作者要感谢ADIR（爱德住所辅助Insuffisants Respiratoires）和基金会去l&#39;Avenir酒店提供的财政支持，以及博士查波斯巴纳比 - 海德编辑稿件。

1。嗅粘膜和嗅球的原代培养<ol><li>此前解剖<ol><li>使介质，50毫升，加入44毫升的无钙的Dulbecco改进的Eagle / Ham氏F12培养基（DMEM/F12），补充用5毫升胎牛血清（FBS）和1毫升青霉素/链霉素的。 </li><li>外套75 厘米的烧瓶用聚-L-赖氨酸（50微克/毫升，1.5微克/厘米2），1小时，在室温下进行。 </li><li>冲洗瓶用PBS 1X。 </li><li>储存在冰箱中的涂层烧瓶达1周。 </li></ol></li><li>解剖<ol><li>安乐死的任何方法都必须通过该机构的动物护理和使用委员会事先批准而由合格的人员进行。 </li><li>选择近交系动物，如大鼠菲舍尔。 </li><li>后大鼠已被证实已经进入深度麻醉用戊巴比妥钠麻醉或其它可注射的形式，如氯胺酮/赛拉嗪，斩首他和r的eMove皮肤。 </li><li>打开颅骨由前至后的部分。 </li><li>取出OB注意避免脑膜。 </li><li>在相同的大鼠，打开鼻腔sagittaly。 </li><li>拆下OM注意避免呼吸道黏膜。注意，OM上皮可通过其泛黄的颜色容易地识别。 </li><li>关于有机质夹层进一步详细的协议可以发现在以前的<a href="https://www.jove.com/video/2762/isolating-nasal-olfactory-stem-cells-from-rodents-or-humans">J。可见。进出口</a> 。出版13。 </li></ol></li><li>文化<ol><li>放置OB在汉克的含0.1％胰蛋​​白酶45分钟，在37℃的缓冲盐溶液（HBSS） </li><li> OM放置在HBSS中含0.05％胶原酶一种45分钟，在37°C。 </li><li>加入2毫升温介质停止酶消化。 </li><li>与介质洗涤细胞并离心它们在300×g离心3分钟。 </li><li> 2样品磨碎毫升培养基用移液管P1000，直到获得均匀的细胞悬浮液。 </li><li>用25毫升温暖的培养基的盘在预涂烧瓶（5-10×10 6细胞/烧瓶）中的单元格。 </li><li>孵化和培养的细胞，在37℃，5％的CO 2。 </li><li>改变一半的培养基，每2天。 </li><li>后在体外 6-8天，查小细胞样本的p75阳性细胞的比例，假定为嗅鞘细胞在啮齿类动物，通过流式细胞仪或免疫细胞化学。对于这些分析，其他标志物可以研究诸如GFAP和S100β。 </li></ol></li><li>流式细胞仪分析<ol><li>如在“细胞移植”（第2.2段）说明收集细胞。 </li><li>在15分钟内于4℃下孵育它们与p75的第一抗体（1:100） </li><li>用2ml PBS-EDTA洗细胞并离心它们在300×g离心3分钟。 </li><li>在15孵育的细胞用PE偶联的抗小鼠二抗分钟，在4°C黑暗（1:200）。 </li><li>用2ml PBS-EDTA洗细胞并离心它们在300×g离心3分钟。 </li><li>重悬在500微升PBS-EDTA的细胞。 </li><li>用流式细胞仪分析10-30×10 3个细胞。 </li></ol></li><li>免疫组化分析<ol><li>板块中，如“文化”（第1.1.2段）描述预涂48小时6孔板的细胞。 </li><li>用PBS洗涤细胞。 </li><li>固定细胞用PFA 4％，持续15分钟，在室温。 </li><li>用PBS洗涤细胞。 </li><li>孵育的细胞用PBS /的Triton X100（0.1％）为15分钟，在室温。 </li><li>用PBS洗涤细胞。 </li><li>孵育的细胞与第一抗体（1:200）：P75，S100β和GFAP过夜，在4℃下</li><li>用PBS洗涤细胞。 </li><li>在1小时在室温下在黑暗中孵育的细胞与第二抗体（1:500）。 </li><li>用PBS洗涤细胞。 </li><li>孵育细胞在R赫斯特10分钟T IN黑暗。 </li><li>用PBS洗涤细胞。 </li><li>用荧光显微镜分析细胞。 </li></ol></li><li>绿色荧光蛋白标记的细胞<ol><li>移植前五天，感染OB和OM培养过夜慢病毒载体携带增强型绿色荧光蛋白（感染复数：20）。 </li><li>支票上的小样本GFP阳性细胞的流式细胞仪在“文化”（1.1节）前面描述的速率移植前。 </li></ol></li></ol> 2。手术和移植<ol><li>喉返神经吻合术<ol><li>所有实验动物必须通过该机构的动物护理和使用委员会事先批准而由合格的人员进行。 </li><li>术前高压灭菌器的所有仪器和保持无菌整个手术过程。 </li><li>通过腹膜内注射氯胺酮盐酸盐（12.5毫克/千克）和盐酸氯丙嗪（麻醉大鼠0.625毫克/千克）。手术前麻醉评估的充分性由脚趾捏。 </li><li>将大鼠在背卧位。 </li><li>用手术刀进行2厘米垂直介质宫颈皮肤切口。 </li><li>下面的内侧白线执行舌骨下肌群的垂直切口。 </li><li>通过使用舌骨下肌群之间自动静态拉钩暴露喉。 </li><li>暴露喉返神经。 </li><li>根据微观控制的，完全切断神经与microscissors在第七气管环的水平。 </li><li>使用显微镜下控制一个点的11.0缝线进行吻合。 这种手术导致喉部的特定马达去神经。 </li></ol></li><li>细胞移植<ol><li>只是在移植前，在37℃用0.05％胰蛋白酶EDTA 5分钟移除菜肴细胞</li><li>加入2毫升温介质停止酶消化。 </li><li>离心机在300×g离心3分钟。 </li><lI&gt;在显微镜下用血细胞计数器计数细胞并根据特定的协议调整细胞浓度。在这个模型中，期望的浓度之间1.2-6×10 6个细胞中加入30μl的DMEM/F12。 </li><li>使细胞的制备与DMEM/F12-Matrige升1:2的混合物（30和60微升在这个实验）在1.5ml管移植之前，将其放在冰上。 为了避免非特异性作用，使用生长因子减少基底膜。 </li><li>只是移植前将灯管保持你的手几秒钟热身基质胶介质混合。 </li><li>废黜混合使用吸管的吻合部位，等到对受损神经的混合自组装。 </li><li>关闭肌肉和皮肤有4.0缝合。 </li><li>单独的房子，并保持在一个加热灯24小时的大鼠。 </li></ol></li></ol> 3。评价<ol><li>喉镜检查<ol><li>通过intraperit大鼠麻醉奥尼尔注射盐酸氯胺酮（12.5毫克/千克）和盐酸氯丙嗪（0.625毫克/千克）。确认大鼠前着手协议麻醉用脚趾捏。 </li><li>将大鼠在背卧位。 </li><li>插入一个30°视频喉镜调整，以提供喉部的最佳视图。确定解剖标志再现了同样的观点为每个记录。 </li><li>使用视频喉镜（5-10序列/动物）录制声带运动。对于每个动物选择连续最大内收和外展最大的三个序列。 </li><li>使用图像分析软件分析喉功能。通过测量最大绑架和声带的最大内收运动之间的差确定的绝对角运动。通过测量运动幅度确定动态得分。通过测量联带运动和paradoxal走势判断功能评分。 </li></ol></li><li>肌电图（EMG） <ol><l我&gt;将电极对大鼠进行电气隔离的腿筋肌肉。 </li><li>通过在“喉返神经吻合术”（2.1节）前述开口的皮肤和肌肉暴露喉部和气管。 </li><li>暴露环甲软骨。 </li><li>开环甲软骨用microscissors，露出环杓后（PCA）的肌肉。 </li><li>在PCA肌肉引入单极针电极（38×0.45mm）内。 </li><li>录制过程中自然通风利用采集系统的PCA肌肉的肌肉电活动。 </li><li>分析的丰富性和同步呼吸方面的肌肉活动。灵感，1期间无韵跟踪，无需增加：0：分析肌电信号，采用以下比例分配一个定性的评分从0-3押韵跟踪与吸气增加，但可怜的跟踪（neurogen），2：押韵更丰富的活动跟踪， 3：押韵跟踪，非常丰富，构成一个i消除干扰图案，类似于一个最大故意活性。 </li><li>揭迷走神经来测量延迟和潜在的持续时间。迷走神经被选为刺激的部位，因为喉返神经可刺激过程中，由于其体积小而损坏。 </li><li>刺激迷走神经与电极。 </li><li>记录电信号在PCA肌肉。测量延迟和潜在的持续时间。如果可能的话使用一个采集模块来记录肌肉活动，延迟和潜在的持续时间，如PowerLab系统系统。 </li></ol></li><li>组织学<ol><li>用戊巴比妥过量安乐死的动物。 </li><li>删除喉返神经的远端残端下的微观控制。 RLN的远侧部分被定义为吻合口和喉之间的部分。注意，有可能找到吻合部位手术后数周，因为11.0缝线是不可吸收的缝线。 </li><li>用0.1M磷酸盐放置RLN的远侧部分中的2％戊二醛（pH值= 7.3），2小时，在4℃下</li><li>精心定位的样本，以实现冠状切片。 </li><li>在磷酸盐缓冲液洗涤样品。 </li><li>切成更小的段（3-4毫米长）和后缀，在室温下60分钟，在1％的二甲胂酸盐缓冲的OsO 4和脱水乙醇。 </li><li>在硅胶模具东方样品并包埋在树脂组成中POLYBED 812，DDSA和MNA，向其中加入加速器BDMA的2％。 </li><li>使用Pyramitome超薄切片系统做半薄横截面（1微米厚）。 </li><li>用0.1％甲苯胺蓝在70℃下染色的1％四硼酸钠的75秒</li><li>分析喉返神经样品计数分析系统。数纤维的数量和通过测定髓鞘的外表面和内边界测量髓神经纤维进行访问。 </li></ol></li><li>移植<ol><li>制备细胞移植及移植如前所述的细胞在“细胞移植”（第2.2节）。 </li><li>分析</li><li>用戊巴比妥过量安乐死的动物。注意：可以使用腔内注射PFA的4％进行了动物的固定。 </li><li>删除喉返神经周围2厘米。使用近端和RLN的远侧部分。 </li><li>固定RLN样品在液氮中。 </li><li>样品储存在-80°C。 </li><li>切割样品纵向使用低温恒温器（10-20微米厚）。 </li><li>分析荧光显微镜下的样品以检测GFP阳性细胞。注意，抗GFP抗体可用于增加荧光发射和执行costainings。 </li></ol></li></ol>

