该研究项目得到了奖号码R21AA017735国家酒精滥用与酒精中毒研究所（YS）。内容完全是作者的责任，并不一定代表美国国立酒精滥用和酒精中毒或美国国立卫生研究院的官方意见。笔者还要感谢里斯蒙哥马利编辑这篇稿子。

1。动物育种与营养肽治疗<ol><li>品种C57BL/mice将雌性小鼠（〜6周岁，平均体重20克）到2小时的男性的笼子。 </li><li>精子塞和/或阴道涂片检查后立刻。 </li><li>如果是正数，指定这个时间点为胚胎第0天（E0）。 </li><li> E7，重量匹配怀孕女性对照组和治疗组（3组）最近被指定为4：1）酒精（乙醇）的流质饮食组（ALC）; 2）对喂养的对照组（PF）; 3）肽治疗组，应该接受腹腔（IP）ADNF-9肽注射酒精暴露流质饮食（ALC/ADNF-9）的旁边。编制的ALC和PF的饮食和腹腔注射过程的细节，可以发现在我们最近的工作4。 </li><li>测量每日饮酒流质饮食可以提供免费访问作为唯一的营养来源。 PF流质饮食个别轭吨ØALC坝每天可以测量。 </li><li>流质饮食，在过去24小时消耗的体积可以记录30毫升毕业于螺丝帽管，并应提供新鲜烹制的饮食，每天从E7到E13。 </li></ol> 2。胎儿解剖显微切割与胎儿大脑<ol><li>牺牲孕鼠E13通过深入实施安乐死的小鼠与CO 2程序由颈椎脱位。 </li><li>用70％乙醇清洁腹部切口。 </li><li>做腹部切口，用无菌剪刀和镊子。 </li><li>一旦腹部是开放的，拿着它用一个镊子和使用其他镊子撕膜及远离子宫子宫解剖。请一定要慢慢做这个程序，以避免穿刺胚胎。 </li><li>从子宫中取出胚胎，并将它们传送到一个​​细胞培养皿（60毫米×15毫米）的Hanks&#39;平衡盐溶液<html> （HBSS），在冰冷的水。 </li><li>从整体胚胎体视显微镜下进行显微切割，解剖胎儿大脑。 </li><li>采用超细显微切割钳，拉断的胚胎在头的上部暴露颅骨皮肤。 </li><li>一旦头骨暴露，并清晰地显示的显微解剖体视显微镜下，然后剥离颅骨从后壁的部分大脑在脊髓和脑干区。 </li><li>一定要一块一块地从后部到前部，头颅骨剥离。 </li><li>一旦胎儿的大脑视觉接触，进行提取。 </li><li>解剖胎儿大脑，从基原基嗅球，后脑的基础上。 </li><li> Postfix的所有解剖胎儿的大脑在4％多聚甲醛为2-3天。您也可以从每个水坝冻结其他胎儿大脑，如果你正在测试他们Western blot法或酶法检测。 </li></ol>NT“&gt; 注：动物用途的程序批准的机构动物护理和使用委员会托莱多大学按照机构的动物护理的指引，在美国国立卫生研究院和使用委员会指南保养和使用实验动物。 3。嵌入胎儿大脑切片明胶<ol><li>准备10％的明胶A级</li><li>填补嵌入模具剥离外半等到明胶凝固。 </li><li>将控制和产前治疗胎儿的大脑侧由端之上的凝固的明胶。这是为了保持一致的条件下在对照组和实验组之间的细胞死亡的免疫检测。 </li><li>添加液体明胶顶部胎儿的大脑，使一个完整的模具（确保明胶是不是太热了）。 </li><li>制备的明胶模具冷藏保存15分钟的胎儿的大脑。 </li><li>剥离嵌入模具塑料预先制作明胶含有胎儿的大脑，然后转移到4％多聚甲醛两天。 </li><li>固定地点及部分胎儿的大脑在明胶vibratome的切片机（莱卡VT 1000S）冠状切片厚度为50微米。该厚度的测试是在我们的研究中，因为它提供了更好的解剖特征。此外，厚度为25μm，也可以进行测试，用这种技术，涉及嵌入胎儿的大脑中，在10％的明胶。需要注意的是冠状切片，使用前脑vibratome的上基底节隆起水平</li><li>收集胎儿的脑切片，于磷酸盐缓冲盐水（PBS）溶液。 </li><li>安装的Superfrost Plus水滑梯胎儿的大脑部分。 </li><li>擦干安装部分为15分钟。 </li><li>将幻灯片（安装与胎儿的大脑部分），在PBS第二天测试TUNEL反应。 </li></ol> 4。德TUNEL反应细胞死亡或凋亡TECTION （TUNEL末端转移酶介导的dUTP缺口末端标记; TDT：脱氧核糖核苷酸末端转移酶）。此法的目的是，以确定细胞死亡。 <ol><li>治疗胎儿的大脑部分的Superfrost幻灯片加蛋白酶K（20微克/毫升），装在5分钟，37°C（300微升的蛋白酶K混合在20毫升PBS）。幻灯片组装在5-Plend开MAILE）。 </li><li>冲洗胎儿的大脑部分（安装在投影片），用PBS三次轨道摇床下5分钟。 </li><li>孵育的部分，用3％H 2 O 2在甲醇中，在室温下和10分钟（3毫升30％H 2 O 2在27毫升100％甲醇）（幻灯片不应该在摇床）。 </li><li>用PBS三次冲洗部分5分钟轨道摇床。 </li><li>部分在透化溶液（0.1％表面活性剂TritonX-100，0.1％的柠檬酸钠）孵育2分钟，在冰上（4℃）（100微升表面活性剂TritonX + 0.1柠檬酸钠+99.9毫升distille二维水流）。 </li><li>在轨道摇床中的5分钟，在PBS中冲洗部分两次。 </li><li>干周围部分幻灯片。 </li><li>绘制屏障的Superfrost Plus幻灯片使用PAP笔。这旨在防止任何泄漏的解决方案，是用于治疗的TUNEL阳性细胞的检测的部分。 </li><li> TUNEL反应混合物（50微升1瓶和2瓶450微升）1小时，在37°C孵育部分将用于培养瓶2溶液中，控制。 </li><li>用PBS三次5分钟轨道摇床下冲洗。 </li><li>干周围组织。 </li><li>孵育部分与转换器-POD 30分钟，在37°C。 </li><li>冲洗部分三次，用Tris盐酸（pH值7.5，0.05 mol）的5分钟。 </li><li>孵育7分钟，在0.05％的3&#39;-3&#39;-二氨基联苯胺四盐酸盐（DAB）和0.003％H 2 O 2（25毫升的Tris盐酸，pH7.5的0.05M的+25μl的3％的H 2 O 2 + 500的部分民建联微升）根据抖Oribtal器在4°C（冰），并在黑暗中（确保做到这一点在新的5 Plend打开邮件的程序）。 </li><li>用PBS三次轨道摇床下5分钟，冲洗部分。 </li><li>保持幻灯片的部分不超过24小时的PBS中，然后在PBS处理他们的尼氏染色，在下一节中详细协议。 </li></ol> 注：DAB是一种致癌物质。使用手套及清洁漂白一次完成所有的实验使用的所有菜肴。 5。尼氏染色显微镜观察胎儿脑切片制备<ol><li> Nissl染色程序应做在生物安全罩。 </li><li>滑动部分，用去离子水冲洗2分钟。 </li><li>孵育2分钟，在70％乙醇的部分。 </li><li>孵育6分钟，95％乙醇中的各节。 </li><li>孵育2分钟，在70％乙醇的部分。 </li><li>冲洗部分在去离子笏ER 2分钟。 </li><li>孵育部分甲酚紫染色3分钟。 </li><li>的部分在去离子水中漂洗2分钟。 </li><li>孵育2分钟，在70％乙醇的部分。 </li><li>孵育在95％乙醇3分钟+ 3毫升冰醋酸部分。 </li><li>孵育2分钟，在95％乙醇部分。 </li><li>孵育的部分在100％乙醇3次，持续2分钟。 </li><li>孵育10分钟三次二甲苯的部分。 </li><li>可以安装使用Permount和盖玻片载玻片幻灯片控股胎儿的大脑部分。 </li><li>一夜之间离开安装滑动前到微观的观察与分析。 </li><li>徕卡直立显微镜下进行显微观察和显微。 </li></ol>

