Name: assessment of ultrastructural neuroplasticity parameters after in utero transduction of the developing mouse brain and spinal cord
Uploaded: 2019-02-26T14:45:00
Description: Watch this Scientific Journal Video about Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord at JoVE.com

作者感谢鲁尔大学波鸿医学院动物设施的同事们的支持和动物护理。

关于动物问题的所有程序都得到了德国联邦各州汉堡和北莱茵-韦斯特法伦的动物伦理机构委员会的批准。 在整个外科过程中使用无菌仪器、防护手套和无菌外套。
1. 子宫传导
<ol><li>在 ph 值7.4 的磷酸盐缓冲盐水 (pbs) 中, 为所需的靶标 (4 x10 11 病毒凝集物μμμl) 准备与腺病毒相关的 1 (aav1) 编码。加入 0.1 mgμμl 快速绿色, 并将 aav1-fast-绿色混合物保持在37°c。</li>
	<li>?...

<ol>
	<li>Blackstad, T. W., Kjaerheim, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+axo-dendritic+synapses+in+the+hippocampal+cortex:+electron+and+light+microscopic+studies+on+the+layer+of+mossy+fibers.">Special axo-dendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of mossy fibers.</a> Journal of Comparative. Neurology. 117, 133159 (1961).</li><li>Hamlyn, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+the+mossy+fibre+endings+in+the+hippocampus+of+the+rabbit.">The fine structure of the mossy fibre endings in the hippocampus of the rabbit.</a> Journal of Anatomy. 96, 112-120 (1962).</li><li>Peters, A., Palay, S., Webster, 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 Fine Structure of the Nervous System. , (1991).</li><li>Rollenhagen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+determinants+of+transmission+at+large+hippocampal+mossy+fiber+synapses.">Structural determinants of transmission at large hippocampal mossy fiber synapses.</a> Journal of Neuroscience. 27, 10434-10444 (2007).</li><li>Lutz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+basic+protein+cleaves+cell+adhesion+molecule+L1+and+promotes+neuritogenesis+and+cell+survival.">Myelin basic protein cleaves cell adhesion molecule L1 and promotes neuritogenesis and cell survival.</a> Journal of Biological Chemistry. 289, 13503-13518 (2014).</li><li>Lutz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+Basic+Protein+Cleaves+Cell+Adhesion+Molecule+L1+and+Improves+Regeneration+After+Injury.">Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Improves Regeneration After Injury.</a> Molecular Neurobiology. 53, 3360-3376 (2016).</li><li>Kraus, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Fragment+of+Adhesion+Molecule+L1+Binds+to+Nuclear+Receptors+to+Regulate+Synaptic+Plasticity+and+Motor+Coordination.">A Fragment of Adhesion Molecule L1 Binds to Nuclear Receptors to Regulate Synaptic Plasticity and Motor Coordination.</a> Molecular Neurobiology. 55, 7164-7178 (2018).</li><li>Andres, K. H., von D&#252;ring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferenzph&#228;nomene+am+osmierten+Pr&#228;parat+f&#252;r+die+systematische+elektronenmikroskopische+Untersuchung.">Interferenzph&#228;nomene am osmierten Pr&#228;parat f&#252;r die systematische elektronenmikroskopische Untersuchung.</a> Mikroskopie. 30, 139 (1974).</li><li>Andres, K. H., von D&#252;ring, M., Hayat, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+phenomenon+on+somium+tetroxide-fixed+specimens+for+systematic+electron+microscopy.">Interference phenomenon on somium tetroxide-fixed specimens for systematic electron microscopy.</a> Principle and Techniques of Electron Microscopy: Biological Appliccations. , 246-261 (1997).</li><li>Andres, K. H., von D&#252;ring, M., Heym, C., Forssmann, W. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+Methods+for+Characterization+of+Brain+Regions.">General Methods for Characterization of Brain Regions.</a> Techniques in Neuroanatomical Research. , 100-108 (1981).