Name: induction of complete transection-type spinal cord injury in mice
Uploaded: 2020-05-06T14:00:00
Description: Watch this Scientific Journal Video about Induction of Complete Transection-Type Spinal Cord Injury in Mice at JoVE.com

这项工作得到了格里菲斯大学国际学生（PhD）津贴的R，佩里交叉基金会赠款JE和JSJ，克莱姆琼斯基金会赠款JSJ和JE，昆士兰州汽车事故保险委员会赠款JSJ和JE的支持。

所有程序都是根据澳大利亚国家健康和医学研究理事会的指导方针，经格里菲斯大学动物伦理委员会（ESK/04/16 AEC 和 MSC/04/18 AEC）的批准进行的。
1. 手术的动物设置程序
<ol><li>麻醉和稳定动物。<ol>
<li>使用8~10周大的雌性C57BL/6只小鼠。在 1 L/min 氧气中使用 5% 异氟兰进行麻醉诱导。为了维持麻醉，在1升/分钟氧气中使用1.5~2%异氟。通过确定尾部和后爪缺乏疼痛反射，确认适当的...

<ol>
	<li>Sharif-Alhoseini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+spinal+cord+injury:+a+systematic+review.">Animal models of spinal cord injury: a systematic review.</a> Spinal Cord. 55 (8), 714-721 (2017).</li><li>Lee, D. H., Lee, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+axon+regeneration+after+spinal+cord+injury.">Animal models of axon regeneration after spinal cord injury.</a> Neuroscience Bulletin. 29 (4), 436-444 (2013).</li><li>Sharif-Alhoseini, M., Rahimi-Movaghar, V., Dionyssiotis, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Topics in Paraplegia. , (2014).</li><li>Talac, R., 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=Animal+models+of+spinal+cord+injury+for+evaluation+of+tissue+engineering+treatment+strategies.">Animal models of spinal cord injury for evaluation of tissue engineering treatment strategies.</a> Biomaterials. 25 (9), 1505-1510 (2004).</li><li>Nakae, 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=The+animal+model+of+spinal+cord+injury+as+an+experimental+pain+model.">The animal model of spinal cord injury as an experimental pain model.</a> Journal of Biomedicine &amp; Biotechnology. 2011, 939023 (2011).</li><li>Kwon, B., Oxland, T., Tetzlaff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+used+in+spinal+cord+regeneration+research.">Animal models used in spinal cord regeneration research.</a> Spine. 27, 1504-1510 (2002).</li><li>Kundi, S., Bicknell, R., Ahmed, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+injury:+current+mammalian+models.">Spinal cord injury: current mammalian models.</a> American Journal of Neuroscience. (4), 1-12 (2013).</li><li>Harrison, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vertebral+landmarks+for+the+identification+of+spinal+cord+segments+in+the+mouse.">Vertebral landmarks for the identification of spinal cord segments in the mouse.</a> Neuroimage. 68, 22-29 (2013).</li><li>Basso, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basso+Mouse+Scale+for+locomotion+detects+differences+in+recovery+after+spinal+cord+injury+in+five+common+mouse+strains.">Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains.</a> Journal of Neurotrauma. 23 (5), 635-659 (2006).</li><li>Seitz, A., Aglow, E., Heber-Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+from+spinal+cord+injury:+a+new+transection+model+in+the+C57Bl/6+mouse.">Recovery from spinal cord injury: a new transection model in the C57Bl/6 mouse.</a> Journal of Neuroscience Research. 67 (3), 337-345 (2002).</li></ol>

这种方法在小鼠T10椎位诱发完全转切型损伤，导致动物完全截瘫，低于损伤水平。总体而言，此方法可导致最小出血、可忽略不计的附带损害和稳定的可重复伤害。与之前公布的无兰胺切除术10的转治方法相比，该方法在直接可视化方面提供了好处，无需操纵脊柱的曲率，更好地控制损伤的完成，增强了控制出血和实现止血的能力。这种方法的优点是，该协议可以修改，用于任?...

