Name: experimental methods for testing the effects of neurotrophic peptide, adnf-9, against alcohol-induced apoptosis during pregnancy in c57bl/6 mice
Uploaded: 2013-04-24T12:00:00
Description: 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

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?...

<ol>
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M., Ge, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+neurotrophic+factor-derived+peptide+prevents+alcohol-induced+apoptosis,+in+part,+through+Bcl2+and+c-Jun+N-terminal+kinase+signaling+pathways+in+fetal+brain+of+C57BL/6+mouse.">Activity-dependent neurotrophic factor-derived peptide prevents alcohol-induced apoptosis, in part, through Bcl2 and c-Jun N-terminal kinase signaling pathways in fetal brain of C57BL/6 mouse.</a> Neuroscience. 202, 465-473 (2012).</li><li>Sari, Y., Zhang, M., Mechref, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+proteins+in+fetal+brains+of+alcohol-treated+prenatally+C57BL/6+mice:+a+proteomic+investigation.">Differential expression of proteins in fetal brains of alcohol-treated prenatally C57BL/6 mice: a proteomic investigation.</a> Electrophoresis. 31, 1-14 (2010).</li><li>Bassan, M., Zamostiano, R., Davidson, A., Pinhasov, A., Giladi, E., Perl, O., Bassan, H., Blat, C., Gibney, G., Glazner, G., 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=Complete+sequence+of+a+novel+protein+containing+a+femtomolar-activity-dependent+neuroprotective+peptide.">Complete sequence of a novel protein containing a femtomolar-activity-dependent neuroprotective peptide.</a> J. Neurochem. 72 (3), 1283-1293 (1999).</li><li>Brenneman, D. E., Hauser, J., Neale, E., Rubinraut, S., Fridkin, M., Davidson, A., 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=Activity-dependent+neurotrophic+factor:+structure-activity+relationships+of+femtomolar-acting+peptides.">Activity-dependent neurotrophic factor: structure-activity relationships of femtomolar-acting peptides.</a> J. Pharmacol. Exp. Ther. 285 (2), 619-627 (1998).</li><li>Spong, C. Y., Abebe, D. T., Gozes, I., Brenneman, D. E., Hill, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+fetal+demise+and+growth+restriction+in+a+mouse+model+of+fetal+alcohol+syndrome.">Prevention of fetal demise and growth restriction in a mouse model of fetal alcohol syndrome.</a> J. Pharmacol. Exp. Ther. 297 (2), 774-779 (2001).</li><li>Sari, Y., 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=Brain+deficits+associated+with+fetal+alcohol+exposure+may+be+protected,+in+part,+by+peptides+derived+from+activity-dependent+neurotrophic+factor+and+activity-dependent+neuroprotective+protein.">Brain deficits associated with fetal alcohol exposure may be protected, in part, by peptides derived from activity-dependent neurotrophic factor and activity-dependent neuroprotective protein.</a> Brain Res. Brain Res. 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. 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=Vasoactive+intestinal+peptide+(VIP)+prevents+neurotoxicity+in+neuronal+cultures:+relevance+to+neuroprotection+in+Parkinson's+disease.">Vasoactive intestinal peptide (VIP) prevents neurotoxicity in neuronal cultures: relevance to neuroprotection in Parkinson's disease.</a> Brain Res. 854 (1-2), 257-262 (2000).</li><li>Steingart, R. A., Solomon, B., Brenneman, D. E., Fridkin, M., 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=VIP+and+peptides+related+to+activity-dependent+neurotrophic+factor+protect+PC12+cells+against+oxidative+stress.">VIP and peptides related to activity-dependent neurotrophic factor protect PC12 cells against oxidative stress.</a> J. Mol. Neurosci. 15 (3), 137-145 (2000).</li></ol>

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>
 </tbody>
</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

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