Name: transplantation of schwann cells inside pvdf-trfe conduits to bridge transected rat spinal cord stumps to promote axon regeneration across the gap
Uploaded: 2017-11-03T12:30:00.000+00:00
Duration: 8 min 5 s
Description: Watch this Scientific Journal Video about Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap at JoVE.com

我们要感谢在迈阿密项目的病毒载体和动物核心, 以治疗瘫痪的生产 lenti-GFP 病毒和提供动物护理, 分别和组织学和成像核心的使用低温, 共焦显微镜,荧光显微镜与立体侦探。资金由一 (09923), 国防部 (W81XWH-14-1-0482) 和 NSF (DMR-1006510) 提供。鹰是克里斯汀·林恩杰出的神经科学教授。

根据 NIH 和美国农业部的指导原则, 女性成年休鼠 (180-200 和 #160; g 体重) 被安置。迈阿密大学机构动物保育和使用委员会 (IACUC) 批准了所有的动物程序. 
 1. 移植前准备 <ol> <li> 导管准备。
	<ol> <li> 在解剖显微镜下使用 #10 刀片将管道长度削减到5毫米. 
		注: 导管内径介于 2.4-2.7 毫米之间;外径介于 2.5-2.8 mm 之间. </li>
		<li> 沿纵向侧轻轻折叠导管 ( 图 1A )。切开四小切口大约0.4 毫米长和至少1毫米从导管的开头和大约1毫米分开使用平直的边缘 Vannas 剪刀 ( 图 1B )。
		<ol> <li> 展开管道 ( 图 1C ), 并沿同一一侧的两个切口之间切开, 从而并排创建两个窗口 ( 图 1D )。确保窗口可以沿未切割的一侧正确打开和关闭. </li>
		</ol> </li> <li> 在10厘米的培养皿中对75% 乙醇中的导管进行25分钟的消毒。用1x 磷酸缓冲盐 (PBS) 冲洗导管一次, 用无菌钳将导管转移到新的 10 cm 培养皿中。用 1x PBS 冲洗导管两次, 每次25分钟. </li>
		<li> 将导管存储在 1x PBS 中直到手术. 
		注: 许多导管可以立即灭菌。将导管存放在单独的无菌1.5 毫升离心管中, 以保持它们不育. </li>
	</ol> </li> <li> 绿色荧光蛋白 (GFP)-雪旺细胞 (SC) 制备 <ol> <li> 从成年雌性费希尔鼠的胫骨神经制备纯化的 SC 培养物, 如先前所描述的 43 。在完成这一步, 板的多聚 l-赖氨酸 (PLL) 涂层 10 cm 培养皿和维护在 D10/3M 介质;这些 SCs 定义为段落 0. 
		注: D10/3M 介质由以下内容组成: 10% 胎牛血清 (FBS)、1% 青霉素-链霉素 (笔/链球菌)、20和 #181; heregulin 佛司可林、2和 #181; Dulbecco 和 #39 的改良鹰和 #39 培养基 (DMEM). </li>
		<li> 每3至4天补充新鲜 D10/3M 培养基 (7 mL/10-cm 培养皿). </li>
		<li> 将 SC 文化分离到70-80% 汇合处。
		<ol> <li> 卸下介质, 然后用汉克和 #39 的平衡盐溶液 (HBSS) 冲洗两次, 不含钙或镁. </li>
			<li> 将5毫升0.25% 胰蛋白酶/EDTA 添加到每 10 cm 培养皿中, 并在室温下孵育5分钟 (RT). </li>
			<li> 在 DMEM 中添加5毫升 D10 培养基 (10% FBS 和1% 支笔/链球菌链球菌) 到50毫升离心管中, 以中和胰蛋白酶. </li>
			<li> 将胰蛋白酶/EDTA 溶液轻轻吸管到培养皿中数次, 以分离 SCs. 从碟中取出电池悬浮液, 并将其放入前一步制备的离心管中。用5毫升的 D10 介质冲洗盘子, 并将其放入离心管中, 使细胞获得最大的收获. </li>
			<li> 离心 370 x g 的单元格为5分钟, 在 4 o c. 将上清液和重的细胞在4毫升 D10/3M 培养基中为每个培养皿分裂. </li>
			<li> 将1毫升的 SC 悬浮液和6毫升的 D10/3M 培养基放入10厘米的培养皿中; 每个培养皿分裂后会产生4种培养皿。这些 SCs 定义为段落 1. </li>