<ol>
	<li>Barraud, P., 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=Neural+crest+origin+of+olfactory+ensheathing+glia.">Neural crest origin of olfactory ensheathing glia.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 21040-21045 (2010).</li><li>Guerout, N., 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=Comparative+gene+expression+profiling+of+olfactory+ensheathing+cells+from+olfactory+bulb+and+olfactory+mucosa.">Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa.</a> Glia. 58, 1570-1580 (1002).</li><li>Honore, 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=Isolation,+characterization,+and+genetic+profiling+of+subpopulations+of+olfactory+ensheathing+cells+from+the+olfactory+bulb.">Isolation, characterization, and genetic profiling of subpopulations of olfactory ensheathing cells from the olfactory bulb.</a> Glia. 60, 404-413 (2012).</li><li>Franssen, E. H., de Bree, F. M., Verhaagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+glia:+their+contribution+to+primary+olfactory+nervous+system+regeneration+and+their+regenerative+potential+following+transplantation+into+the+injured+spinal+cord.">Olfactory ensheathing glia: their contribution to primary olfactory nervous system regeneration and their regenerative potential following transplantation into the injured spinal cord.</a> Brain Res. Rev. 56, 236-258 (2007).</li><li>Lakatos, A., Franklin, R. J., Barnett, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+and+Schwann+cells+differ+in+their+in+vitro+interactions+with+astrocytes.">Olfactory ensheathing cells and Schwann cells differ in their in vitro interactions with astrocytes.</a> Glia. 32, 214-225 (2000).</li><li>Woodhall, E., West, A. K., Chuah, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultured+olfactory+ensheathing+cells+express+nerve+growth+factor,+brain-derived+neurotrophic+factor,+glia+cell+line-derived+neurotrophic+factor+and+their+receptors.">Cultured olfactory ensheathing cells express nerve growth factor, brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor and their receptors.</a> Brain Res. Mol. Brain Res. 88, 203-213 (2001).</li><li>Dombrowski, M. A., Sasaki, M., Lankford, K. L., Kocsis, J. D., Radtke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+and+nodal+formation+of+regenerated+peripheral+nerve+fibers+following+transplantation+of+acutely+prepared+olfactory+ensheathing+cells.">Myelination and nodal formation of regenerated peripheral nerve fibers following transplantation of acutely prepared olfactory ensheathing cells.</a> Brain Res. 1125, 1-8 (2006).</li><li>Guerout, N., 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=Transplantation+of+olfactory+ensheathing+cells+promotes+axonal+regeneration+and+functional+recovery+of+peripheral+nerve+lesion+in+rats.">Transplantation of olfactory ensheathing cells promotes axonal regeneration and functional recovery of peripheral nerve lesion in rats.</a> Muscle Nerve. 43, 543-551 (2011).</li><li>Guntinas-Lichius, O., 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=Transplantation+of+olfactory+ensheathing+cells+stimulates+the+collateral+sprouting+from+axotomized+adult+rat+facial+motoneurons.">Transplantation of olfactory ensheathing cells stimulates the collateral sprouting from axotomized adult rat facial motoneurons.</a> Exp. Neurol. 172, 70-80 (2001).</li><li>Makoukji, 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=Lithium+enhances+remyelination+of+peripheral+nerves.">Lithium enhances remyelination of peripheral nerves.</a> Proc. Natl. Acad. Sci U.S.A. 109, 3973-3978 (2012).</li><li>Guerout, N., 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=Co-transplantation+of+olfactory+ensheathing+cells+from+mucosa+and+bulb+origin+enhances+functional+recovery+after+peripheral+nerve+lesion.">Co-transplantation of olfactory ensheathing cells from mucosa and bulb origin enhances functional recovery after peripheral nerve lesion.</a> PloS one. 6, 22816 (2011).</li><li>Paviot, 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=Efficiency+of+laryngeal+motor+nerve+repair+is+greater+with+bulbar+than+with+mucosal+olfactory+ensheathing+cells.">Efficiency of laryngeal motor nerve repair is greater with bulbar than with mucosal olfactory ensheathing cells.</a> Neurobiol. Dis. 41, 688-694 (2011).</li><li>Girard, S. 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=Isolating+nasal+olfactory+stem+cells+from+rodents+or+humans.">Isolating nasal olfactory stem cells from rodents or humans.</a> J. Vis. Exp. , (2011).</li><li>Nash, H. H., Borke, R. C., Anders, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+of+purification+for+establishing+primary+cultures+of+ensheathing+cells+from+the+adult+olfactory+bulb.">New method of purification for establishing primary cultures of ensheathing cells from the adult olfactory bulb.</a> Glia. 34, 81-87 (2001).</li><li>Bianco, J. I., Perry, C., Harkin, D. G., Mackay-Sim, A., Feron, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin+3+promotes+purification+and+proliferation+of+olfactory+ensheathing+cells+from+human+nose.">Neurotrophin 3 promotes purification and proliferation of olfactory ensheathing cells from human nose.</a> Glia. 45, 111-123 (2004).</li><li>Paviot, A., Bon-Mardion, N., Duclos, C., Marie, J. P., Guerout, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Although+olfactory+ensheathing+cells+have+remarkable+potential+to+sustain+nerve+regeneration,+they+cannot+be+applied+to+a+severe+vagus+nerve+section/resection+model.">Although olfactory ensheathing cells have remarkable potential to sustain nerve regeneration, they cannot be applied to a severe vagus nerve section/resection model.</a> Muscle Nerve. 43, 919-920 (2011).</li></ol>