<ol>
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Rev. 52 (1), 107-118 (2006).</li><li>Brenneman, D. E., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+femtomolar-acting+neuroprotective+peptide.">A femtomolar-acting neuroprotective peptide.</a> J. Clin. Invest. 97 (10), 2299-2307 (1996).</li><li>Zamostiano, R., Pinhasov, A., Gelber, E., Steingart, R. A., Seroussi, E., Giladi, E., Bassan, M., Wollman, Y., Eyre, H. J., Mulley, J. C., Brenneman, D. E., Gozes, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+the+human+activity-dependent+neuroprotective+protein.">Cloning and characterization of the human activity-dependent neuroprotective protein.</a> J. Biol. Chem. 276 (1), 708-714 (2001).</li><li>Offen, D., Sherki, Y., Melamed, E., Fridkin, M., Brenneman, D. 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没有披露。

在这项研究中提出的方法和技术演示的影响，产前暴露于酒精在胎儿大脑和营养肽的作用，对预防这些影响。这些可能提供关于如何学习其他药物滥用或其他有毒化学物质在胎儿的大脑在不同妊娠阶段的信息。 在对于繁殖范式，在某些情况下，精子插头的检测可能不被观察到。在这一点上，它是重要的测试中的幻灯片的阴道涂片在显微镜观察下，它可以提供可能的阳性精子?...

这里描述的实验方法的目标是评估胎儿酒精暴露模型使用几个涉及养殖技术，饲养营养肽，microdissecting，并检测细胞死亡的神经保护作用。从我们的实验室工作涉及的流质饮食，适度饮酒，这可能是人类在长期的酒精量的饮用水范式类似的模型与酒精混合食用1-5。我们和其他人反对的有害影响胎儿酒精暴露，神经保护的三肽衍生物。调查酒精暴露在胚胎阶段，使用动物模型的研究可能会导致潜在神经保护机制识别和允许干预程序的发展。这可能会提供足够的信息在怀孕期间接触酒精的有害影响的神经保护作用和衰减。 产前酒精暴露的影响，可能预防可能涉及治疗用肽已被证明是参与神经保护作用的体外 6,7和体内 1,2,4,8,9孕鼠。在这些肽中，ADNF-9或SAL称为SALLRSIPA来自活性依赖的神经营养因子（ADNF）7,10。另一种肽序列NAPVSIPQ肽，称为NAP，来自活性依赖的神经保护蛋白（ADNP）的6,11，表现出了强有力的保护作用，抗氧化应激伴有酒精暴露12,13。此外，我们最近发现了一种新的合成肽，colivelin，似乎发挥了关键的保护作用，在胎儿酒精暴露模型2。 colivelin由ADNF-9和AGA-（C8R）HNG17（PAGASRLLLLTGEIDLP）的。使用这些肽的重要性是，他们有能力穿越血脑屏障，防止酒精诱导的细胞凋亡的影响和大脑发育赤字的。 实验方法用于测试针对酒精诱导的细胞凋亡的神经保护作用使用C57BL / 6小鼠。为了准确估计胚胎第0 ​​天（E0），因为它可以检测精子塞阴道涂片1-5透露，我们已经采取了2小时的窗口，而不是一夜之间育种。我们已经用流质饮食，因为这被认为是自由访问，而不是通过交付酒精灌胃的路线，这可能会引起应力怀孕小鼠喂养管。另一方面，显微切割可以是具有挑战性由于胎儿脑组织，解剖时可能会遇到在早期胚胎阶段的胎儿大脑的柔软度。在这里，我们将展示可视化技术，以应对所有的挑战，涉及显微切割。此外，也可以是具有挑战性的，因为胎儿的大脑切片，我们已采纳涉及嵌入胎儿大脑中明胶的使用剥离远离嵌入模具的技术。我们已经成功地在自由浮动的部分切割胎儿大脑在50微米厚。这使得我们可以调查任何改动的内容蛋白在胎儿的大脑部分，包括细胞死亡的鉴定。

