of the KO mice was also significantly lower than that of WT littermates. In addition, we found that at 12 h post-TBI the mRNA and protein levels of TNF-α and IL-1β were higher in the KO mice than that in the WT littermates. However, at 24 h post-TBI, the level of TNF-α and IL-1β continually increased in the WT mice but largely declined in the KO mice. These results suggest that the genetic inactivation of A_2AR protects against TBI, which is mainly associated with the suppression of glutamate level.     

Cellular development and myelin production in primary cultures of embryonic mouse brain

The development of cell cultures from embryonic mouse cerebral hemispheres has been followed in detail for periods up to 40 days in culture using a variety of approaches. Functionally well differentiated neurons (shown by receptor binding studies, immunocytochemistry and morphological examination) were found to be abundant early in culture and to form cell contacts with oligodendrocytes characterized both immunocytochemically and morphologically. Myelin-like membranes with the periodicity of classical myelin elaborated by oligodendrocytes were detected only after 30 days in culture when neurones were no longer present. These results are discussed with regard to possible mechanisms of initiation of myelin synthesis.     

Changes of DNA synthesis in brain cortical cells after transplantation of embryonic nervous tissue into the brain of rats after hypoxia

Autoradiographic and biochemical studies with 3H-thymidine have shown that after transplantation of embryonic nervous tissue of rats into the brain of adult rats, intact and subjected to acute hypoxic hypoxia causing mass dystrophy of neurons in the brain cortex of recipients, there occurs stimulation of DNA synthesis in non-nerve cells: glial cells, macrophages and endothelial cells. Stimulation is much more pronounced in the operated hemisphere than in the non-operated one and in intact rats than in hypoxia-subjected ones. On the whole, DNA synthesis was not observed in brain nerve cells except individual neurons located near the wound canal and the transplant.     

Cytoskeletal characterization of a neuroectodermal cell line and embryonic mouse brain

The cytoskeleton of a newly isolated mouse embryo brain-derived cell line and of dissected mesencephalon-rhombencephalon samples of the 10- to 11-day-old mouse embryo, constituted of a ventricular zone only, has been examined by immunocytochemistry, electrophoretic separation and Western blotting techniques. In accordance with their epithelial organization, the ventricular cells contain cytokeratins as main constituents of their cytoskeleton. Vimentin has been also revealed as early as day 10.5 of gestation. The complex cytokeratin polypeptide pattern puts this histological type of epithelia into the category of complex, rather than simple, epithelia and calls for explanations other than keratinization for the presence and functional role of the high-molecular weight, basic keratins. The cytoskeletal composition of the embryo brain-derived gliogenic cell line reflects the vimentin- and cytokeratin-positive character of the ventricular cells.     

Brain lectin-mediated agglutinability of dissociated cells from embryonic and postnatal mouse brain

The distribution of 125I-melatonin binding sites in 6 discrete brain regions of female rats at the estrous stage and the effects of ovariectomy and subsequent 17 beta-estradiol treatment on these binding sites were studied. Specific binding of 125I-melatonin was found in the hypothalamus, medulla-pons, hippocampus, cerebellum, striatum and parietal cortex of the female rats. Ovariectomy produced a large estradiol-reversible decrease in 125I-melatonin binding in the medulla-pons and hypothalamus. In contrast, 125I-melatonin binding sites in the other brain regions were generally unaffected by ovariectomy or estradiol. The estradiol-regulated changes in melatonin binding in the hypothalamus and medulla-pons may reflect the role of a specific estradiol-melatonin interaction in the coordination of the neuroendocrine reproductive axis.     

Isolation of sex-specific cDNAs from fetal mouse brain using mRNA differential display and representational difference analysis

Comparing female and male brain structures reveals a variety of sex differences in many vertebrates. These differences are manifested throughout the brain, in regions such as the hypothalamus, the preoptic area and the amygdala. Some are thought to be induced during the fetal period by the effect of steroid hormones produced in the gonads. It is well-established that fetal androgens, probably through the conversion to estrogen by the enzyme aromatase, masculinize the nervous system and set adult mounting behavior in rodents. However, less is known about molecular mechanisms involved in gender-specific development of the brain. We have taken a broad approach to isolate sex-specific genes from fetal brain. mRNAs from 18.5 days post-coitum (dpc) female and male mouse brain were screened with the classical and the recently developed signal peptide differential display (SPDD) and with representational difference analysis of cDNA (cDNA-RDA). Two sex-specific cDNAs were isolated, F29 and M17, corresponding to the female-specific Xist gene and the male-specific Smcy gene, respectively.     