			<li> 电镀后每3-4 天用新鲜 D10/3M 培养基补充. </li>
			<li> 一旦 SCs 达到70-80% 汇合, 重复步骤1.2.3.1 到1.2.3.6。公务员事务局局长现正通过2条. </li>
		</ol> </li> <li> 传感器的 SCs 曾经达到50% 汇合, 慢矢量编码 GFP 在 D10/3M 介质过夜在30感染的多样性, 如前所述 44 , 45 .</li>
		<li> 第二天和电镀后每3-4 天补充新鲜 D10/3M 培养基. </li>
		<li> 一旦 gfp-scs 达到70-80% 汇合, 重复步骤1.2.3.1 到 1.2.3.5, 但重的 gfp-scs 在6毫升 D10 培养基代替。将 GFP-SC 悬浮液的15和 #181; 1.5 毫升离心管, 加入15和 #181; 0.4% 台盼蓝溶液。轻轻轻拍离心管, 并将10和 #181; L 混合成细胞计数滑动的腔室。
		<ol> <li> 将幻灯片放置到自动单元格计数器中, 并确定单元格的密度. </li>
		</ol> </li> <li> 将 370 x g 的单元格分离为5分钟, 在 4 o c. 将上清液, 重与1.5 毫升的冷冻培养基, 每 3x10^-7 6 gfp-SCs. 向低温小瓶中分配1.5 毫升的 gfp-SC 悬浮液, 冻结在-80 o C 在移动到液态氮气进行贮存之前至少24小时. 
		注: 冷冻培养基: 25% FBS 和8% 二甲基亚砜在 DMEM. </li>
		<li> 在手术前一周解冻 GFP-SCs。
		<ol> <li> 将10毫升 D10 介质添加到50毫升离心管中. </li>
			<li> 将冷冻的 GFP-SCs 在水浴中解冻小瓶 37 o C 直到部分解冻. </li>
			<li> 将 GFP-SC 悬浮液从低温小瓶中取出, 放入1.2.8.1 的50毫升离心管中。反复地吸管解决方案. </li>
			<li> 离心 370 x g 的单元格为5分钟, 在 4 o c. 将上清液和重的细胞在4毫升 D10/3M 培养基中为每瓶解冻. </li>
			<li> 将2毫升的 GFP-SC 悬浮液和5毫升的 D10/3M 培养基放入10厘米的培养皿中; 每个小瓶应产生2种培养皿。GFP-SCs 定义为通道 3. 
			注意: 一个10厘米的培养皿通常在一周后 8x10 6 GFP-SCs 附近生成. </li>
		</ol> </li> <li> 在手术当天, 重复步骤1.2.6。使用步骤1.2.10 中计算的单元格密度. </li>
		<li> 免除等分 3x10^-7 6 GFP-SCs 到无菌1.5 毫升离心管的基础上的细胞密度计算从步骤1.2.9。离心 GFP-SC 等分在 370 x g 为5分钟在 4 o C 和去除上清。添加1毫升的 DMEM/F12 介质到每个分和离心机在 370 x g. 
		注意: 每只动物接受3x10^-7 的 6 gfp-scs. 培养基与血清与 gfp-scs 一起使用直到这一步. </li>
		<li> 将 GFP-SCs 保持在冰上直到移植时间. </li>
	</ol> </li> </ol> 2。完全横断面在胸部8级 (T8) <ol> <li> 动物准备手术 <ol> <li> 麻醉一个雌性 Fischer 鼠 (180-200 和 #160; g) 腹腔注射氯胺酮 (60 毫克/千克和 #160; 体重) 和嗪 (5毫克/千克和 #160; 体重)。检查麻醉深度, 然后再进行下一步骤, 通过观察脚趾的捏和眼睛闪烁的反应. </li>
		<li> 用电动剪刀刮掉老鼠的背部, 用70% 的乙醇擦拭皮肤。将眼部润滑剂应用于双眼。将动物转移到加热垫上的手术台上, 以保持体温在大约 37 o c. 将动物放在一卷上, 这样椎骨就很容易进入. 
		注: 该卷可由纸巾或2在 x2 的纱布, 可以消毒。当 T7-T9 需要平放在轧辊的顶部时, 建议使用较大的卷. </li>
	</ol> </li> <li> T7 到 T9 椎板切除 <ol> <li> 定位 T9-T11 棘突的地标. 
		注意: 与其他段相比, T9、T10 和 T11 之间的间隙较小该地区。在把老鼠放在卷上后, T9-T11 棘的过程会感觉像一个小的三角形肿块通过皮肤. </li>
		<li> 使用 #10 刀片从 T4 到 T11, 使皮肤的中线切口为4到5厘米。用钝端的弯曲剪刀在表面脂肪层做一个小切口解剖脂肪. 
		注: 其他类型的仪器可以用来分离脂肪, 但弯曲的剪刀与钝端, 使脂肪分离最有效和最不侵入的方法. </li>