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent/equipment</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Superfrost Plus Slides</td>
 <td>VWR</td>
 <td>48311-703</td>
 <td></td>
 </tr>
 <tr>
 <td>PAP PEN</td>
 <td>Research Products Int. Corp.</td>
 <td>195506</td>
 <td></td>
 </tr>
 <tr>
 <td>5-Plend Open Mailer </td>
 <td>Heathrow Scientific, LLC</td>
 <td>HS 15983G</td>
 <td></td>
 </tr>
 <tr>
 <td>Peel away embedding molds</td>
 <td>Electron Microscopy Sciences</td>
 <td>70182</td>
 <td></td>
 </tr>
 <tr>
 <td>In situ cell death detection kit, POD</td>
 <td>Roche Diagnostics, Inc</td>
 <td>11684817910</td>
 <td></td>
 </tr>
 <tr>
 <td>Hanks' Balanced Salt Solution</td>
 <td>Invitrogen</td>
 <td>14170-120 </td>
 <td></td>
 </tr>
 <tr>
 <td>Gelatin Type A</td>
 <td>FisherScientific</td>
 <td>G8-500</td>
 <td></td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>W. NUHSBAUM, Inc </td>
 <td>Leica M60, KL 200 LED</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro-Vibratome</td>
 <td>Leica, Inc </td>
 <td>Leica VT 1000S</td>
 <td></td>
 </tr>
 <tr>
 <td>Moria Ultra Fine Forceps </td>
 <td>Fine Science Tools</td>
 <td>11370-40</td>
 <td>2 pairs</td>
 </tr>
 <tr>
 <td>Graduate 30 ml feeding tubes</td>
 <td>Dyets, Inc </td>
 <td>900012 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vitamin Mix </td>
 <td>Bio-Serv. Inc. </td>
 <td>F8031 </td>
 <td></td>
 </tr>
 <tr>
 <td>Mineral Mix</td>
 <td>Bio-Serv. Inc. </td>
 <td>F8598 </td>
 <td></td>
 </tr>
 <tr>
 <td>Maltose Dextrin</td>
 <td>Bio-Serv. Inc. </td>
 <td>3650 </td>
 <td></td>
 </tr>
 <tr>
 <td>Ethyl alcohol 190 Proof, 5 gallons</td>
 <td>111000190-SS05 </td>
 <td>Pharmco-AAPER</td>
 <td></td>
 </tr>
 <tr>
 <td>Upright microscope</td>
 <td>W. NUHSBAUM, Inc</td>
 <td>LEICA DM 4000 </td>
 <td></td>
 </tr>
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</table>

experimental methods for testing the effects of neurotrophic peptide, adnf-9, against alcohol-induced apoptosis during pregnancy in c57bl/6 mice

