我们要感谢为这项工作的健康研究（CIHR）加拿大研究所的资金支持，Krembil家庭基金会的主席亥尔波特在神经修复和再生，菲利普和佩吉DeZwirek，和戈登姚明的贡献，图2 。克劳斯Zweckberger资金由从"德意志Forschungsgesellschaft"（DFG）的资助。

注:以下实验方案被批准为大学健康网络（多伦多，加拿大）的动物保护委员会，并按照既定的指导，关心和准备使用的动物保健加拿大议会实验动物的政策。 1.颈椎动脉瘤夹挫伤/压缩​​模式<ol><li>术前高压灭菌仪器和通过将仪器在70％的酒精浴保持无菌条件下整个surgcial过程中。 </li><li>麻醉Wistar大鼠（250-270克）与氧的结合（O 2），一氧化二氮（N 2 O）（1:1），和1.8-2.2％异氟醚，并且通过气体麻醉面罩支持自主呼吸。麻醉诱导开始用5％异氟醚1分钟，减少事后。前给予疼痛刺激（ 如在爪子）开始手术，麻醉的控制深度。适用于脂肪药膏在眼睛干涩避免和防止以后感染。 </li><li>把老鼠到加热坐垫（37°C）和固定头部到立体定位框架。 </li><li>剃手术区周围的颈椎和消毒用碘伏和70％的酒精。 </li><li>使中线切口从颈椎（C2）延伸到胸椎椎体2（T2）的突出突脊的上方。 </li><li>通过脊椎肌肉直接在中线外层切（避免出血）在头尾方向，并进一步剖析更深的肌肉层直言，直到你到达棘突和椎板。插入拉钩。 </li><li>定向的椎体T2观察目标水平椎板切除的突出棘突。鉴定和显微外科制备选定薄片​​后，通过ligamenti flavae切割以松开薄层和棘突。最后，CUT斯达康通过与骨推子横向于脊髓的薄片和除去那些轻轻，避免了脊髓本身的任何压缩。 注意:最常见的水平C5 / 6，C6 / 7或C7 / T1。围手术期死亡率增加了更多喙损伤水平。从椎旁静脉窦出血​​是常见的，并且可以通过用海绵小心压缩处理。 </li><li>插入剪辑的精神创伤线前，确定新出现的神经根，从削波（特别是在一级C5 / 6），放过他们。 </li><li>为了确保平稳剪辑感应，松开从椎体的背侧腹侧硬膜用钩，并准备一个走廊的剪辑。 </li><li>最后，将打开夹子，让它捕捉关（快速闭合），实现了挫伤。夹子闭合力和夹闭合的持续时间决定了创伤的强度和压缩的程度。常用的有间15-35克和clippi夹力吴持续时间如 1分钟。 （ 图1）。 </li><li>除去夹子后，适应的肌肉在2层和闭合伤口。 </li><li>停止麻醉并让动物唤醒在你连续观察，直到它重新获得足够的意识的胸骨斜卧。最后，把老鼠在一个笼子里，并按照手术后的治疗指南。 </li><li>由于动物的这种类型的损伤的严重程度斗争，必须特别注意​​术后护理: <ol><li>施用止痛药（丁丙诺啡和美洛昔康3天，5天，分别与根据临床症状）。 </li><li>得到附加的盐水溶液皮下3天（每天2次，5-10毫升（ml））。 </li><li>提前2天内提供在饮用水抗生素和直到后7天手术（ 例如 Moxifloxacine） </li><li>挤压膀胱，每日2-3次，直到膀胱功能的恢复是不变LY明显。 </li><li>每天观察神经功能缺损及手术动物的生理状态至少一次。 </li></ol></li></ol> 2.注射的SAP与NPC（伤后14天） <ol><li>在1.1中描述诱导麻醉。到1.3，固定的大鼠的头在立体定位框架，取出线圈或卷绕片段和消毒伤口并用聚维酮碘和70％乙醇的手术区域。 </li><li>小心解剖椎旁肌肉，插入拉钩，从硬脑膜显微镜除去疤痕组织，并重新露出病灶部位。 </li><li>以1％的浓度制备的SAP（重量/体积），以执行一个细胞外基质的凝胶。 QL6的SAP具有生理上相容的pH值，并且不需要在注射前进行缓冲。在脊髓SAP的可视化，使用QL6（QL6-FITC）的荧光衍生物。 </li><li>注入的SAP（5微升（μL）到中心的病变，分布在2份，每份中线2.5微升双边。用Hamilton注射器连接到立体定位框架用微玻璃毛细管（100微米（μm）的外径（OD））。用锋利的针尖小心打开硬脑膜，并插入玻璃毛细管立体定位2毫米（毫米）到创伤脊髓。 </li><li>注射体积的1/3后，取出针至1.5mm的深度，并经过进一步的1/3至1毫米。注射的总体积的后，并除去注射器之前，等待5分钟，以稳定的凝胶形成。 </li><li>要生成的NPC，用成年鼠红色荧光蛋白（或YFP阳性鼠，绿色）和隔离，并从paraventrical区4,18,21培养他们。 </li><li>评估用台盼蓝染色的NPC指示90％的活细胞的〜在细胞悬浮液中存在的生存能力。稀释的细胞在生长培养基中（50×103活细胞/微升），然后将它们用于细胞移植。 </li><li>提出四2微升（微升8总武lume明，含有4×10 5个的NPC）脊柱内注射双边为2mm喙和损伤部位的尾部。开口硬脑膜后，插入汉密尔顿微玻璃毛细管1.5毫米脊髓的背侧表面下方，并注入2微升的细胞悬浮液。选择喷射率在约0.5微升/分钟（分钟）。 </li><li>在每次注射并除去毛细管出来的帘线之前结束时，等待至少1分钟，使组织伸展，以适应新的细胞体积。 （ 图2） </li></ol> 3.植入硬膜下的泵生长因子应用<ol><li>为了丰富用生长因子的脑脊髓液（CSF），支持细胞存活，使用微渗透泵在整个7-14天子durally稀释的生长因子，以0.5微升/小时的稀释率，0.04的导管直径厘米外径，以及100微升的贮存体积。 </li><li>选择首选生长因子（例如，脑源性生长因子（BDGF），表皮生长因子（EGF），成纤维细胞生长因子（FGF）），补油泵6-10小时之前注入，并平衡泵的水浴中在37℃。 </li><li>立即NPC注射后，准备皮下凹陷放置泵。优选的位置是thoraco-腹部区域避免由于泵本身主要局部不适的横向侧面。 </li><li>获得的生长因子的最高浓度，确保了导管的开口前端部附近的病变部位。因此，进行跳跃椎板切除相邻的上或下的水平。例如，如果在SCI是C7 / T1，执行小椎板C5的。 </li><li>把泵入皮下凹槽，缩短导管必要的长度，并具有若干缝（6.0）上的椎旁肌避免任何运动相关脱位固定。 </li><li>用锋利的尖端张开硬脑膜（ 例如 ，在C5）后一针，引入导管进入硬膜下腔，并让它在没有任何抵抗，没有伤及脊髓骶管的方向滑行。 如 C6的跳过椎板提供固定导管和稳定的额外点。 </li><li>确保导管运行平稳，不折。 </li><li>关闭的肌肉层和皮肤，用缝线或夹子。 （ 图3） </li><li>停止麻醉并让动物唤醒在你连续观察，直到它重新获得足够的意识的胸骨斜卧。最后，把它们放回在一个笼子里，并按照手术后的治疗指南。 </li><li>提供特殊的术后治疗，尽管，动物可能在这个时间点回收的部分: <ol><li>施用止痛药（Buprenorthine和美洛昔康3天，5天，分别与根据临床症状）。 </li><li>在水瓶提前2天，直到提供抗生素后7天的手术（ 例如 Moxifloxacine）。 </li><li>继续挤压膀胱，如果还是必要的。 </li><li>给予额外皮下流体，如果大鼠出现脱水。 </li><li>保持每天观察手术动物的神经功能和生理条件的至少一次。 </li><li>辖免疫抑制治疗前2天NPC注射到晚上7天后transplanation（米诺环素），直到牺牲（SANDIMMUNE），分别为。 </li></ol></li></ol> 4.组织评估<ol><li>牺牲动物在观察时间结束时（ 例如 4周SCI后）在深度麻醉（5％异氟醚为2-3分钟），并用50ml冷的（4℃）的盐水，随后150毫升冷的4 transcardially灌注它们％多聚甲醛的0.1M磷酸盐缓冲盐水（PBS）。 </li><li>取出脊髓，并把它在4％多聚甲醛的0.1M磷酸盐缓冲盐水（PBS）中24小时。 </li><li&gt;把在25％蔗糖溶液的脊髓进行冷冻保护48小时。 </li><li>提供纵向冷冻切片的厚度为&lt;30微米。 </li><li>对于所有细胞的细胞核提供了一个背景使用的DAPI（1:1000）免疫组化染色。红色荧光蛋白阳性的NPC出现红色，QL-6 FITC显示为绿色，并且都因此不需要进行特殊染色。 （ 图4）。 </li><li>为扫描电子显微镜（SEM）让样品浸泡在戊二醛在4℃下2小时，在10％的增量步骤的乙醇缓慢脱水它们为5分钟，并将其放置在一个加压的液体CO 2的虹吸1小时。用溅射镀膜机大衣支架黄金。采取与日立S-3400N扫描电子显微镜图像。 （ 图5） </li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+trasplantation+of+adult+neural+presursor+cells+promotes+remyelination+and+functional+neurological+recovery+after+spinal+cord+injury.">Delayed trasplantation of adult neural presursor cells promotes remyelination and functional neurological recovery after spinal cord injury.</a> J Neurosci. 26 (13), 3377-3389 (2006).</li><li>Burdick, J. A., Ward, M., Liang, E., Young, M. J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+neurite+outgrowth+by+neurotrophins+delivered+from+degradable+hydrogels.">Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels.</a> Biomaterials. 27, 452-459 (2006).</li></ol>