		<li> 使用 #10 刀片的背面定位 T7 T9 棘突。用钝钳夹住 T4 的肌肉, 在椎骨两侧的肌肉上做一个切口, 从 T6-T10。使切口尽可能靠近椎骨, 以使伤口干净, 对动物造成最小的伤害. </li>
		<li> 通过切割 T6 和 T7、T7 和 T8、T8 和 T9、T9 和 T10 的肌肉和韧带, 将每个棘突从 T7 分离到 T9. </li>
		<li> 使用钳将叶片上的任何肌肉和韧带从 T7 移到 T9。尽可能地移除肌肉和韧带, 直到从 T7 到 T9 的横向过程之间的间隙是可见的. </li>
		<li> 用钳移除 T9 棘过程。在 T9 和 T10 过程之间的小开口处, 用钝钳和轻轻提起 T8 棘突, 提升 T9 叶片. </li>
		<li> 从开口和移动 rostrally 中取出叶片, 钳在 T9。尽可能地侧移叶片;椎板切除后的背根应可见. </li>
		<li> 一旦移除叶片, 重复 T8 和 T7 的过程. 
		注意: 在椎板切除术中, 使用吸收矛去除血液, 同时继续与椎板切除。如果出现过量出血, 请在出血部位放置一个压缩海绵, 加盐水以帮助血液凝固. </li>
	</ol> </li> <li> 横断在 T8 <ol> <li> 将牵引器放在 T7 T9。一旦所有的叶片被移除, 确保横向过程之间的间隙是可见的, 特别是在 T8。还要检查两侧的骨骼沿 T7 至 T9 之间的长度, 以确保没有向外凸出的骨骼碎片. 
		注: T8 的这一缺口对执行完全横断面很重要。沿长度的椎骨应该被删除, 以便更容易插入导管. </li>
		<li> 使用倾斜的弹簧剪刀将背部和腹根从 T8 的上方和下方切开。在脊髓中加入生理盐水, 等待血液凝结. #160; 注意: 此步骤对于正确插入管道非常重要. </li>
		<li> 将倾斜的弹簧剪刀放置在 T8 的横突之间的间隙上, 使一剪完全切断脊髓。将一小块压缩泡沫放入产生的 2-2. 5 mm 间隙, 并立即将盐水添加到该区域. </li>
	</ol> </li> </ol> 3。管道插入 <ol> <li> 在等待断线的残肢到达止血时, 从 1x PBS 中取出导管。将吸收三角形切割成细长的长片, 并将它们放入管道中以去除多余的 PBS。检查预窗口是否已打开. </li>
	<li> 使用细长的吸收三角形去除生理盐水和血液。用 microspatula 抬起两根脊髓, 确保良好的分离。用 microspatula 轻轻地抬起侧的残肢, 用窗户朝自己的车窗滑动导管。确保整个树桩插入, 并没有多余的出血到管道. </li>
	<li> 轻轻抬起尾端, 然后将导管的另一端滑过树桩。确保将整个残肢插入, 并且窗口在背表面 ( 图 2A ). </li>
</ol> 4。雪旺细胞/注射基质 (见材料表) 准备和注射 <ol> <li> 在等待被割断的绳子到达止血时, 把 GFP-SC 颗粒上面的培养基除去 (步骤1.2 中准备)。重10和 #181 的 GFP-SCs; 冷 DMEM/F12。添加10和 #181; 冷注射凝胶对细胞悬液的影响, 并通过反复移。将 GFP-SC/DMEM/F12/injectable 基质混合物放在冰上, 直到导管插入完成. </li>
	<li> 在确保背表面上的窗口打开 ( 图 2A ) 后, 注入20和 #181; 通过预窗口之一的 GFP-SC/DMEM/F12/injectable 矩阵混合的 l. 46 使用西方印迹加载提示和微。如果 SC/DMEM/F12/injectable 基质混合物应溢导管, 则用吸收三角形除去多余的。注射后关闭窗户;不需要缝合 ( 图 2B ). </li>
</ol> 5。伤口闭合和术后护理 <ol> <li> 缝合肌肉层和表面脂肪层。清洁皮肤与70% 乙醇, 然后主食 ( 例如, 使用伤口剪辑) 皮肤关闭。将动物置于热调节的孵化器中, 直到它们恢复知觉。把动物转移回笼子里给水提供一个长的喂养管和放置在床上用品的食物颗粒, 方便访问. </li>
	<li> 每天两次进行丁丙诺啡 (0.1 毫克/千克体重) 3 天皮下手术后立即开始, 以减轻疼痛。注射庆大霉素 (5 毫克/千克体重) 每天一次, 7 天后立即手术, 以防止和减少感染。每天注射10毫升的林格铃声溶液皮下注射两次, 为期7天, 用于水合. </li>
	<li> 每天手动清空膀胱两次, 直到膀胱功能恢复。如果发生膀胱感染, 然后管理10毫升生理盐水皮下注射, 直到尿液变得清晰。如果两天没有改善, 注射庆大霉素 (5 毫克/千克体重, 每天一次) 皮下注射, 直到尿液变得清晰. </li>
</ol>