</li><li>Palay, S. L., McGee-Russel, S. M., Gordon, S., Grillo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+of+neural+tissues+or+electron+microscopy+by+perfusion+with+solutions+of+osmium+tetroxide.">Fixation of neural tissues or electron microscopy by perfusion with solutions of osmium tetroxide.</a> Journal of Cell Biology. 12, 385-410 (1962).</li><li>Webster, H. F., Collins, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+osmium+tetroxide+and+glutaraldehyde+perfusion+fixation+for+the+electron+microscopic+study+on+the+normal+rat+peripheral+nervous+system.">Comparison of osmium tetroxide and glutaraldehyde perfusion fixation for the electron microscopic study on the normal rat peripheral nervous system.</a> Journal of Neuropathology and Experimental Neurology. 1, 109-126 (1964).</li><li>Andres, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Methodik+der+Perfusionsfixierung+des+Zentralnervensystems+von+S&#228;ugern.">Zur Methodik der Perfusionsfixierung des Zentralnervensystems von S&#228;ugern.</a> Mikroskopie. 21, 169 (1967).</li><li>Forssmann, W. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+par+perfusion+pour+la+microscopie+&#233;lectronique.+Essai.+De+g&#233;n&#233;ralisation.">Fixation par perfusion pour la microscopie &#233;lectronique. Essai. De g&#233;n&#233;ralisation.</a> Journal de Microscopie. 6, 279-304 (1967).</li><li>Descarries, L., Schr&#246;der, 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=Fixation+du+tissue+nerveux+par+perfusion+&#224;+grand+debit.">Fixation du tissue nerveux par perfusion &#224; grand debit.</a> Journal de Microscopie. 7, 281-286 (1968).</li><li>Langford, L. A., Coggeshall, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+potassium+ferricyanide+in+neural+fixation.">The use of potassium ferricyanide in neural fixation.</a> Anatomical Records. 197, 297-303 (1980).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calretinin-positive+L5a+pyramidal+neurons+in+the+development+of+the+paralemniscal+pathway+in+the+barrel+cortex.">Calretinin-positive L5a pyramidal neurons in the development of the paralemniscal pathway in the barrel cortex.</a> Molecular Brain. 7, 84 (2014).</li><li>Lee, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilins+regulate+synaptic+plasticity+and+mitochondrial+calcium+homeostasis+in+the+hippocampal+mossy+fiber+pathway.">Presenilins regulate synaptic plasticity and mitochondrial calcium homeostasis in the hippocampal mossy fiber pathway.</a> Molecular Neurodegeneration. 12, 48 (2017).</li><li>LoTurco, J., Manent, J. B., Sidiqi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+and+improved+tools+for+in+utero+electroporation+studies+of+developing+cerebral+cortex.">New and improved tools for in utero electroporation studies of developing cerebral cortex.</a> Cerebral Cortex. , 120-125 (2009).</li><li>dal Maschio, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+and+site-directed+in+utero+electroporation+by+a+triple-electrode+probe.">High-performance and site-directed in utero electroporation by a triple-electrode probe.</a> Nature Communications. 3, 960 (2012).</li><li>Nishiyama, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+and+regulated+gene+expression+in+murine+Purkinje+cells+by+in+utero+electroporation.">Selective and regulated gene expression in murine Purkinje cells by in utero electroporation.</a> European Journal of Neuroscience. 36, 2867-2876 (2012).</li><li>Takeo, Y. H., Kakegawa, W., Miura, E., Yuzaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RORalpha+regulates+multiple+aspects+of+dendrite+development+in+cerebellar+purkinje+cells+in+vivo.">RORalpha regulates multiple aspects of dendrite development in cerebellar purkinje cells in vivo.</a> Journal of Neuroscience. 35, 12518-12534 (2015).</li></ol>