Covidien supplied the QL6 SAP for this study.

此协议已被开发，以使读者对大鼠进行颈椎损伤模型，并使用同的SAP与NPC促进子宫颈脊髓损伤后更好的恢复的组合的治疗方法。 特别是在相对于其他宫颈创伤模型，如在（半）-transection模型或重降和震荡模型，夹子挫伤/压缩​​模型代表两个主要病理生理机制创伤 - 挫伤和压缩在，因此，模拟最佳临床病症。虽然它是公认的用于在大鼠和小鼠胸椎7-11的用途，它最近?...

脊髓损伤的50％以上都与颈椎。在临床上设置两个主要病理生理学机制的描述:脊髓的初始挫伤，随后，引起骨骨折，出血或组织肿胀正在进行压缩。 动脉瘤夹挫伤/压缩​​模型模拟两者的病理生理机制:捕捉剪辑产生挫伤和削波的持续时间表示所述压缩元件，承认其在引起骨骨折，出血或组织临床设置中的压缩肿胀最后显著更长。所用动脉瘤夹由一环状弹簧保证精确和可重现的剪裁力修改。尤其是在比较半侧横断或挫伤模型，这种动脉瘤夹模型模拟最好的临床设置。虽然患者的胸部损伤截瘫，从大多数颈椎病患者注射吃亏uries是四肢瘫痪，完全依赖。颈髓的解剖结构，然而，示出相比于胸椎或腰椎显著的差异，并且因此，被寻址特别是在此协议。 髓内腔和疤痕组织的发展，是恢复和再生障碍。为了克服这些障碍，利用支架材料是一种很有前途的方法。自组装肽可直接注射到病灶的中心。在那里，他们组装成纳米纤维支架桥接腔，并通过减少炎症和组织瘢痕改善抑制环境。而刚性材料造成相当大的损害植入期间脊髓，流体肽可以安全且没有严重的额外伤害注入。 改善与自组装肽的抑制环境干细胞移植之前，因此，支持小区integration，分化最后，功能恢复，颈脊髓损伤后。

<table border="0" cellpadding="0" cellspacing="0" width="541">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:268px;">Company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aneurysmal clip</td>
			<td>SharpTech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical microscope</td>
			<td>Leica</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro injection system</td>
			<td>World Precision Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small animal stereotaxic instrument</td>
			<td>David Kopf Instruments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamilton syringe</td>
			<td>Hamilton company</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Subdural pumps</td>
			<td>Alzet osmotic micro pump 1007D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical instrument</td>
			<td>Fine Science tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane USP</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% Sodium Chloride injection USP</td>
			<td>Baxter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7.5% Povidone iodine</td>
			<td>Purdue Pharma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% Isopropyl alcohol USP</td>
			<td>GreenField Ethanol Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QL6 SAP</td>
			<td>Covidien</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.4% Trypan blue</td>
			<td>Gibco</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platelet-Derived Growth Factor (PDGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF)</td>
			<td>Sigma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibroblast Growth Factor (FGF)</td>
			<td>Sigma</td>
		</tr>
	</tbody>
</table>

synergetic use of neural precursor cells and self-assembling peptides in experimental cervical spinal cord injury