脊髓损伤 （SCI） 是一个复杂的医疗问题，导致健康和生活方式的急剧变化。SCI没有治愈的方法，SCI的病理生理学也没有被彻底理解。动物SCI模型，特别是啮齿动物模型，为试验新疗法提供了宝贵的工具，并已被用于探索SCI几十年。迄今为止，超过72%的临床前SCI研究采用了老鼠模型，而只有16%的人使用过小鼠1。虽然大鼠由于体型较大，而且倾向于形成类似于人类SCI的蛀牙，传统上是研究新疗法的首选模型动物，但老鼠（包括许多转基因小鼠模型）现在正更频繁地用于研究SCI 2的细胞和分子机制。小鼠模型在比大鼠更容易处理、更快的生殖速率和更低的成本方面提供了额外的好处;小鼠也表现出高度的基因组相似性与人类1，2，3。小鼠模型的主要缺点是尺寸明显缩小，这给创建和治疗脊髓损伤的外科手术带来了挑战。
现有文献中存在差距，突出表明需要一种坚固且可重复的手术方案，以在小鼠模型中诱导稳定的 SCI。因此，我们提供了一种新颖和精确的手术方法，以克服这些限制。该协议提供了深入的指南，以诱导小鼠的转切型损伤，因为这种损伤类型已被确认为最合适的研究再生和退行性的变化后，伤害6，以及神经可塑性，神经电路和组织工程方法7。我们选择诱导损伤在较低的胸腔区域，因为胸腔水平SCI是最常用的文献1。

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			<td>Consumable</td>
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			<td>Reusable</td>
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			<td>Reusable</td>
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			<td height="21" style="height:21px;width:449px;">Cotton swabs (5pcs)</td>
			<td style="width:243px;">Multigate</td>
			<td style="width:161px;">21-893</td>
			<td>Consumable</td>
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			<td height="21" style="height:21px;width:449px;">Dremel Micro</td>
			<td style="width:243px;">DREMEL</td>
			<td style="width:161px;">8050-N/18</td>
			<td>Cordless rotary tool</td>
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		<tr height="20">
			<td height="20" style="height:21px;width:449px;">Dressing forceps fine</td>
			<td style="width:243px;">Multigate</td>
			<td style="width:161px;">06-306</td>
			<td>Single use disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Drill bits</td>
			<td style="width:243px;">Kemmer Pr&#228;zision</td>
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			<td>Reusable</td>
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			<td height="21" style="height:21px;width:449px;">Dumont #7b forceps</td>
			<td style="width:243px;">Fine Science Tools</td>
			<td style="width:161px;">11270-20</td>
			<td>Reusable</td>
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			<td height="21" style="height:21px;width:449px;">Dumont tweezers, style 5</td>
			<td style="width:243px;">ProSciTech</td>
			<td style="width:161px;">T05-822</td>
			<td>Reusable</td>
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			<td height="21" style="height:21px;width:449px;">Fur trimmer</td>
			<td style="width:243px;">WAHL</td>
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			<td>Zero Overlap Hair Trimmer</td>
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			<td height="21" style="height:21px;width:449px;">Iris scissors, Ti, sharp tips, straight, 90mm</td>
			<td style="width:243px;">ProSciTech</td>
			<td style="width:161px;">TY-3032</td>
			<td>Reusable</td>
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			<td height="21" style="height:21px;width:449px;">Isoflurane isothesia NXT 250</td>
			<td style="width:243px;">Provet</td>
			<td style="width:161px;">ISOF 00 HS</td>
			<td>Anaesthetic agent</td>
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			<td height="21" style="height:21px;width:449px;">Colibri Retractor - 4cm</td>
			<td style="width:243px;">Fine Science Tools</td>
			<td>17000-04</td>
			<td>Reusable</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Scalpel handle</td>
			<td style="width:243px;">ProSciTech</td>
			<td style="width:161px;">T133</td>
			<td>Reusable</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Signature latex surgical gloves size 7.5</td>
			<td style="width:243px;">Medline</td>
			<td style="width:161px;">MSG5475</td>
			<td>Consumable</td>
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			<td height="21" style="height:21px;width:449px;">Sodium Chloride 0.9%</td>
			<td style="width:243px;">STS</td>
			<td>PHA19042005</td>
			<td>Consumable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Sterile Dressing Pack</td>
			<td style="width:243px;">Multigate</td>
			<td style="width:161px;">08-709</td>
			<td>Single use disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Sterile Fluid Impervious Drape 60x60 cm</td>
			<td style="width:243px;">Multigate</td>
			<td style="width:161px;">29-220</td>
			<td>Single use disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Surgical spirit 100 mL</td>
			<td style="width:243px;">Provet</td>
			<td style="width:161px;"># SURG SP</td>
			<td>Consumable</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Suture Material - SILK BLK 45CM 5/0 FS-2</td>
			<td style="width:243px;">Johnson &#38; Johnson Medical</td>
			<td style="width:161px;">682G</td>
			<td>Silk Suture</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Suture Material - Vicryl 70CM 5-0 S/A FS-2</td>
			<td style="width:243px;">Johnson &#38; Johnson Medical</td>
			<td style="width:161px;">VCP421H</td>
			<td>Vicryl Suture</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:449px;">Temgesic 0.3 mg in 1 mL, x 5 ampoules (class S8 drug)</td>
			<td style="width:243px;">Provet</td>
			<td style="width:161px;">TEMG I</td>
			<td>Post-operative care drug</td>
		</tr>
	</tbody>
</table>