在子宫转导的一个关键的步骤是注射程序。精确注射到脑室或其他感兴趣的领域需要经验和动手技巧。微毛细管尖端越薄, 可能发生的组织损伤越少;然而, 这是以增加注射压力为代价的。与子宫电穿孔19、20、21、22 相比, 在子宫转导后注射胚胎的存活率非常高。所有子宫角的胚胎都可以注射, 即使胚胎?...

任何光子都不能穿透电子深度等级的超薄组织标本。与光学显微镜技术相比, 这在捕获精细结构的纳米分辨率图像方面具有宝贵的优势。例如, tem 允许细胞内细胞器的可视化, 如线粒体、黑色素体和各种类型的分泌颗粒、微管、微丝、纤毛、微绒毛和细胞间连接 (细胞表面)(专业), 特别是突触在神经系统1,2,3,4。本方法研究的总体目标是结合子宫内的最先进技术, 对产前干预后发育过程中神经可塑性变化的超微结构识别。病毒编码的感兴趣的蛋白质在子宫中被转化为中枢神经系统5,6,7, 包括脊髓6。例如, 在子宫转导联合 tem 已被用来研究细胞粘附分子 l1 对 l1 缺乏小鼠运动学习可塑性的影响, 特别是在 l1 和核受体蛋白之间的相互作用在小脑神经元7。
神经可塑性参数的分析需要关于神经系统中最小区域定位的精确信息。因此, 它是足够的描述超微结构细节和他们的确切地形方向相对于其他结构。在本研究中, 提出了一种基于光镜和电子显微镜的具体准备方法, 旨在对不同的形态区域进行详细的研究。这种方法结合了组织操作的几种技术, 从小鼠大脑和脊髓的子宫转导开始, 然后是灌注固定, 霉菌包埋, 并处理组织的 tem。在透射电镜组织的嵌入和处理之间的一个重要步骤是组织的记录, 使用干涉光反射技术, 允许精确的微照片和低放大倍率的记录组织标本8,9,10。在目前的方法中, 这项技术使研究人员能够在准备 tem 之前检查神经组织表面和标本切片剖面的地形和结构细节。
一个特殊的框架, 用于分割整个大脑对应于立体定向坐标。该框架有利于神经组织中区域的形态三维重建, 可用于形态分析。可视化剖面的宏观图被指定为地形坐标, 按顺序编号的部分在组织地图集中生成地图。
根据上述组织地图集的地图, 在树脂处理后, 将嵌入组织切割成含有选定区域的超薄切片 (& lt;70 nm)。超薄切片采用透射电镜, 以获得其内容物的可塑性参数 (例如突触波或轴突纤维的横截面剖面区域) 的高分辨率图像, 以及与复合物内相邻结构的接触的图像神经皮。
通过本文所述的方法, 从可视化宏观结构到微观和纳米结构的平稳过渡, 使发育中神经的子宫传导后的形态神经元可塑性进行比较深入的研究系统。