作者没有什么可透露的。

建立有效的横断面模型的最关键的一步是在一个或两个切口切断脊髓。2-2. 5 毫米之间的间隙之间的侧和尾部脊髓残肢应存在于横断现场。三最可能的原因, 这样的差距没有出现的是 (1) 背部/腹根没有被正确删除, (2) 腹侧硬脑膜没有被充分删除, 和/或 (3) 该动物没有正确放置在她下面的轧辊。
在树桩之间进行有效的导管插入: (1) 导管的直径应根据实验中所用动物的特定种类和年龄而定制;(2) 叶片必须横向移除, 直到横向过程之间的间隙可以可视化;(3) 根必须除去, (4) 在第一次尝试时, 应完成在树桩之间的导管插入。如果需要多次尝试, 这可能会导致在树桩的水肿, 进一步复杂化的任务, 导管插入之间的树桩和造成额外的伤害。
要执行有效的 GFP-SC/DMEM/F12/injectable 基质引入导管, 确保: (1) 脊髓不流血前导管插入之间的树桩。如果管道内有液体在树桩之间插入, 使用一小块吸收矛通过预窗口去除它。(2) 确保导管在两个脊髓残肢井上滑动, (3) 背预窗打开。注射20µl 的 SC/可注射基质混合物应足以填补管道。从预窗口溢出是一个很好的衡量有效的移植。
这一过程的局限性是: (1) 没有恢复硬脑膜, (2) 没有控制的运动的导管/sc 移植后完成的手术, 和 (3) 没有其他方法, 如果有泄漏时, 注射 sc/注射基质混合物。
这一过程的优点是结合使用两个结构管道与注射基质。任何用可弯曲的材料制成的导管, 都可以在这个手术过程中使用。任何可注射的选择基质也可用于本程序;温度敏感的凝胶是可取的, 因为它能够凝胶原位和轮廓形状的树桩创建一个无缝的接口。具有更复杂的内部结构的导管可以使用, 而不是空心内部。药物, 生长因子, 小分子, 或任何细胞类型可以纳入结构化导管或注射基质或两者都增加移植生存, 提供神经保护立即损伤后, 减少炎症, 促进轴突再生, 促进血管生成。

脊髓损伤修复是一个复杂而富有挑战性的问题, 需要一个组合治疗策略, 例如, 使用细胞和生物材料为移植细胞功能和轴突提供有利的微环境损伤部位再生。半 1,2,3,4,,5,6,7,8 和完全横断9 ,11,12,131415161718 、20、21、22模型经常用于评估 biomaterial-based 桥接疗法的效果。使用半模型的优点是它为桥接结构提供了更稳定的比完全横断面。然而, 在半模型中, 由于无组织的存在, 很难证明轴突再生是应用治疗方法的结果。完全横断面模型是显示轴突再生的最严格的方法。
各种天然和合成材料已被研究用于注射凝胶, 预先凝胶放置在挫伤或半模型, 或作为一个结构的管道到半或完整的横断面模型 (详细的审查23,24,25).原位可注射基质/sc 合剂的凝胶性在移植物和轴突交叉的主绳之间创建一个更宽松的接口26,27与 pre-gelled 基质/sc 种植体相比5,18,19,28.原位胶凝允许矩阵在不规则的主机接口周围进行轮廓, 而更刚性和结构化的导管或较不模压的预先凝胶不能。与注射基质相比, 结构化导管通常提供接触引导和植入稳定性。这里介绍的协议描述了一个手术过程, 既利用了可注射基底膜基质的优势 (例如,基质, 参见材料表,称为可注射基质), 以及结构化管道评价轴突再生的最严格的脊髓损伤模型。
纺聚 vinylidenedifluoride-乙烯 (PVDF-TrFE) 对齐的纤维空心导管用于我们的实验方法。PVDF-TrFE 是一种压电聚合物, 它在机械变形时产生瞬态电荷, 并被证明可以促进突起扩展和轴突再生,无论是体外29、30还是在体内31. 静电纺丝是一种常用的脚手架制作方法, 可以使用各种可控性能的聚合物快速生成可靠的纤维支架, 如纤维对齐、纤维直径和脚手架的厚度神经和其他应用程序32,33,34。
大量研究大鼠 SCs 移植到脊髓损伤部位已显示治疗效果5,9,18,19,20,,21 ,26。这些移植是神经保护的组织周围的病变, 减少病变腔大小, 并促进轴突再生到病变/移植部位和鞘再生轴突。人体 SCs 可以 autologously 移植, 比其他大多数神经相关细胞24的优势。经外周神经活检, SCs 可以分离和纯化, 并将激增到预期的数量, 移植到人类。自体 SC 移植治疗脊髓损伤患者已被证明是安全的在伊朗35,36,37,38, 中国39,40, 以及美国41,42。众所周知, SCs 分泌大量的神经营养因子和胞外基质蛋白对轴突生长起重要作用, 并在周围神经损伤后的轴突再生中起着必不可少的功能。我们的目标是描述一种方法, 可以研究导管设计, 以改善大鼠脊髓横断模型中 SC 移植的结果。