induction of complete transection-type spinal cord injury in mice

脊髓损伤 （SCI） 在很大程度上导致不可逆转和永久性的功能丧失，最常见的是创伤的结果。正在研究一些治疗方案，如细胞移植方法，以克服SCI引起的使人衰弱的残疾。大多数临床前动物试验是在SCI的啮齿动物模型中进行的。虽然SCI的老鼠模型已经被广泛使用，但老鼠模型受到的关注较少，尽管老鼠模型比老鼠模型有显著的优势。老鼠体积小，比老鼠低动物维护成本，许多转基因小鼠模型的提供对许多类型的研究都有利。诱导动物的可重复和精确伤害是SCI研究的主要挑战，在小型啮齿动物中，需要高精度手术。过去十年来，转切型损伤模型一直是移植治疗研究中常用的损伤模型，但不存在诱导小鼠完全转切型损伤的标准化方法。我们开发了一种手术方案，用于诱导胸椎10级（T10）的C57BL/6小鼠出现完全转切型损伤。该程序使用小尖端钻头代替角膜来精确去除层，之后使用带圆切刃的薄刀片诱导脊髓转液。这种方法导致小啮齿动物的可重复转切型损伤，具有最小的附带肌肉和骨骼损伤，因此最大限度地减少混淆因素，特别是在行为功能结果分析的地方。

图 1中描述的方法涉及小鼠的充分稳定（图 1A）和脊柱和准视组织（图1B） 的可视化。旋转过程和层压可以清楚地可视化与最小的肌肉解剖和失血（图1C，高亮区）。精细尖端钻孔按图 1D所示执行，以创建如矩形所示的拉明切除术窗口。由此产生的拉明切除术窗口是明确的，并允许直接...

Watch this Scientific Journal Video about Induction of Complete Transection-Type Spinal Cord Injury in Mice at JoVE.com

Induction of Complete Transection-Type Spinal Cord Injury in Mice

该协议描述了如何创建精确的拉明切除术，在小鼠模型中诱导稳定的转节型脊髓损伤，为脊髓损伤研究提供最小的附带损伤。

小鼠完全转型脊髓损伤的诱导

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...