<table border="0" cellpadding="0" cellspacing="0" style="width:969px;" width="968">
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		<tr height="40">
			<td height="40" style="height:40px;width:257px;">2,4,6-Tris(dimethyl-aminomethyl)phenol</td>
			<td style="width:167px;">Serva</td>
			<td style="width:123px;">36975</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">26Gx 1&#39;&#39; needle</td>
			<td style="width:167px;">Henke-Sass, Wolf GmbH</td>
			<td style="width:123px;"></td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td>410 Anaesthesia Unit for air pump</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">8323102</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="414">
			<td>Adeno-associated virus serotype 1 (AAV1)</td>
			<td>UKE (Viral Core Facility)</td>
			<td>-</td>
			<td>For references and target areas of AAV1 see: https://www.addgene.org/viral-vectors/aav/aav-guide/ and also: Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Kwon I, Schaffer DV. Pharm Res. 2008 Mar;25(3):489-99. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Burger C, Gorbatyuk OS, Velardo MJ, Peden CS, Williams P, Zolotukhin S, Reier PJ, Mandel RJ, Muzyczka N. Mol. Ther. 2004 Aug;10(2):302-17. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. McCarty DM, Monahan PE, Samulski RJ. Gene Ther. 2001 Aug;8(16):1248-54.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Agarose</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:123px;">A9414</td>
			<td style="width:423px;">low gelling agarose</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Air Pump</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">Eheim 100</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Araldite</td>
			<td style="width:167px;">CIBA-GEIGY</td>
			<td style="width:123px;">23857.9</td>
			<td style="width:423px;">resin for embedding of tissue</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">aspirator tune assemblies</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:123px;">A5177-5EA</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Breathing Mask Mouse Anodized Aluminium</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">buprenorphine</td>
			<td style="width:167px;">Temgesic</td>
			<td style="width:123px;">ampules</td>
			<td style="width:423px;">painkiller</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">capillaries</td>
			<td style="width:167px;">Science-Products</td>
			<td style="width:123px;">GB100TF-10</td>
			<td style="width:423px;">with fillament</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Dodecenylsuccinic anhydride</td>
			<td style="width:167px;">Fluka</td>
			<td style="width:123px;">44160</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Dumont tweezers (#3, 12 cm, straight, 0.2 x 0.12 mm)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">11203-23</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">electric shaver</td>
			<td style="width:167px;">Phillips</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Ethicon sutures (Ethilon, 6-0 and 3-0)</td>
			<td style="width:167px;">Ethicon</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">polyamide</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">eye lubricant</td>
			<td style="width:167px;">Bepanthene</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:257px;">Fast Green</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:123px;">F7252</td>
			<td style="width:423px;">for visualization of injected liquids</td>
		</tr>
		<tr height="133">
			<td height="133" style="height:133px;width:257px;">Gas Routing Switch 4/2 connectors</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">8433020</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;width:257px;">halsted Mosquito hemostatic forceps (12.5 cm, straight)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">13011-12</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:257px;">Heparin-Natrium</td>
			<td style="width:167px;">Ratiopharm</td>
			<td style="width:123px;"></td>
			<td style="width:423px;">25 000 I.E./5 ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Induction box for mice 
			with horizontally moving lid. 
			Inner dimensions: LxBxH: 155x115x130 mm. 
			Wall thickness: 6 mm</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">iris forceps (10cm, curved, serrated)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">14007-14</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">iris scissors (11cm, straight, tungsten carbide)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">14501-14</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">Isofluran OP Tisch, electrically heated, sm 
			Outer dimensions: 257x110x18 mm. 
			Heating area: 190x90 mm 
			The removal of the isoflurane escaping 
			the breathing mask is downwards in compliance with the 
			regulations</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">isoflurane (Attane)</td>
			<td style="width:167px;">JD medical</td>
			<td style="width:123px;"></td>
			<td style="width:423px;">inhalation anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:257px;">LED RGB lights</td>
			<td style="width:167px;">Cameo</td>
			<td style="width:123px;">CLQS15RGBW</td>
			<td style="width:423px;">LEDs 2 x 15 W</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Light microscope Basic DM E</td>
			<td style="width:167px;">Leica</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">4x (N.A. 0.1 &#8734;/-), 10x (N.A. 0.22 &#8734;/0.17), 40x (N.A. 0.65 &#8734;/0.17), 100x (N.A. 1.25 &#8734;/0.17) objectives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">micropipette puller</td>
			<td style="width:167px;">Science-Products</td>
			<td style="width:123px;">P-97</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Mosquito hemostatic forceps (12.5cm, curved)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">13010-12</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Nickel grids, 200 mesh</td>
			<td style="width:167px;">Ted Pella</td>
			<td style="width:123px;">1GC200</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Osmium (VIII)-oxid</td>
			<td style="width:167px;">Degussa</td>
			<td style="width:123px;">73219</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Propylene oxide</td>
			<td style="width:167px;">Fluka</td>
			<td style="width:123px;">82320</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">razor blades</td>
			<td style="width:167px;">Schick</td>
			<td style="width:123px;">87-10489</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Sodium pentobarbital (Narcoren)</td>
			<td style="width:167px;">Merial GmbH</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TC01mR 1-Channal temperature controller with feedback</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Technovit 4004 two components glue</td>
			<td style="width:167px;">Kulzer</td>
			<td style="width:123px;"></td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:257px;">Telemacrodevice</td>
			<td style="width:167px;">Canon</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">Canon Spiegelreflex Kamera EOS2000D, EF-S 18-55 mm f/3.5-5.6 IS STM Objective, Extension below 150 mm, Manual Extension Tube 7 mm ring, 14 mm ring, 28 mm ring, Macro reverse ring (58 mm), Canon copy stand.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Thermopuller P-97</td>
			<td style="width:167px;">Sutter Instruments</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">thin vibrating razor blade device</td>
			<td style="width:167px;">Krup</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">with Szabo thin blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">toluidine blue</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td align="right">89640</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Transmission electron microscope C20</td>
			<td style="width:167px;">Phillips</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">up to 200 kV</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:257px;">Tygon 6/4 Tubing material for connection of all parts 
			Outer diameter: 6mm 
			Inner diameter: 4mm 
			Wa 
			ll thickness: 1mm</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Ultracut E</td>
			<td style="width:167px;">Reichert-Jung</td>
			<td style="width:123px;">-</td>
			<td style="width:423px;">ultramicrotome</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:257px;">Univentor Scavenger</td>
			<td style="width:167px;">Biomedical Instruments (Univentor)</td>
			<td style="width:123px;">8338001</td>
			<td style="width:423px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:257px;">Vannas scissors (8 cm, straight)</td>
			<td style="width:167px;">FST</td>
			<td style="width:123px;">15009-08</td>
			<td style="width:423px;"></td>
		</tr>
	</tbody>
</table>

assessment of ultrastructural neuroplasticity parameters after in utero transduction of the developing mouse brain and spinal cord

