我们想感谢与解剖和文化技术有益的建议A. Hadjantonakis（斯隆 - 凯特林研究所）的实验室和L. Niswander（科罗拉多州丹佛U）的实验室。这项工作已经支持NRSA NS059562 JDG和RO1NS05897 MER L. Niswander和J.纳多（系统生物学研究所）合作。

1。培养基的制备 - 所有试剂都列于表1 <ol><li>解冻雄性大鼠血清在37 ° C。 </li><li>热灭活30分钟大鼠血清在55 ° C </li><li>离心机在10K大鼠血清在室温下5分钟</li><li>免费酚红的DMEM /高糖取出上清和混合50:50 </li><li>使用0.45微米注射器过滤消毒媒体</li><li>至少应做好媒体/胚胎1毫升</li></ol> 2。从怀孕坝子宫隔离<ol><li>使用机构的动物护理和使用委员会（IACUC）的批准程序，怀孕的大坝是先麻醉，然后由颈椎脱位处死</li><li>消毒用70％乙醇的腹部</li><li>捏一小块皮肤路径，沿中线乳头线以上，与大钳和开放的大剪刀钳下方的腹部。要小心，不要破坏任何内部结构小号</li><li>使用剪刀横向减少对鼠标两侧的初始开放，使整个腹部开放</li><li>肠子和多余的脂肪垫，往往可以掩盖子宫，这些应该被移到搁置，子宫和卵巢的顶部，可以在每边观察</li><li>捏低于卵巢子宫顶部，并用剪刀从脂肪中分离出来的子宫，子宫的一端切割到其他卵巢</li><li>电梯在培养皿中，用生理盐水子宫钳和地点</li></ol> 3。去除胚胎从子宫<ol><li>用生理盐水冲洗子宫，以消除任何多余的血液和修剪任何多余的脂肪从外面</li><li>转移到一个干净菜室温。 Tyrodes生理盐水以改善可视化</li><li>使用立体显微镜，单独使用或如有必要，罚款或者小剪刀钳（＃5）每个胚胎剥离子宫除了仔细。 </li> &lt;李&gt;插入的子宫壁和蜕膜和剥离子宫壁之间的空间精细镊子，小心不要刺破胚胎</li></ol> 4。从胚胎中去除蜕膜<ol><li>东方蜕膜，使较暗的部分（胎盘）正面临着远离你</li><li>沿较轻的部分分离蜕膜暗部分与您钳切口，注意不要剪得太深</li><li>完成从卵黄囊顶部的胎盘分离，小心不要撕破ectoplacental锥（EPC）。此时，你应该能够看到卵黄囊Reicherts膜</li><li>继续清除不小心捅破卵黄囊整个蜕膜（较轻的部分） </li><li>轻轻一捏，从EPC脱皮删除Reicherts。如果EPC损坏或从卵黄囊分离，胚胎将无法正常打开E8.5 - E9.5 </li><li&gt;随着Reicherts移除胚胎应通过卵黄囊容易看到，并可以通过计数somities上演。 </li></ol> 5。设立文化系统<ol><li>剪下一个塑料移液管，用剪刀增加开放的直径，使胚胎可以在不损害卵黄囊吸管</li><li>填写1毫升每胚胎媒体清洁滚瓶。根据滚瓶型使用最多3-6胚胎可以装载在每个瓶子。 </li><li>传输使用切吸管媒体充满滚瓶，尽可能少Tyrodes超过的胚胎，以免冲淡媒体</li><li>将玻璃辊瓶橡胶塞（塞） </li><li>插入辊文化鼓滚瓶，密封插头和鼓之间形成</li><li>一旦所有的瓶子都受到高度重视，并在任何鼓空槽已密封瓦特第i个橡胶瓶塞，激活滚筒</li><li>打开CO 2和O 2坦克，约2 PSI调整，使出口阀是每秒释放一个泡沫。 E8.5 - E10的胚胎，20％O 2和5％的CO 2，应使用。 </li><li>确保孵化器的设置为37 ° C和覆盖或关闭的孵化器，使胚胎从光屏蔽。 </li><li>应定期检查胚胎的somities此外，转弯的进展，和神经管闭合的程度，以评估其发展</li><li>经过2-3小时，在文化，药理抑制剂或其他治疗，可以添加到媒体</li><li>研究设计应包括控制，如车辆或类似物的研究试剂无效。 </li></ol> 6。评估后全胚胎培养的发展<ol><li>关闭煤气，停止辊，从孵化器中删除瓶</li><li>转移胚胎到台氏在评价体视显微镜下的培养皿或PBS </li><li>卵黄囊应该会出现气球样，心跳应该是可见的的和心率应每分钟120次，流通，应明显</li><li>如有必要，照片胚胎卵黄囊删除前</li><li>从胚胎中取出切割孔附近的EPC和翻转，头部和臀部的胚胎卵黄囊和羊膜卵黄囊。 </li><li>切断脐带，可以从胚胎中分离出来的卵黄囊，卵黄囊可用于基因分型的胚胎，如果有必要的。 </li><li>检查胚胎体节数和神经管缺陷，如开放的颅褶皱，不完全封闭的脸和/或一个开放的尾鳍neuropore的，。胚胎可以根据Theiler多个形态发生变化的分类<a href="http://genex.hgu.mrc.ac.uk/Databases/Anatomy/MAstaging.html">（http://www.emouseatlas.org/ EMAP / home.html</a>做为）同样，一个摄影记录上演胚胎发育是有益的。 </li></ol> 7。注释<ol><li>一个高品质的夹层是必要的，因为任何划痕或卵黄囊的泪水会阻止成功的转折点和抑制的正常发展。也可以被损坏的EPC在拆除赖克特的膜（RM），如果EPC甚至部分从卵黄囊分离，胚胎不能正常发育。然而，不完全切除的RM可引起胚胎粘在一起和/或RM细胞可长满文化和收缩卵黄囊的扩张，这两个会妨碍正常的发展。 </li><li>清洁和锋利的钳，获得良好的解剖秘密，因为沉闷或弯曲钳使程序明显更加困难。文书上的蛋白质沉淀，会导致组织损伤。 </li><li>清洁，无菌的玻璃瓶也是必不可少的，以防止坚持侧壁和被破坏胚胎。在实验开始之前，瓶应SCRubbed用肥皂和水，在DH 2 O冲洗，并在每个瓶子的水，直到水沸腾的少量微波。瓶，然后用70％乙醇冲洗，并允许空气干燥。 </li><li>尽可能快地工作，因为胚胎迟早可以转移到文化传媒和温暖至37 ° C的更好的生存。 </li><li>完成解剖任何胚胎转移前滚瓶的整个的垃圾，使每个胚胎在室温下是相同的时间量，以避免造成任何差异在胚胎发育</li><li>保持无菌的环境，以防止细菌污染的文化。我们的文化实验已经执行无抗生素，但许多实验室自己的媒体中添加抗生素，以防止细菌生长。媒介应该出现在实验结束后，在实验开始时一样清楚。如果媒体已变得混浊或有一个可见的沉淀，有可能是一个CON混染，感染了你的文化。 </li><li>无效的气体交换，也可以延迟胚胎的发育，所以要确保有一个可靠的稳压器，确保连续投放。 </li><li>胚胎发育在体外培养慢约50％，因此，它是必不可少的计数体节，以确保您已达到适当的发展阶段，为完成实验。 </li></ol> 8。代表性的成果： 前胚胎的外观和后滚子文化是如图1所示。在解剖时，应千方百计配置（图1A，D）的尾巴后面的头部皱褶胚胎。在培养36小时后，胚胎应该有完成的转折点，使他们在C型弯曲，胎儿的位置，尾巴在前面的头（图1B，C，E，F）。 RhoA蛋白激酶抑制剂（Y - 27632），收敛扩展已知的抑制剂在药理操纵神经胚（Ybot - 冈萨雷斯，2007年），缩短沿喙尾椎轴（图1E，F）的胚胎和抑制颅神经倍封闭的结果。我们的数据显示，逐步增加剂量的Y - 27632损害颅折封（图2A），并缩短身体的纵轴（图2B），下游RhoA的颅神经胚形成和收敛扩展信号的作用是一致的。 <img alt="图1" src="/files/ftp_upload/3132/3132fig1.jpg" /> 图1。文化胚胎和操纵的药理抑制剂Y - 27632的外观。 （A，D）解剖前全胚胎培养胚胎在E8.5（B，E）在E10与卵黄囊仍完好无损，辊培养36小时后的胚胎。 （C，F）卵黄囊已被删除，说明成功的转折和神经管闭合。与Rho激酶抑制剂Y - 27632（E，F）处理的胚胎未能接受适当的收敛延伸，并说明一个体轴缩短。 <img alt="图2" src="/files/ftp_upload/3132/3132fig2.jpg" /> 图2。RhoA蛋白激酶抑制剂对颅神经管闭合和轴伸长。 （一）胚胎的百分比，能够成功地完成其颅褶皱（％NTC =百分比神经管闭合）相比，Y - 27632的剂量添加到培养基。 （二）奥迪囊泡和前肢之间的距离显着减少的Y - 27632剂量的增加（P &lt;0.05）。

