Name: synergetic use of neural precursor cells and self-assembling peptides in experimental cervical spinal cord injury
Uploaded: 2015-02-23T13:00:00.000+00:00
Duration: 11 min 57 s
Description: 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

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><tbody><tr><td>Aneurysmal clip</td><td>SharpTech</td><td></td><td></td></tr><tr><td>Surgical microscope</td><td>Leica</td><td></td><td></td></tr><tr><td>Micro injection system</td><td>World Precision Instruments, Inc.</td><td></td><td></td></tr><tr><td>Small animal stereotaxic instrument</td><td>David Kopf Instruments</td><td></td><td></td></tr><tr><td>Hamilton syringe</td><td>Hamilton company</td><td></td><td></td></tr><tr><td>Subdural pumps</td><td><a href="http://www.alzet.com" target="_blank"><img src="/img/new_window.png" /> Alzet osmotic micro pump 1007D</a></td><td></td><td></td></tr><tr><td>Surgical instrument</td><td>Fine Science tools</td><td></td><td></td></tr><tr><td>Isoflurane USP</td><td>Pharmaceutical Partners of Canada Inc.</td><td></td><td></td></tr><tr><td>0.9% Sodium Chloride injection USP</td><td>Baxter</td><td></td><td></td></tr><tr><td>7.5% Povidone iodine</td><td>Purdue Pharma</td><td></td><td></td></tr><tr><td>70% Isopropyl alcohol USP</td><td>GreenField Ethanol Inc.</td><td></td><td></td></tr><tr><td>QL6 SAP</td><td>Covidien</td><td></td><td></td></tr><tr><td>0.4% Trypan blue</td><td>Gibco</td><td></td><td></td></tr><tr><td>Platelet-Derived Growth Factor (PDGF)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Epidermal Growth Factor (EGF)</td><td>Sigma</td><td></td><td></td></tr><tr><td>Fibroblast Growth Factor (FGF)</td><td>Sigma</td><td></td><td></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

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

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 ...

神经科学

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 ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

三维组织工程化排列的星形胶质细胞网络, 以概括发展机制和促进神经系统再生

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

生物工程

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 ...

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

解剖学启发的三维微组织工程神经网络用于神经系统重建，调制和建模

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

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

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 injury. The complete transection model, although not as clinically relevant as the contusion model, is the most rigorous method to evaluate axon regeneration. In the contusion model, it is difficult to distinguish regenerated from sprouted or spared axons due to the presence of remaining tissue post injury. In the complete transection model, a bridging method is necessary to fill the gap and create continuity from the rostral to the caudal stumps in order to evaluate the effectiveness of the treatments. A reliable bridging surgery is essential to test outcome measures by reducing the variability due to the surgical method. The protocols described here are used to prepare Schwann cells (SCs) and conduits prior to transplantation, complete transection of the spinal cord at thoracic level 8 (T8), insert the conduit, and transplant SCs into the conduit. This approach also uses in situ gelling of an injectable basement membrane matrix with SC transplantation that allows improved axon growth across the rostral and caudal interfaces with the host tissue.

This article describes a technique to insert a hollow conduit between the spinal cord stumps after complete transection and fill with Schwann cells (SCs) and injectable basement membrane matrix in order to bridge and promote axon regeneration across the gap.

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 ...

A Neurosphere Assay to Evaluate Endogenous Neural Stem Cell Activation in a Mouse Model of Minimal Spinal Cord Injury

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied in vitro using the neurosphere assay. This colony-forming assay is a powerful tool to study the response of NSCs to exogenous factors in a dish; however, this can also be used to study the effect of in vivo manipulations with the proper understanding of the strengths and limitations of the assay. One manipulation of the clinical interest is the effect of injury on endogenous NSC activation. Current models of spinal cord injury provide a challenge to study this as the severity of common contusion, compression, and transection models cause the destruction of the NSC niche at the site of the injury where the stem cells reside. Here, we describe a minimal injury model that causes localized damage at the superficial dorsolateral surface of the lower thoracic level (T7/8) of the adult mouse spinal cord. This injury model spares the central canal at the level of injury and permits analysis of the NSCs that reside at the level of the lesion at various time points following injury. Here, we show how the neurosphere assay can be utilized to study the activation of the two distinct, lineally-related, populations of NSCs that reside in the spinal cord periventricular region - primitive and definitive NSCs (pNSCs and dNSCs, respectively). We demonstrate how to isolate and culture these NSCs from the periventricular region at the level of injury and the white matter injury site. Our post-surgical spinal cord dissections show increased numbers of pNSC and dNSC-derived neurospheres from the periventricular region of injured cords compared to controls, speaking to their activation via injury. Furthermore, following injury, dNSC-derived neurospheres can be isolated from the injury site &#8212; demonstrating the ability of NSCs to migrate from their periventricular niche to sites of injury.

Here, we demonstrate the performance of a minimal spinal cord injury model in an adult mouse that spares the central canal niche housing endogenous neural stem cells (NSCs). We show how the neurosphere assay can be used to quantify activation and migration of definitive and primitive NSCs following injury.

小鼠脊髓损伤模型中内源性神经干细胞活化的干细胞检测

Neural stem cells (NSCs) in the adult mammalian spinal cord are a relatively mitotically quiescent population of periventricular cells that can be studied ...

Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in the US, and it represents an immeasurable public health concern. Research has been conducted to provide effective therapy; however, SCI is still considered incurable due to the complex nature of the injury site. A variety of strategies, including drug delivery, cell transplantation, and injectable biomaterials, are investigated, but one strategy alone limits their efficacy for regeneration. As such, combinatorial therapies have recently gained attention that can target multifaceted features of the injury. It has been shown that extracellular matrices (ECM) may increase the efficacy of cell transplantation for SCI. To this end, 3D hydrogels consisting of decellularized spinal cords (dSCs) and sciatic nerves (dSNs) were developed at different ratios and characterized. Histological analysis of dSCs and dSNs confirmed the removal of cellular and nuclear components, and native tissue architectures were retained after decellularization. Afterward, composite hydrogels were created at different volumetric ratios and subjected to analyses of turbidity gelation kinetics, mechanical properties, and embedded human adipose-derived stem cell (hASC) viability. No significant differences in mechanical properties were found among the different ratios of hydrogels and decellularized spinal cord matrices. Human ASCs embedded in the gels remained viable throughout the 14-day culture. This study provides a means of generating tissue-engineered combinatorial hydrogels that present nerve-specific ECM and pro-regenerative mesenchymal stem cells. This platform can provide new insights into neuro-regenerative strategies after SCI with future investigations.

In this study, nerve-mimetic composite hydrogels were developed and characterized that can be utilized to investigate and capitalize on the pro-regenerative behavior of adipose-derived stem cells for spinal cord injury repair.

使用干细胞递送开发脊髓损伤的组合疗法

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in ...