脊髓损伤（SCI）导致严重的神经功能障碍和心理，经济和社会后果的患者和他们的家庭。在临床上，脊髓损伤的50％以上的影响颈椎1。作为原发性损伤的结果，二次机制，包括炎症，细胞凋亡，和脱髓鞘级联发生最终导致组织结疤髓内腔2,3和发展。均代表物理和化学障碍细胞移植，集成和再生。因此，塑造抑制环境和桥接腔营造一个有利的环境对细胞移植和再生是一种很有前途的治疗目标4。这里，子宫颈癌的SCI的使用的动脉瘤夹挫伤/压缩​​模型进行说明。这种模式比其他实验模型更临床相关的，因为完全横断或电源线的破裂是罕见的。此外，在试比较n至重量下降模型，它特别损害鼻背列，则脊髓的圆周压缩出现有利的。夹子闭合力和持续时间可以调整，以达到不同的损伤程度。环形的弹簧有利于精确校准和夹子力恒定。在生理条件下，合成的自组装肽（SAP）的自组装成纳米纤维，因此，呼吁在SCI 5的应用程序。它们可以直接注射到病灶减少损坏的线。 SAP的具有生物相容性的结构支架架设桥梁髓内腔，因此，装备损坏的电源线用于再生治疗。 K2（QL）6K2（QL6）是由Dong 等一升。6引入相对于其他的肽的新型的SAP，QL6自我组装成β片在中性pH SCI后60.14天，急性期，SAP的后被注入到病变和神经前体细胞（NPC）的中心是麟蹄反恐执行局到相邻背列。为了支持细胞存活，移植是结合的生长因子通过渗透微泵连续7天连续硬膜下施用。

<html>当执行上述的过程中，将得到一个SAP的支架桥接所述腔，并提供一种改进的抑制环境的，较少的组织瘢痕和增加鼻咽癌存活。 图4示出了在获得的大鼠脊髓的纵剖面损伤部位6周SCI后4周QL6 SAP注入和NPC移植后。 QL6肽成功注入脊髓，聚集在震中和半影扩散rostro-尾端。电子显微镜成像进一步示出，在图5中，1％的组件（重量/体积）QL6肽内2小时在PBS中稀释溶液的纳米纤维支?...

Watch this Scientific Journal Video about Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury at JoVE.com

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

在实验颈脊髓损伤协同使用神经前体细胞，并自组装肽的

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

synergetic-use-neural-precursor-cells-self-assembling-peptides

Research

JoVE Journal

Medicine

9.2K 次观看.  University Health Network, Krembil Neuroscience Centre. Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established. 

视频: Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

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

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 ...

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Controlled Cervical Laceration Injury in Mice

Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.

A novel technique to create a reproducible in vivo model of cervical spinal cord laceration injury in the mouse is described. This technique is based on spine stabilization by fixation of the cervical facets and laceration of the spinal cord using an oscillating blade with an accuracy of &plusmn;0.01 mm.

控制宫颈裂伤损伤小鼠

Use of genetically modified mice enhances our  understanding of molecular mechanisms underlying several neurological disorders  such as a spinal cord ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

评价呼吸肌慢性脊髓损伤个人使用呼吸电机控制评估（RMCA）

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

A Murine Model of Cervical Spinal Cord Injury to Study Post-lesional Respiratory Neuroplasticity

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed to improve/ameliorate the respiratory failure following high cervical spinal cord injury (SCI). Here we propose a murine pre-clinical model of high SCI at the cervical 2 (C2) metameric level to study diverse post-lesional respiratory neuroplasticity. The technique consists of a surgical partial injury at the C2 level, which will induce a hemiparalysis of the diaphragm due to a deafferentation of the phrenic motoneurons from the respiratory centers located in the brainstem. The contralateral side of the injury remains intact and allows the animal recovery. Unlike other SCIs which affect the locomotor function (at the thoracic and lumbar level), the respiratory function does not require animal motivation and the quantification of the deficit/recovery can be easily performed (diaphragm and phrenic nerve recordings, whole body ventilation). This pre-clinical C2 SCI model is a powerful, useful, and reliable pre-clinical model to study various respiratory and non-respiratory neuroplasticity events at different levels (molecular to physiology) and to test diverse putative therapeutic strategies which might improve the respiration in SCI patients.

Respiratory failure is the leading cause of death following a cervical spinal cord injury. Having a reproducible, quantifiable, and reliable pre-clinical animal model of respiratory failure induced by a partial cervical injury will help to understand the subsequent respiratory and non-respiratory neuroplasticity and allow testing putative repair strategies.

颈脊髓损伤的小鼠模型研究后皮损呼吸神经可塑性

A cervical spinal cord injury induces permanent paralysis, and often leads to respiratory distress. To date, no efficient therapeutics have been developed ...

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 ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

脊髓压迫损伤的校准钳型

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...