<ol>
	<li>Cockroft, D. L., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+culture+of+post+implantation+embryos.">Dissection and culture of post implantation embryos.</a> Mammalian Postimplantation Embryos A Practical Approach. , (1990).</li><li>Gray, J. D., Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+insights+into+folate+supplementation+from+Crooked+tail+and+other+NTD-prone+mutant+mice.">Mechanistic insights into folate supplementation from Crooked tail and other NTD-prone mutant mice.</a> Birth Defects Res. A. Clin. Mol. Teratol. 85, 314-321 (2009).</li><li>Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Atlas of Mouse Development. , (1992).</li><li>Ybot-Gonzalez, P., Savery, D., Gerrelli, D., Signore, M., Mitchell, C. E., Faux, C. H., Greene, N. D., Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+extension,+planar-cell-polarity+signalling+and+initiation+of+mouse+neural+tube+closure.">Convergent extension, planar-cell-polarity signalling and initiation of mouse neural tube closure.</a> Development. 134, 789-799 (2007).</li><li>Ross, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-environment+interactions,+folate+metabolism+and+the+embryonic+nervous+system.">Gene-environment interactions, folate metabolism and the embryonic nervous system.</a> Wiley Interdiscip Rev Syst Biol Med. 2, 471-480 (2010).</li></ol>

没有利益冲突的声明。

那些在其发展的内在胚胎分离孕产妇，胎儿宫内因素的能力，是研究胚胎发育的各个阶段的重要工具。在这里，我们的RhoA蛋白激酶对颅神经胚形成前宫内的小分子抑制剂，从而消除产妇代谢药物的变量，分析了影响。这药理操纵对颅神经胚形成和融合扩展产生深远的影响。这种化合物的敏感性，可以比较不同的遗传鼠标突变体。这里介绍的方法也可以适用于其他发展中的分子途径的研究，允许使用的各种试剂的胚胎细胞功能的直接操纵。