调查产前酒精暴露在早期胚胎阶段的胎儿大脑发育的影响的实验设计是具有挑战性的。这主要是由于胎儿的大脑和他们测定产前暴露于酒精引起的细胞凋亡的切片显微切割的难度。这里所描述的实验提供从小鼠胎儿脑组织中繁殖的细胞死亡的识别的可视化技术。本研究采用C57BL / 6小鼠为动物模型，为研究胎儿酒精暴露和营养肽对酒精诱导的细胞凋亡的作用。养殖由2小时抠图窗口，以确定确切的胚胎年龄阶段。建立了胎儿酒精暴露模型已被用于这项研究，以确定影响胎儿大脑的产前酒精暴露。这涉及到免费使用酒精或对喂流质饮食，孕鼠营养的唯一来源。 为了研究细胞凋亡，胎儿的大脑是第一款嵌入式明胶使用剥离远离模具，以方便他们的切片vibratome的装置。嵌入并固定在多聚甲醛的胎儿大脑容易切片，和自由浮动部分可以安装在测定细胞凋亡或细胞死亡的Superfrost加幻灯片。 转移酶介导的缺口末端标记; TDT：脱氧核糖核苷酸末端转移酶（TUNEL）检测已被用来确定细胞死亡或凋亡细胞。值得注意的是，APoptosis和细胞介导的​​细胞毒性，其特征在于DNA片段。因此，可视化的TUNEL阳性细胞的细胞死亡或细胞凋亡的指标。 这里的实验设计提供信息使用酒精的作用研究建立流质饮食和营养神经肽的作用，在怀孕期间胎儿的大脑。这涉及孕鼠的繁殖和饲养，胎儿大脑microdissecting，并决定细胞凋亡。总之，这些视觉和文本的技术可能是胎儿大脑有害物质调查产前暴露源。

这里所描述的实验方法表明，有2小时的繁殖窗口是必不可少的，以判断准确的妊娠阶段。在E13的年龄，我们已经展示了清扫的胚胎。我们还展示了胎儿大脑的显微解剖E13。该过程包括几个阶段（交流）， 图1中所描述的。胎儿大脑的解剖从基原基嗅球，后脑（ 图1）的基极。胎儿的大脑，然后固定在4％多聚甲醛固定，进行免疫化学检测。固定后，在10％明胶嵌入胎儿大脑?...

Watch this Scientific Journal Video about Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice at JoVE.com

Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

这里重点提出的实验设计，研究酒精暴露在怀孕期间胎儿大脑细胞凋亡和应用营养神经肽的影响。从养殖到胎儿大脑的集合的详细描述。细胞凋亡的测定技术进行详细说明。

实验方法测试的影响，在怀孕期间对酒精诱导的细胞凋亡在C57BL / 6小鼠神经营养肽，ADNF-9

Experimental designs for investigating the effects of prenatal alcohol exposure during early embryonic stages in fetal brain growth are challenging. This ...

experimental-methods-for-testing-effects-neurotrophic-peptide-adnf-9

Research

JoVE Journal

Neuroscience

11.6K 次观看.  University of Toledo. 这里重点提出的实验设计，研究酒精暴露在怀孕期间胎儿大脑细胞凋亡和应用营养神经肽的影响。从养殖到胎儿大脑的集合的详细描述。细胞凋亡的测定技术进行详细说明。

视频: Experimental Methods for Testing the Effects of Neurotrophic Peptide, ADNF-9, Against Alcohol-induced Apoptosis during Pregnancy in C57BL/6 Mice

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

在哺乳动物的中枢神经系统视神经横断后的实验操作方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

科研

神经科学

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

器官型小脑文化：细胞凋亡的挑战和检测

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.
Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Cortex-，Hippocampus-，Thalamus-，下丘脑 - ，横向隔核 - 纹状体专用<em&gt;在子宫内</em&gt;电穿孔的C57BL / 6小鼠

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

实验模型研究酸性后处理对脑缺血的神经保护作用

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

Sagittal Plane Kinematic Gait Analysis in C57BL/6 Mice Subjected to MOG35-55 Induced Experimental Autoimmune Encephalomyelitis

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the application of these techniques to identify gait deficits in a mouse model of MS, known as experimental autoimmune encephalomyelitis (EAE). Paralysis and motor deficits in mice subjected to EAE are typically assessed using a clinical scoring scale. However, this scale yields only ordinal data that provides little information about the precise nature of the motor deficits. EAE disease severity has also been assessed by rotarod performance, which provides a measure of general motor coordination. By contrast, kinematic gait analysis of the hind limb in the sagittal plane generates highly precise information about how movement is impaired. To perform this procedure, reflective markers are placed on a hind limb to detect joint movement while a mouse is walking on a treadmill. Motion analysis software is used to measure movement of the markers during walking. Kinematic gait parameters are then derived from the resultant data. We show how these gait parameters can be used to quantify impaired movements of the hip, knee, and ankle joints in EAE. These techniques may be used to better understand disease mechanisms and identify potential treatments for MS and other neurodegenerative disorders that impair mobility.

