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...

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&#39;s modified Eagle&#39;s medium: nutrient mixture F-12</td>
			<td>ThermoFisher Scientific</td>
			<td>11039-021</td>
			<td>&#34;DMEM/F12&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>ThermoFisher Scientific</td>
			<td>15140-122</td>
			<td>&#34;Pen/Strep&#34; in protcol.</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH300-70-03</td>
			<td>&#34;FBS&#34; 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&#38;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>&#34;PLL&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium</td>
			<td>ThermoFisher Scientific</td>
			<td>11965-092</td>
			<td>&#34;DMEM&#34; in protocol.</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution</td>
			<td>ThermoFisher Scientific</td>
			<td>14170-112</td>
			<td>&#34;HBSS&#34; 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>&#34;Compressed foam&#34; 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>&#34;Injectable matrix&#34; 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?...

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

Research

JoVE Journal

Medicine

6.9K 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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

在大鼠脊髓损伤后的急性和慢性触觉测试

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

测量和制止在弱视治疗

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

控制皮层影响模型创伤性脑损伤

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

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 ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

主动脉阻塞后脊髓缺血大鼠模型中的可再生电机缺陷

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

A Clinical Trial Assessing the Safety, Efficacy, and Delivery of Olive-Oil-Based Three-Chamber Bags for Parenteral Nutrition

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based three-chamber bags (3CBs) and soybean-oil-based compounded bags (CoBs) in hospitalized adult patients. We designed a multicenter, randomized, prospective, open-label, noninferiority protocol to compare the efficacy, safety, and distribution of olive-oil-based 3CBs and soybean-oil-based CoB formulations in adult Chinese patients requiring PN during surgical intervention. Subjects were randomized to receive either one of the study treatments using an interactive voice or web-based recognition system in accordance with the randomization code. Randomization was further stratified based on the study site and surgical category. Both treatment groups received similar amounts of calories and protein. In addition, the two study treatments contained a similar composition of the amino-acid component. The only difference between the two PN formulations was the lipid constitution. The duration of administration of study treatments was a minimum of 5 days up to a maximum of 14 days after the surgical procedure. The primary efficacy endpoint was serum prealbumin levels on day 5 of the study. Noninferiority was proved if the anti-log of the lower bound of the 95% confidence interval (CI) of the treatment difference was at least 0.80. Other efficacy measures included treatment preparation time; duration to achieve tolerability of oral nutrition; associated infectious complications; length of hospitalization; and laboratory assessment of markers of nutrition, inflammation, metabolism, and oxidative stress. A total of 458 patients were enrolled in the study. The results showed that olive-oil-based 3CBs were non-inferior to soybean-based CoBs, besides being well tolerated. The infection rate was found to be significantly lower in the olive-oil-based 3CB group. Thus, this study may be used as a reference for future research on lipid emulsion and 3CBs.

Here, we present a protocol for a study comparing the efficacy, safety, and delivery of olive-oil-based 3CB and soybean-oil-based CoB formulations in adults requiring parenteral nutrition. The results revealed that olive-oil-based 3CBs is non-inferior and well tolerated compared to soybean formulations.

一项临床试验，评估基于橄榄油的三腔袋的亲子营养的安全性、有效性和交付

Limited evidence exists to precisely estimate efficacy and safety differences between parenteral nutrition (PN) prepared using olive-oil-based ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

改良异位异体下肢骨皮瓣模型在大鼠转化血管化复合同种异体移植研究中的应用

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

A Rat Lung Transplantation Model of Warm Ischemia/Reperfusion Injury: Optimizations to Improve Outcomes

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods for choosing appropriate cuff sizes for the pulmonary vein (PV), pulmonary artery (PA), or bronchus (Br) are varied, thus making the determination of proper cuff size during rat lung transplantation an exercise of trial and error. By standardizing the cuffing technique to use the smallest effective cuff appropriate for the size of the vessel or bronchus, one can make the transplantation procedure safer, faster, and more successful. Since diameters of the PV, PA, and Br are related to the body weight of the rat, we present a strategy to choosing an appropriate size using a weight-based guide. Since lung volume is also related to body weight, we recommend that this relationship should also be considered when choosing the proper volume of air for donor lung inflation during warm ischemia as well as for the proper volume of PBS to be instilled during bronchoalveolar lavage (BAL) fluid collection. We also describe methods for 4th intercostal space dissection, wound closure, and sample collection from both the native and transplanted lobes.

Here, we present optimizations to a rat lung transplantation model that serve to improve outcomes. We provide a size guide for cuffs based on body weight, a measurement strategy to ascertain the 4th intercostal space, and methods of wound closure and BAL (bronchoalveolar lavage) fluid and tissue collection.

温缺血/再灌注损伤的大鼠肺移植模型:改善结局的优化

From our experience with rat lung transplantation, we have found several areas for improvement. Information in the existing literature regarding methods ...