<table><tbody><tr><td>Cryogenic vials</td><td>ThermoFisher Scientific</td><td>5000-0020</td><td></td></tr><tr><td>10 cm Petri dish</td><td>VWR</td><td>25382-428</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium: nutrient mixture F-12</td><td>ThermoFisher Scientific</td><td>11039-021</td><td>&quot;DMEM/F12&quot; in protocol.</td></tr><tr><td>Penicillin-streptomycin</td><td>ThermoFisher Scientific</td><td>15140-122</td><td>&quot;Pen/Strep&quot; in protcol.</td></tr><tr><td>Fetal bovine serum</td><td>Hyclone</td><td>SH300-70-03</td><td>&quot;FBS&quot; in protocol.</td></tr><tr><td>Pituitary extract</td><td>Biomedical Technologies</td><td>BT-215</td><td></td></tr><tr><td>Forskolin</td><td>Sigma-Aldrich</td><td>F6886</td><td></td></tr><tr><td>Heregulin</td><td>R&amp;amp;D Systems</td><td>396-HB/CF</td><td></td></tr><tr><td>Poly L-lysine</td><td>Sigma-Aldrich</td><td>P2636</td><td>&quot;PLL&quot; in protocol.</td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium</td><td>ThermoFisher Scientific</td><td>11965-092</td><td>&quot;DMEM&quot; in protocol.</td></tr><tr><td>Hank&#039;s balanced salt solution</td><td>ThermoFisher Scientific</td><td>14170-112</td><td>&quot;HBSS&quot; in protocol.</td></tr><tr><td>Tryspin-EDTA</td><td>ThermoFisher Scientific</td><td>15400-054</td><td></td></tr><tr><td>Female Fischer rat (160-180g)</td><td>Envigo</td><td></td><td></td></tr><tr><td>Vannas scissor, straight</td><td>FST</td><td>15018-10</td><td></td></tr><tr><td>Ketamine</td><td>Vedco Inc</td><td>5098976106</td><td>100 mg/ml</td></tr><tr><td>Xylazine</td><td>Lloyd Inc</td><td>AnaSed</td><td>20 mg/ml</td></tr><tr><td>Gentamycin</td><td>APP Pharmaceuticals</td><td>NDC 63323-010-02</td><td>Can be any brand of choice.</td></tr><tr><td>Micro Spatula</td><td>FST</td><td>10089-11</td><td>Can be any brand of choice.</td></tr><tr><td>Curved scissors with blunt end</td><td>FST</td><td>14017-18</td><td>Can be any brand of choice.</td></tr><tr><td>Blunt forceps</td><td>FST</td><td>11006-12</td><td>Can be any brand of choice.</td></tr><tr><td>rongeur</td><td>FST</td><td>16121-14</td><td>Can be any brand of choice.</td></tr><tr><td>Angled spring scissors</td><td>FST</td><td>15006-09</td><td>Can be any brand of choice.</td></tr><tr><td>Absorption triangles</td><td>FST</td><td>18105-03</td><td>Can be any brand of choice.</td></tr><tr><td>Gelfoam</td><td>Henry Schein</td><td>9083300</td><td>&quot;Compressed foam&quot; in protocol.</td></tr><tr><td>#10 blades</td><td>Sklar</td><td>06-3010</td><td>Can be any brand of choice.</td></tr><tr><td>Matrigel</td><td>Corning</td><td>354234</td><td>&quot;Injectable matrix&quot; in protocol.</td></tr><tr><td>Chicken anti-green fluorescent protein antibody</td><td>Millipore</td><td>AB16901</td><td></td></tr><tr><td>Mouse RT97 hybridoma antibody</td><td>DSHB</td><td>RT97</td><td></td></tr><tr><td>Rabbit anti-neurofilament antibody</td><td>Encor Biotechnology, Inc</td><td>PRCA-NF-H</td><td></td></tr><tr><td>Polyclonal Rabbit anti-Glial Fibrillary Acidic Protein antibody</td><td>Dako</td><td>Z033401</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-chicken IgG (H+L)</td><td>ThermoFisher Scientific</td><td>A-11039</td><td></td></tr><tr><td>Alexa Fluor 546 goat anti-rabbit IgG (H+L)</td><td>ThermoFisher Scientific</td><td>A-11035</td><td></td></tr><tr><td>Alexa Fluor 647 goat anti-rabbit IgG (H+L)</td><td>ThermoFisher Scientific</td><td>A-21244</td><td></td></tr><tr><td>Alexa Fluor 647 goat anti-mouse IgG (H+L)</td><td>ThermoFisher Scientific</td><td>A-21236</td><td></td></tr><tr><td>Confocal Microscopy</td><td>Nikon</td><td>clsi</td><td></td></tr></tbody></table>

transplantation of schwann cells inside pvdf-trfe conduits to bridge transected rat spinal cord stumps to promote axon regeneration across the gap