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这里介绍的技术使嗅鞘细胞，以研究细胞移植在周围神经损伤模型一个有用的模型。细胞培养协议相对简单，可以很容易地进行。在另一方面，外科手术，在喉返神经的特定部分/吻合，需要经验的，必须由合格的人员。 在这个协议中描述的方法强调重要因素集中，以便获得可能的最佳结果。首先，有机质分解过程中，我们建议仔细解剖，以获得唯一的嗅觉，而不是呼吸道上?...

主嗅觉系统包括两个不同的部分;嗅粘膜（OM）在中枢神经系统外周神经系统和嗅球（OB）。主嗅觉系统的特征在于主嗅觉神经元（PON）的自我更新整个生命中哺乳动物物种的能力。这种能力是由可能的，因为神经干细胞在OM的存在。 PON的生长和再生从OM到OB是由专门的神经胶质细胞称为嗅鞘细胞（嗅鞘细胞）促进。嗅鞘细胞是其从OM的PON到OB 1的神经发生创造了有利的微环境神经嵴细胞。因此，嗅鞘细胞可以在OM和OB构成细胞2,3的不同亚群被发现。嗅鞘细胞的不同属性有铅科学家将其用于细胞移植在几个神经系统病变范式4。事实上，嗅鞘细胞产生生长因子，减少胶质瘢痕，公关表参轴突再生，并且可以自由交融与星形胶质细胞5,6。然而，这些研究绝大多数是基于对脊髓损伤（SCI），其中一些已经用于嗅鞘细胞周围神经损伤（PNI）7,8之后。 虽然周围神经系统有很大的容量神经损伤后再生，科室齐全诱发异常的轴突再生。事实上，面部或喉返神经（RLN）完全横断后误传的轴突产生肌肉cocontractions叫联带运动。因此，它是最重要的，提出PNI的一个模型，不仅量化轴突再生长，但也评估肌肉收缩的效率。在文献中描述了最常见的模式是基于面部神经病变9,10。在这个模型中，功能性评估是基于对晶须运动10的恢复。然而，它是复杂的，以证明MOV的效率对此语句和歧视的肌肉cocontractions现象。在这里我们提出基于一个喉返神经损伤模型。这种模式不仅允许轴突再生和声带运动，但也是这些运动细胞移植11,12后的效率和功能的评价。该协议提供了一个喉返神经节/吻合模型一步步的程序，以文化和嗅鞘细胞移植从OM和OB和术后评估动物。

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transplantation of olfactory ensheathing cells to evaluate functional recovery after peripheral nerve injury

<html>嗅鞘细胞（嗅鞘细胞）是神经嵴细胞，使增长的主要嗅觉神经元和再生。事实上，主嗅觉系统的特征在于，其以产生新的神经细胞即使在成年动物的能力。这种特殊的能力，部分原因是嗅鞘细胞的存在而对神经营造良好的微环境。嗅鞘细胞的这种特性已被用于细胞移植，如脊髓损伤模型。虽然外周神经系统有较大容量的神经损伤比对中枢神经系统后再生，完整的章节后，面部喉神经切断术的过程中，特别轴突再生长诱导错误路由。具体来说，喉返神经（RLN）的全切片诱导导致声带联带运动异常轴突再生。在这种特定的模型中，我们发现，嗅鞘细胞移植有效地增加轴突再生。 的同时存在于嗅黏膜（OM-嗅鞘细胞）和嗅球（OB-嗅鞘细胞）亚群的几个ve_content“&gt;嗅鞘细胞构成的。我们在座细胞移植的基础上在利用嗅鞘细胞的这些不同亚群的典范喉返神经损伤模型，利用这种模式，OB-嗅鞘细胞和OM-嗅鞘细胞的原代培养的喉返神经第吻合后移植于基底膜。术后2个月，我们评估移植的动物通过互补分析的基础上videolaryngoscopy，肌电图（EMG）和组织学研究。首先，videolaryngoscopy使我们能够评估喉功能，特别是肌肉cocontractions现象，然后，肌电图的分析表明丰富性和肌肉的活动同步。最后，基于甲苯胺蓝染色的组织学研究中所允许的数量和分布的量化有髓纤维。 总之，我们在这里描述了如何分离，培养，IDEntify和嗅鞘细胞移植从OM和OB RLN段吻合术，以及如何评估和分析轴突的再生和喉功能这些移植细胞的效率之后。

插图与控制和神经支配（节/吻合）动物被选为结果可能会因执行（OM，OB或OM + OB）细胞移植而有所不同。 细胞培养 细胞粘附迅速到塑料表面，成为分隔成细胞，长形或锥形，三角形，多极，或纺锤状的平行分割行（ 图1A和1B）。经过8 天的体外流式细胞分析表明p75的阳性细胞的比率，在初级OB 71％，并且在初级培养物的OM 13％（ <strong...