该协议提供精确的手术控制损伤，诱导小鼠完全脊髓转染，具有高可重复性和非常高的存活率。使用细钻对骨损伤最大限度地减少对周围组织的损伤，使损伤和治疗反应得到更准确的建模。为了进行拉明切除术，在确认麻醉的8至10周大雌性C57黑色6小鼠对踏板反射反应不足后，剃光背部毛皮，将手术区域暴露在背脊椎上，用浸泡在波维酮碘防腐液和手术精神中的无菌棉签拭子对剃光区域进行消毒。使用手术刀，在T10椎骨水平上进行垂直中线切口，并使用直钳将皮肤从底层筋膜上抬起来，以方便缩回器放置。要暴露T9到T11椎骨的脊椎，使用手术刀的钝边在皮下组织和下皮筋膜上做一个小中线切口，并使用细尖非锋利的钳子钝解剖和反射筋膜。要暴露肌酸，使用手术刀的钝尖分裂背部躯干和准针肌肉沿T9的脊椎到T11椎骨，并使用钝细尖钳钝解剖肌肉层，以暴露椎骨的肌骨。使用相同的钳子在 T10 椎骨的横向过程周围制作小口袋，并在创建的口袋内的横向过程下钩住弯曲钳子的爪，以稳定 T10 椎体。用温暖的盐水彻底冲洗T10层，并使用棉签轻轻擦拭骨质表面，注意侧表面没有肌肉或韧带附件。要双边间打破层，请使用直径 0.55 毫米、7 毫米长度的钻头尖端，沿着钻头沿 T10/T11 椎间空间跟踪从 T9/T10 椎间空间到 T10/T11 椎间空间的垂直路径。追踪路径后，打开钻头，缓慢而小心地在 T10 椎骨的右层上制作一个垂直沟槽，使用弯曲的钳子保持椎骨稳定。在层压左侧重复此过程后，用温暖的盐水灌溉组织，以洗去剩余的骨碎片。用一只手保持良好的脊柱抓地力对于这一步至关重要。使用钻头变得舒适和自信也很重要。接下来，使用倾斜细尖钳抓住旋转过程，并拆下由双边钻孔分隔的层压的整个支脚段。然后，灌溉和拭子，以允许脊髓在拉明切除术窗口内清晰可视化。要诱导外露脊髓内的脊髓损伤，请使用窄圆切边刀片切开脊髓中心拉明切除术窗口的中心，注意扫描脊柱的横向凹槽，以诱发完全的转膜损伤。要确认转节损伤的完整性，请使用钝细尖端钳子移除转流位点上剩余的连接。一旦在转口站点上实现了血侧，释放弯曲的力p对T10椎骨的抓地力，并沿中线将解剖肌肉的边缘聚集在一起，以达到良好的定位。使用 5 / 0 多糖蛋白 910 可吸收缝合线，将肌合在层中，确保脊柱的自然曲率不会导致缝合线的任何张力或打开缝合线。然后，使用5/0不可吸收的丝质缝合来关闭皮下组织和皮肤，注意在关闭之前皮肤下没有出血、血块或碎屑。为了评估这种诱导小鼠转切型脊髓损伤的方法是否可重复和一致，可以通过免疫理论分析损伤脊髓，并可对小鼠进行行为测试。对星细胞标记的免疫标记，胶质纤维酸蛋白，有利于划定完整的脊髓的边界。在此纵向部分中，可以如预期的那样在线桩之间可视化位于的伤害点。在平均最小距离为 550.4、正负 17.3 微米时，可以在转流位点诱导一致的大小缺陷。部署开放场测试的巴索小鼠量表的行为数据显示，受伤的小鼠在受伤后没有表现出后肢运动。因此，该协议产生一个完整和可靠的转节类型损伤，导致完全丧失功能低于伤害水平，不会导致自发逆转的瘫痪。注意用稳定的手在直线上钻孔，因为一旦钻错了，修复拉明切除术窗口可能非常棘手。脊髓损伤诱导后，可以应用各种治疗方案，包括药物输送和细胞移植，以确定改善脊髓损伤恢复的潜在疗法。

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

侧向液压冲击：创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

脊髓损伤 wistar 大鼠跑步机的作业训练

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

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

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 ...

Establishing a Mouse Contusion Spinal Cord Injury Model Based on a Minimally Invasive Technique

基于微创技术的小鼠挫伤脊髓损伤模型的建立

Using minimally invasive methods to model spinal cord injury (SCI) can minimize behavioral and histological differences between experimental animals, ...