<table border="1"><tbody><tr><th>名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>体视显微镜解剖</td><td>徕卡</td><td> M165 </td><td></td></tr><tr><td>精密孵化器</td><td> BTC工程（英国） </td><td> BTC 001 </td><td></td></tr><tr><td>旋转瓶文化单位</td><td> BTC </td><td> BTC 003 </td><td></td></tr><tr><td>旋转装置的玻璃瓶</td><td> BTC </td><td> BTC 005 </td><td></td></tr><tr><td>硅橡胶塞</td><td> BTC </td><td> BTC 007 </td><td></td></tr><tr><td>硅橡胶软木</td><td> BTC </td><td> BTC 008 </td><td></td></tr><tr><td>气体鼓泡入口</td><td> BTC </td><td> BTC 012 </td><td></td></tr><tr><td>气体鼓泡入口陷阱</td><td> BTC </td><td> BTC 013 </td><td></td></tr><tr><td>气体鼓泡插座</td><td> BTC </td><td> BTC 014 </td><td></td></tr><tr><td>气瓶</td><td> TechAir </td><td></td><td> 20％O2，5％的CO 2 </td></tr><tr><td>燃气调压器</td><td> TechAir </td><td></td><td></td></tr><tr><td>雄性大鼠血清</td><td>哈伦实验室</td><td> 4520 </td><td></td></tr><tr><td> W / O型的DMEM酚红</td><td> Invitrogen公司</td><td> 31053 </td><td></td></tr><tr><td> 10毫升注射器</td><td>屋宇署</td><td> 309604 </td><td></td></tr><tr><td>注射器过滤器</td><td> NALGENE </td><td> 190-2545 </td><td></td></tr><tr><td>大解剖剪刀（先端钝） </td><td> VWR </td><td> 82027-594 </td><td></td></tr><tr><td>超细波恩剪刀</td><td>精细（FST）的科学工具</td><td> 14084-08 </td><td></td></tr><tr><tD&gt; Dumot＃5镊子</td><td>福斯特</td><td> 11252-50 </td><td></td></tr><tr><td>台氏盐水</td><td></td><td></td><td>如1990年在Cockroft描述</td></tr><tr><td> 10厘米的一次性培养皿</td><td>屋宇署</td><td> 351029 </td><td></td></tr></tbody></table>

neural tube closure in mouse whole embryo culture

遗传小鼠模型是研究哺乳动物的神经管闭合的重要工具（灰色和罗斯，2009年，罗斯，2010）。然而，小鼠胚胎在子宫内的研究是有限的，我们无法直接药理操纵试剂利益产妇代谢的影响，在隔离的胚胎。一个小分子，重组蛋白，或siRNA，这些物质传递的母亲，无论是通过饮食或注射使用将受这些不稳定的化合物，以各种身体的防御系统，可以防止他们到达胚胎。全胚胎培养的调查，可用于单独的产妇从发展的内在胎儿的影响。 在这里，我们目前培养小鼠胚胎滚筒孵化设备，允许正常解剖后神经管闭合“（克罗克特，1990年）在使用高浓缩铀的媒体的方法。一旦在文化，胚胎可以被操纵在体外培养技术，否则不会有可能的， 如果胚胎在子宫内仍然使用传统。胚胎的兄弟姐妹可以在不同时间点收集，研究不同方面的神经胚发生，E7 - 7.5（神经板的形成，神经胚启动前）E9.5 - 10（颅倍和尾鳍neuropore结束封闭，考夫曼，1992年）。在这个协议中，我们展示了解剖胚胎颅神经胚形成的研究最佳的时间点，我们的方法。胚胎将被解剖E8.5（约10-12 somities），神经管闭合后启动，但前胚胎车削和颅神经倍关闭，并在文化一直维持到E10（26-28 somities），当颅神经胚应该是完整的的。

Watch this Scientific Journal Video about Neural Tube Closure in Mouse Whole Embryo Culture at JoVE.com

Neural Tube Closure in Mouse Whole Embryo Culture

允许直接药理操纵小鼠胚胎神经胚绕过产妇代谢的方法描述。可适应的技术，来研究神经胚形成的不同方面，不同的时间点和药理代理人。

在小鼠全胚胎培养的神经管封闭

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

neural-tube-closure-in-mouse-whole-embryo-culture

Research

JoVE Journal

Neuroscience

21.4K 次观看.  Weill Cornell Medical College. 允许直接药理操纵小鼠胚胎神经胚绕过产妇代谢的方法描述。可适应的技术，来研究神经胚形成的不同方面，不同的时间点和药理代理人。

视频: Neural Tube Closure in Mouse Whole Embryo Culture

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学：

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

建立小鼠胚胎神经干细胞培养神经球含量

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.
Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.

本地化的蛋白质三维神经干细胞培养：一个混合的可视化方法

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

检测内源NADH的荧光双光子成像的小鼠大脑皮层Microregional缺氧

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

从小鼠胚胎神经管的神经嵴细胞的分离和培养

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...