Watch this Scientific Journal Video about Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury at JoVE.com

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

嗅鞘细胞（嗅鞘细胞）是神经嵴细胞，使初级嗅觉神经元的生长。此特定属性可用于蜂窝移植。我们在座细胞移植的基础上在喉上神经损伤模型中使用嗅鞘细胞的模型。

嗅鞘细胞移植周围神经损伤后，以评估功能恢复

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

transplantation-olfactory-ensheathing-cells-to-evaluate-functional

Research

JoVE Journal

Neuroscience

11.7K 次观看.  University of Rouen. 嗅鞘细胞（嗅鞘细胞）是神经嵴细胞，使初级嗅觉神经元的生长。此特定属性可用于蜂窝移植。我们在座细胞移植的基础上在喉上神经损伤模型中使用嗅鞘细胞的模型。 

视频: Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

科研

神经科学

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

背根神经节神经元和区分脂肪干细胞：一种<em&gt;在体外</em&gt;共培养模型来研究周围神经再生

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

活的嗅觉途径的功能评价

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

评估大鼠急性中风后脑恢复的临床前模型

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

从体外感觉和运动神经中纯化成纤维细胞和施万细胞

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递：对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Automated Gait Analysis to Assess Functional Recovery in Rodents with Peripheral Nerve or Spinal Cord Contusion Injury

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and a lot of comparative data has been published in the literature. This includes a multitude of assessment methods to study functional recovery following nerve injury and repair. Besides evaluation of nerve regeneration by means of histology, electrophysiology, and other in vivo and in vitro assessment techniques, functional recovery is the most important criterion to determine the degree of neural regeneration. Automated gait analysis allows recording of a vast quantity of gait-related parameters such as Paw Print Area and Paw Swing Speed as well as measures of inter-limb coordination. Additionally, the method provides digital data of the rats&#8217; paws after neuronal damage and during nerve regeneration, adding to our understanding of how peripheral and central nervous injuries affect their locomotor behavior. Besides the predominantly used sciatic nerve injury model, other models of peripheral nerve injury such as the femoral nerve can be studied by means of this method. In addition to injuries of the peripheral nervous systems, lesions of the central nervous system, e.g., spinal cord contusion can be evaluated. Valid and reproducible data assessment is strongly dependent on meticulous adjustment of the hard- and software settings prior to data acquisition. Additionally, proper training of the experimental animals is of crucial importance. This work aims to illustrate the use of computerized automated gait analysis to assess functional recovery in different animal models of peripheral nerve injury as well as spinal cord contusion injury. It also emphasizes the method&#8217;s limitations, e.g., evaluation of nerve regeneration in rats with sciatic nerve neurotmesis due to limited functional recovery. Therefore, this protocol is thought to help researchers interested in peripheral and central nervous injuries to assess functional recovery in rodent models.

Automated gait analysis is a feasible tool to evaluate functional recovery in rodent models of peripheral nerve injury and spinal cord contusion injury. While it requires only one setup to assess locomotor function in various experimental models, meticulous hard- and soft-ware adjustment and training of the animals is highly important.

自动步态分析，评估带外周神经或脊髓挫伤的啮齿动物的功能恢复

Peripheral and central nerve injuries are mostly studied in rodents, especially rats, given the fact that these animal models are both cost-effective and ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Motor Dual-Tasks for Gait Analysis and Evaluation in Post-Stroke Patients

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask gait analysis consisted of a single walking task (Task 0), a simple motor dual-task (water-holding, Task 1), and a complex motor dual-task (crossing obstacles, Task 2). The task of crossing obstacles was considered to be equivalent to the combination of a simple walking task and a complex motor task as it involved more nervous system, skeletal movement, and cognitive resources. To eliminate heterogeneity in the results of the gait analysis of the stroke patients, the dual-task gait cost values were calculated for various kinematic parameters. The major differences were observed in the proximal joint angles, especially in the angles of the trunk, pelvis, and hip joints, which were significantly larger in the dual motor tasks than in the single walking task. This research protocol aims to provide a basis for the clinical diagnosis of gait function and an in-depth study of motor control in stroke patients with motor control deficits through the analyses of dual-motor walking tasks.

This paper presents a protocol specifically for dual motor task gait analysis in stroke patients with motor control deficits.

用于中风后患者步态分析和评估的运动双任务

Eighteen stroke patients were recruited for this study involving the evaluation of cognition and walking ability and multitask gait analysis. Multitask ...