在大鼠脊髓损伤的各种模型中, 钝挫伤模型是最常用的, 因为它是最常见的类型的人脊髓损伤。完整的横断面模型, 虽然与钝挫伤模型没有临床相关性, 但却是评价轴突再生最严格的方法。在钝挫伤模型中, 由于存在残余的组织损伤, 很难区分再生与发芽或幸免的轴突。在完整的横断面模型中, 需要一个桥接方法来填补间隙, 并从侧到尾端以建立连续性, 以评估治疗效果。一个可靠的搭桥手术是必要的测试结果的措施, 减少因手术方法的变异性。此处所述的协议用于在移植前制备雪旺细胞 (scs) 和导管, 在胸椎8级 (T8) 上完全横断脊髓, 插入导管, 并将 SCs 移植到导管中。这种方法也使用原位胶凝剂基底膜基质与 SC 移植, 允许改善轴突生长横跨侧和骶管接口与宿主组织。

使用这种手术技术的目的是评估使用的结构导管和注射基质, 最大限度地发挥 SC 功能后移植到完成断脊髓。移植后三周, 动物被灌入4% 甲醛, 脊柱被严重解剖, 并固定在同一定影液中, 用于另24小时。然后解剖脊髓, 将低温矢状切面的样品放入30% 蔗糖溶液中冻。从另一组解剖脊髓的 SC 桥中间分离出来的1毫米厚的横断面被放进戊二醛固定剂中, 用于塑料切片。样品被服从于电子显微镜准备的日程表由贝茨et al.详述47. 低温矢状切面 immunostained 与 GFP、胶质纤维酸性蛋白 (胶质细胞)、重链丝 (RT97) 和中等链丝 (NF), 其次是继发抗体, 包括goat-anti-chicken-488, goat-anti-rabbit-546, goat-anti-mouse-647 和山羊-兔 647, 分别。GFP-SCs 均匀分布在管道内 (图 3A; 由黄色线指示的内壁)。轴突再生观察 (图 3B), 并且与 GFP-SCs (图 3A和图 3C) 的存在密切相关, 这表明该技术成功地在结构化管道中提供了 SC 桥, 并在促进轴突再生沿侧和尾部树桩之间的桥梁。血管和髓轴突也被发现在 SC 桥的中心 (图 3D)。更多细节的影响, SCs 与纺 PVDF-TrFE 纤维导管的轴突再生可以找到在我们最近发布的工作31。
其他的结果措施可以执行, 包括: 定量轴突再生的直线样条法, 描述在我们最近的工作31和量化的髓轴突和在塑料横断面的船只数量。为塑料切片准备的样品可以进一步分段为透射电子显微镜, 以量化髓轴突的数量。cryoprotected 脊髓标本也可以截面, 而不是矢切片和 immunostained, 以量化轴突再生和存在的 GFP-SCs. 行为测试也可以在适当的时间间隔对动物进行, 而动物们正在被维护。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56077/56077fig1.jpg"> 
图 1: 管道中的窗口准备沿 5 mm 导管 (A) 的纵轴折叠一侧。切开四切口大约0.4 毫米长和至少1毫米从导管的开头 (B)。每个切口大约1毫米分开。通过展开管道, 可观察到4平行切口 (C)。沿侧-尾轴的两个切口之间进行切割, 以创建一个可以通过提起襟翼打开的窗口。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56077/56077fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56077/56077fig2.jpg"> 
图 2: SC/DMEM/F12/matrix 注入后的窗口关闭在将导管放置在侧和尾端 (A) 之间后, 通过折叠每个襟翼打开窗口。在注入后关闭窗口 (B)。缩放条 = 1 mm<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56077/56077fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56077/56077fig3.jpg"> 
图 3: 在对齐纤维 PVDF-TrFE 导管中, 用 GFP-SCs 移植的脊柱的矢状切面共聚焦荧光图像和一个塑料横断面的明亮场图像.在管道内的 SC 桥的概述, 其中内壁是由黄线和 immunostained 的 GFP 和胶质纤维酸性蛋白 (胶质), 以可视化的移植 SCs 和宿主脊髓星形胶质细胞分别。再生轴突被标记为 RT97 (重链丝) (A, B) 和中链丝 (NF, C) 抗体。轴突在 (A) 中不可见, 因为它们与 GFP-SCs 的密切关联, 在 mid-conduit 区域 (C) 中观察到放大率较高。轴突是不可见的侧树桩由于不完全扫描在深度的组织切片时, 成像的共焦显微镜。血管 (标记为 v 在d) 和髓轴突 (标记为 MA 在d) 在 mid-conduit 区域在一个塑料部分被观察了。放大和刻度条: A、B (10x、200µm);C (20x, 100 µm);D (63x, 25 µm)。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56077/56077fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap at JoVE.com

Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

本文介绍了一项技术, 插入一个中空导管之间的脊髓残肢完全横断面和填充雪旺细胞 (SCs) 和可注射基底膜基质, 以桥梁和促进轴突再生跨越的差距。

氟 TrFE 导管内许旺细胞移植桥断大鼠脊髓残肢促进轴突再生跨越间隙

Among various models for spinal cord injury in rats, the contusion model is the most often used because it is the most common type of human spinal cord ...

transplantation-schwann-cells-inside-pvdf-trfe-conduits-to-bridge

Universidad San Sebastian

Research

JoVE Journal

Medicine

7.0K 次观看.  University of Miami Miller School of Medicine. 本文介绍了一项技术, 插入一个中空导管之间的脊髓残肢完全横断面和填充雪旺细胞 (SCs) 和可注射基底膜基质, 以桥梁和促进轴突再生跨越的差距。

视频: Transplantation of Schwann Cells Inside PVDF-TrFE Conduits to Bridge Transected Rat Spinal Cord Stumps to Promote Axon Regeneration Across the Gap

Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) cases 1 and is the ultimate cause of death in patients with the motor neuron disorder, amyotrophic laterals sclerosis (ALS) 2.
ALS is a devastating neurological disorder that is characterized by relatively rapid degeneration of upper and lower motor neurons. Patients ultimately succumb to the disease on average 2-5 years following diagnosis because of respiratory paralysis due to loss of phrenic motor neuron innnervation of the diaphragm 3. The vast majority of cases are sporadic, while 10% are of the familial form. Approximately twenty percent of familial cases are linked to various point mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene on chromosome 21 4. Transgenic mice 4,5 and rats 6 carrying mutant human SOD1 genes (G93A, G37R, G86R, G85R) have been generated, and, despite the existence of other animal models of motor neuron loss, are currently the most highly used models of the disease.
Spinal cord injury (SCI) is a heterogeneous set of conditions resulting from physical trauma to the spinal cord, with functional outcome varying according to the type, location and severity of the injury 7. Nevertheless, approximately half of human SCI cases affect cervical regions, resulting in debilitating respiratory dysfunction due to phrenic motor neuron loss and injury to descending bulbospinal respiratory axons 1. A number of animal models of SCI have been developed, with the most commonly used and clinically-relevant being the contusion 8.
Transplantation of various classes of neural precursor cells (NPCs) is a promising therapeutic strategy for treatment of traumatic CNS injuries and neurodegeneration, including ALS and SCI, because of the ability to replace lost or dysfunctional CNS cell types, provide neuroprotection, and deliver gene factors of interest 9.
Animal models of both ALS and SCI can model many clinically-relevant aspects of these diseases, including phrenic motor neuron loss and consequent respiratory compromise 10,11. In order to evaluate the efficacy of NPC-based strategies on respiratory function in these animal models of ALS and SCI, cellular interventions must be specifically directed to regions containing therapeutically relevant targets such as phrenic motor neurons. We provide a detailed protocol for multi-segmental, intraspinal transplantation of NPCs into the cervical spinal cord ventral gray matter of neurodegenerative models such as SOD1G93A mice and rats, as well as spinal cord injured rats and mice 11.

Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.

椎管内针对肌萎缩性脊髓侧索硬化症和外伤性脊髓损伤颈腹角细胞移植

Respiratory compromise due to phrenic motor neuron loss is a debilitating consequence of a large proportion of human traumatic spinal cord injury (SCI) ...

科研

医学

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.

结合周边神经移植和矩阵调制修复受伤的大鼠脊髓

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

生物学

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

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

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 ...

神经科学

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord injury (SCI), and can form functional relays to re-connect spinal cord segments above and below a lesion. Previous studies grafting neural stem cells have been limited by incomplete graft survival within the spinal cord lesion cavity. Further, tracking of graft cell survival, differentiation, and process extension had not been optimized. Finally, in previous studies, cultured rat NSCs were typically reported to differentiate into glia when grafted to the injured spinal cord, rather than neurons, unless fate was driven to a specific cell type. To address these issues, we developed new methods to improve the survival, integration and differentiation of NSCs to sites of even severe SCI. NSCs were freshly isolated from embryonic day 14 spinal cord (E14) from a stable transgenic Fischer 344 rat line expressing green fluorescent protein (GFP) and were embedded into a fibrin matrix containing growth factors; this formulation aimed to retain grafted cells in the lesion cavity and support cell survival. NSCs in the fibrin/growth factor cocktail were implanted two weeks after thoracic level-3 (T3) complete spinal cord transections, thereby avoiding peak periods of inflammation. Resulting grafts completely filled the lesion cavity and differentiated into both neurons, which extended axons into the host spinal cord over remarkably long distances, and glia. Grafts of cultured human NSCs expressing GFP resulted in similar findings. Thus, methods are defined for improving neural stem cell grafting, survival and analysis of in vivo findings.