本研究结合子宫转导和透射电子显微镜 (tem), 旨在精确地对地形结构中的超微结构参数进行形态测量分析, 这是受感兴趣的蛋白质影响的通过病毒转移引入生物体。这种组合方法允许从宏观结构到超微结构识别的顺利过渡, 通过遵循组织地图集中的地形导航地图。子宫内转染组织的高分辨率电子显微镜揭示了神经 pil 的精细超微结构及其可塑性参数, 如横截面突触 bouton 区、内突触囊和线粒体的数量。bouton 剖面、突触线长度、横截面轴突区、髓鞘厚度、髓鞘层数和线粒体剖面横截面区。对这些参数的分析揭示了对神经系统区域超微结构可塑性变化的重要洞察, 这些变化受到基因结构病毒转移的影响。这种组合方法不仅可以用来研究基因工程生物分子和药物对神经元可塑性的直接影响, 而且还为研究子宫内神经元可塑性的抢救提供了可能性 (例如, 在神经退行性疾病)。

为了对小鼠进行可靠、快速的麻醉, 考虑了许多安全参数, 并且麻醉单元的优化工作空间被证明是足够的 (图 1a)。该装置旨在精确控制液体异氟烷和环境空气的混合物, 并对小动物 (如小鼠和大鼠) 进行成功的手术。空气和异氟醚根据所需的设置混合在蒸发器中, 并通过面罩或通过面罩传送到动物体内 (图 1a</stron...

Watch this Scientific Journal Video about Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord at JoVE.com