Kinematic gait analysis in the sagittal plane yields highly precise information about how movement is executed. We describe the application of these techniques to identify gait deficits for mice subjected to autoimmune-mediated demyelination. These methods may also be used to characterize gait deficits for other mouse models featuring impaired locomotion.

MOG35-55 诱发自身免疫性脑脊髓炎 C57BL/6 小鼠矢状面运动步态分析

Kinematic gait analysis in the sagittal plane has frequently been used to characterize motor deficits in multiple sclerosis (MS). We describe the ...

A Simple Approach to Induce Experimental Autoimmune Neuritis in C57BL/6 Mice for Functional and Neuropathological Assessments

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by immunizing mice with neurogenic peptides to direct an inflammatory attack toward components of the peripheral nervous system (PNS). Recent advances have enabled the induction of EAN in the relatively resistant C57BL/6 mouse line using myelin protein zero (P0)106-125 or P0180-199 peptides delivered in adjuvant combined with the injection of pertussis toxin. The ability to induce EAN in the C57BL/6 strain allows for the use of the numerous genetic tools that exist on this mouse background, and thus allows the sophisticated study of disease pathogenesis and interrogation of the mechanistic action of novel therapeutics in combination with transgenic approaches. In this study, we demonstrate a simple approach to successfully induce EAN using the P0180-199 peptide in C57BL/6 mice. We also outline a protocol for the assessment of functional deficits that occur in this model, accompanied by an array of neuropathological features. Thus, this model is a powerful experimental model to study the pathogenesis of human peripheral demyelinating neuropathies, and to determine the efficacy of potential therapies that aim to promote myelin repair and protect against nerve damage in autoimmune neuritis.

This report outlines a simple approach to successfully induce experimental autoimmune neuritis (EAN) using the myelin protein zero (P0)180-199 peptide in combination with Freund&#39;s complete adjuvant and pertussis toxin. We present a sophisticated paradigm capable of accurately assessing the extent of functional deficits and neuropathology that occur in this EAN.

C57BL/6 小鼠自身免疫性神经炎的功能和病理评价的简易方法

Experimental autoimmune neuritis (EAN) is a well-appreciated experimental model of autoimmune peripheral demyelinating diseases. EAN disease is induced by ...

Cochlear Implant Surgery and Electrically-evoked Auditory Brainstem Response Recordings in C57BL/6 Mice

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of hearing. Improvement of the implant technology is needed if CI users are to perceive music and perform in more natural environments, such as hearing out a voice with competing talkers, reflections, and other sounds. Such improvement requires experimental animals to better understand the mechanisms of electric stimulation in the cochlea and its responses in the whole auditory system. The mouse is an increasingly attractive model due to the many genetic models available. However, the limited use of this species as a CI model is mainly due to the difficulty of implanting small electrode arrays. More details about the surgical procedure are therefore of great interest to expand the use of mice in CI research.
In this report, we describe in detail the protocol for acute deafening and cochlear implantation of an electrode array in the C57BL/6 mouse strain. We demonstrate the functional efficacy of this procedure with electrically-evoked auditory brainstem response (eABR) and show examples of facial nerve stimulation. Finally, we also discuss the importance of including a deafening procedure when using a normally hearing animal. This mouse model provides a powerful opportunity to study genetic and neurobiological mechanisms that would be of relevance for CI users.

Animal models of cochlear implants can advance knowledge of the technological bases of treating permanent sensorineural hearing loss with electrical stimulation. This study presents a surgical protocol for acute deafening and cochlear implantation of an electrode array in mice as well as the functional assessment with auditory brainstem response.

c57bl6 小鼠耳蜗植入术与电唤起脑干反应记录

Cochlear implants (CIs) are neuroprosthetic devices that can provide a sense of hearing to deaf people. However, a CI cannot restore all aspects of ...