Fibrin matrices containing growth factors were used to retain grafted neural stem cells into sites of complete spinal cord transection. Grafted cells completely filled the lesion cavity and differentiated into multiple neural cell types, including neurons that extended axons into host spinal cord over long distances.

促进存活神经干细胞与纤维蛋白和生长因子鸡尾酒和分化的严重脊髓损伤后

Neural stem cells (NSCs) can self-renew and differentiate into neurons and glia. Transplanted NSCs can replace lost neurons and glia after spinal cord ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

无线电遥测系统，以监测大鼠脊髓横断和胚胎神经干细胞移植在心血管功能

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the spinal cord, tumor resections, or tissue defects resulting from traumatic accidents can aid in facilitating general tissue repair as well as regenerative growth of nerve fibers into and beyond the affected area. Two experimental treatment strategies are presented: (1) implantation of a novel microconnector device into an acutely and completely transected thoracic rat spinal cord to readapt severed spinal cord tissue stumps, and (2) polyethylene glycol filling of the SCI site in chronically lesioned rats after scar resection. The chronic spinal cord lesion in this model is a complete spinal cord transection which was inflicted 5 weeks before treatment. Both methods have recently achieved very promising outcomes and promoted axonal regrowth, beneficial cellular invasion and functional improvements in rodent models of spinal cord injury.
The mechanical microconnector system (mMS) is a multi-channel system composed of polymethylmethacrylate (PMMA) with an outlet tubing system to apply negative pressure to the mMS lumen thus pulling the spinal cord stumps into the honeycomb-structured holes. After its implantation into the 1 mm tissue gap the tissue is sucked into the device. Furthermore, the inner walls of the mMS are microstructured for better tissue adhesion.
In the case of the chronic spinal cord injury approach, spinal cord tissue - including the scar-filled lesion area - is resected over an area of 4 mm in length. After the microsurgical scar resection the resulting cavity is filled with polyethylene glycol (PEG 600) which was found to provide an excellent substratum for cellular invasion, revascularization, axonal regeneration and even compact remyelination in vivo.

Severe spinal cord injuries often result in tissue defects. Two possibilities are described to successfully bridge such gaps to promote tissue adaptation, regenerative responses and functional improvement in rats via implantation of a mechanical microconnector system after acute injury and five weeks after complete spinal cord transection.

实验策略急，慢性损伤后桥的损伤脊髓组织大差距

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Sheath-Preserving Optic Nerve Transection in Rats to Assess Axon Regeneration and Interventions Targeting the Retinal Ganglion Cell Axon

Retinal ganglion cell (RGC) axons converge at the optic nerve head to convey visual information from the retina to the brain. Pathologies such as glaucoma, trauma, and ischemic optic neuropathies injure RGC axons, disrupt transmission of visual stimuli, and cause vision loss. Animal models simulating RGC axon injury include optic nerve crush and transection paradigms. Each of these models has inherent advantages and disadvantages. An optic nerve crush is generally less severe than a transection and can be used to assay axon regeneration across the lesion site. However, differences in crush force and duration can affect tissue responses, resulting in variable reproducibility and lesion completeness. With optic nerve transection, there is a severe and reproducible injury that completely lesions all axons. However, transecting the optic nerve dramatically alters the blood brain barrier by violating the optic nerve sheath, exposing the optic nerve to the peripheral environment. Moreover, regeneration beyond a transection site cannot be assessed without reapposing the cut nerve ends. Furthermore, distinct degenerative changes and cellular pathways are activated by either a crush or transection injury.
The method described here incorporates the advantages of both optic nerve crush and transection models while mitigating the disadvantages. Hydrostatic pressure delivered into the optic nerve by microinjection completely transects the optic nerve while maintaining the integrity of the optic nerve sheath. The transected optic nerve ends are reapposed to allow for axon regeneration assays. A potential limitation of this method is the inability to visualize the complete transection, a potential source of variability. However, visual confirmation that the visible portion of the optic nerve has been transected is indicative of a complete optic nerve transection with 90-95% success. This method could be applied to assess axon regeneration promoting strategies in a transection model or investigate interventions that target the axonal compartments.

This protocol describes an optic nerve transection that preserves the optic nerve sheath in rats. Hydrostatic pressure from microinjections into the optic nerve generate a complete transection, allowing for sutureless reapposition of the transected optic nerve ends and direct targeting of the axonal compartment in a transection model.