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

透射电子显微镜和子宫转导的结合是研究神经系统发育过程中精细超微结构形态变化的有力方法。这种组合方法可以深入了解神经可塑性相对于其地形表现的结构细节的变化。

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

我们的联合方法有助于回答超结构神经可塑性参数在子宫内新生神经系统的早期发育干预后如何变化。我们方法的主要优点是获取组织图集中预定义坐标系中宏-中微和纳米结构的高分辨率图像。我们的方法的含义是它对先天性神经退行性疾病的转化治疗潜力。例如，在胚胎阶段的治疗和抢救，在子宫转导技术中使用这种治疗和抢救。我们的方法可以应用于任何其他模型。例如，可以更改小鼠物种以及感兴趣的区域。首次尝试我们方法的研究人员需要深入的手术训练，以及如何为电子显微成像组织做准备的知识。首先，将含有四倍10的定制毛细管尖端装到11种与腺相关的病毒1型涂层的病毒颗粒，用于所需目标，并贴上快速绿色染料的标签到吸气管上。在毛细管中加入15微升标记的腺相关病毒颗粒。接下来，确认在麻醉胚胎14.5怀孕小鼠中对头趾捏没有反应，并对皮肤进行消毒。使用弯曲锯齿状虹膜钳和直钨硬质合金剪刀，使皮肤切口沿线膜介质。使用直杜蒙钳子抓住腹膜壁，继续沿着线阿尔巴与直Vannas剪刀，并放置一块芬斯特石蜡膜在腹部开口，并使用勺子状装置暴露子宫角，而不会损害角内的胚胎。在子宫角上涂抹几滴37摄氏度的PBS后，检查胚胎的子宫囊内有无损伤或畸形。将胚胎小心地在囊内转动，直到达到每次注射所需的位置。当胚胎就位时，将一到两个微升的快速绿色标记病毒颗粒溶液注入每个胚胎。所有胚胎注射后，将子宫角放回腹腔，并在腹部加入几滴37摄氏度的PBS。然后使用聚酰胺 6-0 大小的缝合线关闭内面壁和聚酰胺 3-0 大小的缝合线和 Halsted 的蚊子止血钳关闭皮肤。要嵌入远程微距摄影感兴趣的孤立组织，请将组织样本放入具有可重现剖面角度的特殊框架中，并记录坐标。用30摄氏度3%的加糖填充框架，直到组织被覆盖，用塑料盖盖住框架，直到加糖硬化。当红糖变硬时，使用远程宏图形设备对框架内的嵌入式组织及其坐标进行成像。当阿加罗斯凝固后，将加糖嵌入的组织转移到框架中，并具有与第一帧坐标对应的切削间隙。使用具有薄振动剃须刀刀片的设备，将嵌入的组织切割成适当实验厚度的部分。然后在 PBS 中对每个组织部分进行图像图像，然后将图像收集到文件夹中。为了准备传输电子显微镜的组织样本，在生物安全柜中，用两次30分钟的PBS洗涤组织部分，随后在室温下用2%水性三氧化二氮溶液进行两小时的孵育。在孵育结束时，每次洗涤在PBS中清洗两次，洗涤30分钟，然后按指示连续10至15分钟乙醇浸入液脱水。70%乙醇浸入后，将每个样品放入一个黑色的盘中，在LED RGB灯下以70%乙醇成像样品，以45度角从左右两侧对样品进行应用。通过收集文件夹中的序列样本图像，创建具有坐标的剖面图像图集。用两个30分钟100%乙醇孵化来处理标本，然后两个30分钟100%丙烯氧化物孵育，均在室温下。当样品孵育时，以一对一和一对二的比例将新鲜准备好的树脂与氧化丙烯混合，并在两种溶液中加入3%的加速器。第二次丙烯氧化物孵育后，将样品转移到一至二树脂丙烯氧化物嵌入介质溶液中，在室温下两小时，随后在室温下将一对一树脂丙烯氧化物嵌入介质溶液中孵育两小时。在第二次孵育结束时，将组织转移到扁平聚丙烯培养皿中，并在65至85摄氏度下用含有3%加速器的新鲜树脂固化嵌入组织，12至24小时。然后将组织冷却至室温，然后从聚丙烯盘上去除树脂嵌入的试样。要映射感兴趣区域，请从以前准备的图像图集中选择包含感兴趣区域的图像。将感兴趣区域的边界绘制到剖面图像上，光学地将这些边界叠加到显微镜下的嵌入式试样上，并使用一英寸 26 量表针将这些区域边界划到树脂试样上。然后，将样品加热到 95 摄氏度，以软化树脂。接下来，从烤箱中取出温暖的树脂样品，并使用剃刀刀片来切除感兴趣的区域。将试样粘附在适当口径的丙烯酸玻璃固定杆上，并修剪安装的试样，进行半薄和超薄的分切。使用超微子获取半薄 0.7 微米和超薄 70 纳米部分，收集玻璃载体上的半薄部分和镍栅格上的超薄部分。在 PBS 中用 1% 的托卢伊丁蓝色染色半薄部分 4 分钟，随后在去维化水中多次洗涤，然后通过光显微镜检查部分。然后，通过180千伏的透射电子显微镜直接评估超薄部分，无需进一步修改。切片后，组织切片通过远程微距摄影进行成像，正如刚刚演示的。在渗透和乙醇浸入后，通过干扰光远程扫描再次对各部分进行成像，为每种组织表面显示一种特定颜色的图案。在额外的脱水和树脂嵌入后，选择感兴趣的区域，并进一步处理传输电子显微镜。在海马和小脑中，与其他突触相比，青苔纤维突触表现出独特的超结构特性，包括大型前突触。在他们的横截面剖面剖面图中，这些牙带含有大量的囊泡和线粒体，并且经常包围几个树突状的脊椎。在脊髓中，后脑膜含有许多由寡头纤维制成的斧质纤维。在横向部分，可以在脊髓的两个后角之间找到背角。在传输电子显微镜图像上，横截面髓轴子出现圆形，轴子周围的骨髓护套被组织在拉梅莱。编制的图集和超结构参数的微图传输电子显微镜图像，不仅有助于比较不同处理条件下不同部分的形态神经可塑性，而且有助于同一区域内参数的比较。请记住，在开始子宫转导之前，要进行彻底的计划和准备，并确保在出现并发症时有材料和仪器的备份。按照我们的方法，可以添加其他技术，如电子显微镜前的免疫金标签，用于对感兴趣的特定分子进行超结构跟踪。从整个生物体的整体视图开始，并放大分子地形，我们能够研究和操纵组织功能和形态之间的关系。三氧化二氮是危险的。使用这种试剂时，请务必在发动机罩下工作，并戴上防护眼镜和手手套。

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

科研

发育生物学

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

内皮细胞迁移评估暴露于有毒化学品后，

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

珠嫁接到发展鸡胚四肢识别信号转导途径影响基因表达

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

产妇血管重构的评估在怀孕期间在小鼠子宫

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

发育小鼠垂体的解剖和冠状切片制备

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

小鼠宫内移植中静脉和羊膜内植入的研究

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

发展中雏鸡光学顶盖中切向细胞迁移的可视化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...