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Elevated spinal fluid protein levels can either be the result of diffusion of plasma protein across an altered blood-brain barrier or intrathecal synthesis. An optimized testing protocol is presented in this article that helps to discriminate both cases and provides quantitative measurements of intrathecally synthesized proteins.

小鼠体内合成蛋白的定量测量

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the ...

Murine Corneal Nerve Abrasion for the Induction of Neurotrophic Keratitis

To begin corneal abrasion, place an anesthetized mouse on a table. Apply a drop of artificial tear on the eye undergoing the surgery. Apply a drop of ocular gel on the contralateral eye.Next, position the mouse on its side on a heated plate. Gently use a cotton swab to absorb the artificial tear, and brush aside the mouse's eyelashes without making contact with the eye. Turn on the ocular burr, and carefully open the mouse's eyelid with two fingers.Simultaneously shield the vibrancy to prevent them from entangling in the burr. Locate the pupil or the eye center and gently apply the ocular burr to the eye's surface in circular motions until the epithelium is removed. Alternatively, perform about 20 circular movements on the cornea.Now apply a drop of fluorescent staining solution on the abraided eye. After 20 seconds, dab the eye with a tissue to absorb the solution. Then rinse it with a drop of artificial tear.Finally, absorb the artificial tear with a tissue, and illuminate the eye using a blue cobalt lamp. Prior to the surgery, the basal epithelium was intact and the nerve fibers in the sub basal plexus formed a vortex. The burr tip removed the outer corneal layer, leaving the stroma and endothelium intact with a visible gap at the basal epithelium and sub basal plexus.Regeneration of the epithelium occurred over a week post-surgery. However, the vortex remained absent from the corneal center.

小鼠角膜神经磨损诱导神经营养性角膜炎

用于行为研究的 C57BL/6 小鼠的青少年社会压力范式

Social Defeat Stress Model for Adolescent C57BL&#47;6 Male and Female Mice

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female rodents is needed to understand its effects on ongoing brain development and behavioral outcomes. The chronic social defeat stress paradigm (CSDS) has been widely used to model social stress in adult C57BL/6 male mice by leveraging on the aggressive behavior displayed by an adult male rodent to an intruder invading its territory. An advantage of this paradigm is that it allows to categorize defeated mice into resilient and susceptible groups based on their individual differences in social behavior 24 h after the last defeat session. Implementing this model in adolescent C57BL/6 mice has been challenging because adult or adolescent mice do not typically attack early adolescent male or female mice and because adolescence is a short period of life, encompassing discreet temporal windows of vulnerability. This limitation was overcome by adapting an accelerated version of the CSDS to be used for adolescent male and female mice. This 4-day stress paradigm with 2 physical attack sessions per day uses a C57BL/6 male adult to prime the CD-1 mouse for aggressiveness such that it readily attacks the male or female adolescent mouse. This model was termed accelerated social defeat stress (AcSD) for adolescent mice. Adolescent exposure to AcSD induces social avoidance 24 h later in both males and females, but only in a subset of defeated mice. This vulnerability occurs despite the number of attacks being consistent across sessions between resilient and susceptible groups. The AcSD model is short enough to allow exposure during discrete periods within adolescence, allows the segregation of mice according to the presence or absence of social avoidance behavior, and is the first model available to study social defeat stress in adolescent C57BL/6 female mice.

作者聚焦：了解青少年社交逆境对小鼠神经发育的影响

We developed an accelerated social defeat stress model for adolescent C57BL/6 mice, which works in both males and females and allows exposure during discrete adolescent periods. Exposure to this model induces social avoidance, but only in a subset of defeated male and female mice.

青少年C57BL/6雄性和雌性小鼠的社交失败应激模型

Social adversity in adolescence is prevalent and can negatively impact mental health trajectories. Modeling social stress in adolescent male and female ...