Composition and synthesis of polyunsaturated fatty acyl groups in the embryonic brain of the trisomy-16 mouse

The trisomy-16 (T-16) mouse is considered to be a promising model for human trisomy-21 (T-21) (Down's syndrome, DS). Therefore, the fatty acyl (PUFA) compositions of phosphoglycerides in embryonic brains (days E-17 and E-18) of T-16 mice have been compared with those of balanced heterozygotic embryos from the same litters, in order to determine whether similar abnormalities are present as have been found in foetal DS brain (Brooksbank et al., 1985, J. Neurochem. 44, 869-874). The analyses revealed that the ratio of (n-3) to (n-6) PUFA was significantly increased in ethanolamine (EPG) and in choline phosphoglycerides, as it is in EPG in the foetal T-21 brain. However, the abnormality was not so marked in the murine as in the human trisomy, and the (n-3)/(n-6) ratio in EPG was primarily elevated on account of decreased proportions of 20:4(n-6) and 22:4(n-6), there being no significant increase in (n-3) PUFA. The PUFA composition of the phosphoglycerides of the corresponding trisomic and balanced placentae was also determined, but no relevant differences could be discerned between the genetically different tissues. As 6-desaturase, the rate-limiting enzyme system in PUFA synthesis, reacts more readily with (n-3) than with (n-6) substrates, the shift in (n-3)/(n-6) ratio of PUFA might be related to an alteration in 6-desaturase activity in trisomy. Comparison of the specific activity of 6-desaturase in fresh brain homogenates of T-16 embryos with those from balanced litter-mates revealed, however, no differences.     

Axonogenesis in the medaka embryonic brain

In order to know the general pattern of axonogenesis in vertebrates, we examined axonogenesis in the embryonic brain of a teleost fish, medaka (Oryzias latipes), and the results were compared with previous studies in zebrafish and mouse. The axons and somata were stained immunocytochemically using antibodies to a cell surface marker (HNK-1) and acetylated tubulin and visualized by retrograde and anterograde labeling with a lipophilic dye. The fiber systems developed correlating with the organization of the longitudinal and transverse subdivisions of the embryonic brain. The first axons extended from the synencephalic tegmentum, forming the first fiber tract (fasciculus longitudinalis medialis) in the ventral longitudinal zone of the neural rod, 38 hours after fertilization. In the neural tube, throughout the entire brain two pairs of longitudinal fiber systems, one ventral series and one dorsal or intermediate series, and four pairs of transverse fiber tracts in the rostral brain were formed sequentially during the first 16 hours of axon production. In one of the dorsal longitudinal tracts, its branch retracted and disappeared at later stages. One of the transverse tracts was found to course in the telencephalon and hypothalamus. The overall pattern of the longitudinal fiber systems in medaka brain is similar to that in mouse, but apparently different from that in zebrafish. We propose that a ventral tract reported in zebrafish partially belongs to the dorsal fiber system, and that the longitudinal fiber systems in all vertebrate brains pass through a common layout defined by conserved genetic and developmental programs.     

小鼠胰腺干细胞饲养层条件下的连续传代培养

背景：胰腺干细胞具有分裂与高度分化潜能的特性,可以在体外进行成功分离和培养,但体外如何对其进行有效扩增是亟待解决的问题。 目的：在胎鼠成纤维细胞饲养层条件下体外传代培养小鼠胰腺干细胞。 设计、时间及地点：细胞学体外观察,于2007-07/2008-01在广西中医学院基础医学院细胞无菌培养室完成。 材料：SPF级新生昆明小鼠20只,孕14 d昆明小鼠多只,均购自广西中医学院实验动物中心。 方法：取SPF级新生昆明小鼠的胰腺组织,Ⅴ型胶原酶消化,未消化完全的组织块自然沉降后,收集上层的含细胞离散液,离心弃上清,加入含角质细胞生长因子、碱性成纤维细胞生长因子的无血清低糖DMEM培养基,在经多聚赖氨酸溶液处理的24孔板中培养。取孕14 d昆明小鼠,剖腹取胎鼠,去除头部及内脏,将躯干及四肢采用组织块胰蛋白酶消化法分离培养成纤维细胞,调整密度为5×108 L-1接种于24孔板中,传至第3代经丝裂霉素适当处理制备饲养层细胞。取原代培养5 d的胰腺干细胞,按30个/cm2密度种入铺有饲养层细胞的孔板中,当胰腺干细胞铺满小孔底部面积的80%时继续传代。 主要观察指标：倒置显微镜下观察细胞形态变化,原代培养48 h对细胞进行碱性磷酸酶染色,通过免疫组织化学染色鉴定胰腺干细胞特异分子标志物巢蛋白的表达。分别于传代后2,3,4,5 d检测碱性磷酸酶、巢蛋白和胰十二指肠同源盒基因1的表达。 结果：原代培养的来源于胰腺组织的细胞中,可见一些大、圆、单个核的细胞,胞浆折光性强,核浆比例大,呈附壁生长,碱性磷酸酶染色呈阳性,表达胰腺干细胞特异性分子标志巢蛋白,且具有活跃的分裂增殖能力。在小鼠胚胎成纤维细胞饲养层条件下对胰腺干细胞进行体外传代培养,可连续传至第3代,各代胰腺干细胞仍保持大、圆、单个核、核浆比例大及增殖能力强等特性,传代后各时间点碱性磷酸酶、巢蛋白染色均呈阳性,胰十二指肠同源盒基因1蛋白染色呈阴性反应,可保持未分化状态。 结论：以小鼠胚胎成纤维细胞作为饲养层,经丝裂霉素C处理后可分泌供胰腺干细胞生长所需的因子,并抑制胰腺干细胞的自主分化,体外连续传至第3代仍能够保持较好的生长特性、高度增殖能力及未分化状态。