这项工作是由国际脊髓研究信托与信托Rosetrees支持。

伦理学声明：所有的程序均符合由英国内政部的指导方针和动物（科学程序）1986年法令。 1.手术和伤害注意：使用大鼠于220 g重的可以帮助术后恢复，如果医生发现的动物往往失去自己的术前体重的10％以上。 <ol><li>手术前，测试动物的前爪偏好与缸饲养试验15或蒙托亚staicase测试24。最好，切金字塔/科委对应的主导前爪。 注：对于前两天手术，将以下物品放入笼子，让老鼠熟悉，这将是从手术中恢复过程中有益的食物：水凝胶包，湿捣烂，和高热量的食物补充剂。可以从不同的供应商，这些食品补充剂无菌版本。 </li><li>麻醉女L存器罩住大鼠（诱导和维持5％1〜2％）中的氧，用异氟烷（在200和250g体重）（流速：1.5升/分钟）。注：麻醉剂的选择是很重要的。吸入麻醉剂允许更快调节麻醉深度大于氯胺酮，这也是常用的。异氟烷允许更好地控制呼吸频率和深度。麻醉剂的选择是异氟醚。注意：使用大鼠于220 g重的可以帮助术后恢复，如果医生发现的动物往往失去自己的术前体重的10％以上。 </li><li>辖卡洛芬皮下作为镇痛时将动物麻醉，以减轻炎症和提供镇痛。注：卡洛芬是在锥体束术根据我们的兽医的意见，给出我们的实验室。在手术过程中的阿片类药物可能会导致进一步的呼吸抑制。 </li><li>使用家庭手术过程中保持这些动物在37℃下吸热层系统和直肠温度探头。 </li><li>确认完全麻醉通过检查的爪子掐撤回反射和瞬目反射。 注：可选：为了对抗期间手术，气管插管和通风呼吸问题可能是可行的。然而，要在手术过程中被移位记住，气管的需要。 </li><li>一旦麻醉，将大鼠在仰卧位，剃腹侧颈部和用1％的氯己定擦拭和/或酒精擦拭消毒它。应用无菌手术毯在动物和保持区域无菌在任何时候。 </li><li>一旦麻醉，将大鼠在仰卧位，剃腹侧颈部和用1％的氯己定和酒精擦拭消毒它。应用无菌手术毯在动物和保持区域无菌在任何时候。 注意：任选的是，老鼠的头部可在手术过程中被固定在仰卧位置的立体框架来禁用头部， 即流动性枕骨。 </li><li>使从下巴一个2至3厘米长的中线切口，以使用无菌手术刀（＃10）胸骨几乎喙端。可选，适用于小斗牛犬夹收回皮肤。 </li><li>钝解剖组织的上层，如腺（ 如颌下腺和腮腺）和肌肉使用反向动作与钝剪刀和齿钳覆盖气管。始终保持对中线。该组织应该很容易分开。 </li><li>一旦气管露出，取代它向一侧。中线是两个白色的脂肪垫下方可见，标志着中线的喙结束。 </li><li>喙的白色脂肪垫，钝解剖组织，直到头骨（的basioccipital骨）的腹面为止。 </li><li>插入长齿拉钩（齿长度5.5毫米或16毫米）至保持移位到一侧（最多1厘米）的气管和头骨基部露出。气管位移OB结构呼吸。 注：对于一些动物，松开拉钩在多次手术中去除应变的气管， 例如 2分钟，每隔10分钟。钩拉钩可使用的长齿拉钩代替，以减少位移的应变。 </li><li>仔细烧灼小可见血管的喙去年底对喉部，避免出血。 注意：为了防止烧灼喉返神经血管精心拉离组织壁他们烧灼之前。 </li><li>调整显微镜的手术的其余部分。 </li><li>取下骨膜覆盖用细镊子的颅底。调整拉钩来实现对钻孔的最佳视图。 </li><li>感觉basioccipital骨细镊子的表面凹凸不平，并指出海拔在中线，它涵盖了基底动脉。吻侧，头骨变得提出一个轻微的凸起。 </li><li>根据哪一方病变需要，钻约为1mm横向到中侧运动的中线孔。 注：请记住，老鼠是在仰卧位，其左边可能是在调查右手边！ </li><li>一旦孔被制成，放大朝具有小垂直向上和向下-运动用钻头在中线直到基底动脉清晰可见和放大侧向至少2毫米。 </li><li>确保基底动脉清晰可见。金字塔可以通过其稍稍鼓出的形状（ 图2）和由接壤的血管来鉴定。 </li><li>用细镊子取出任何剩余的骨头碎片。 </li><li>申请一滴盐酸多沙普仑，呼吸兴奋剂，在舌头上。 </li><li>用26号针头纵向打开硬脑膜和细镊子横向到基底动脉。沉浸在脑脊液（CSF）和棉签的任何出血。 </li><li>避免仁州Ry的基底动脉，使切割大约1.5毫米宽，横跨金字塔为0.5mm垂直于基底动脉（ 图2）来中断CST纤维深与Vannas弹簧剪刀的宽度。 注：标记microscissors事先在0.5毫米的提示。一旦硬脑膜打开基底动脉可以稍微移动到一侧允许金字塔更好的访问。 </li><li>重复使用26号针头的尖端，以确保病变包括靠近所述基底动脉的纤维切断。 </li><li>停止与光压力棉签应用于出血。任选地，该切口可覆盖有明胶海绵。 </li><li>取出拉钩，更换组织，只缝合皮肤3-0薇乔缝线。 </li><li>手术后，保持动物在培养箱在32℃下，直到完全清醒和管理用5ml盐水皮下，如果需要的话。不要让动物无人值守。 </li><li>给Carporfen皮下作为抗炎和作为镇痛用于手术后疼痛1天术后。 </li><li>手术后至少一次之后，每周密切一周监控动物。 </li><li>至于做手术，地方水化凝胶包，泥婴儿食品，湿捣烂干州城笼地板上后需要。提供水瓶长嘴（缺少球轴承阀门）。 </li></ol>

<ol>
	<li>Lemon, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descending+pathways+in+motor+control.">Descending pathways in motor control.</a> Annual review of neuroscience. 31, 195-218 (2008).</li><li>Lemon, R. N., Griffiths, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparing+the+function+of+the+corticospinal+system+in+different+species:+organizational+differences+for+motor+specialization.">Comparing the function of the corticospinal system in different species: organizational differences for motor specialization.</a> Muscle &amp; nerve. 32, 261-279 (2005).</li><li>Starkey, M. L., 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=Assessing+behavioural+function+following+a+pyramidotomy+lesion+of+the+corticospinal+tract+in+adult+mice.">Assessing behavioural function following a pyramidotomy lesion of the corticospinal tract in adult mice.</a> Experimental neurology. 195, 524-539 (2005).</li><li>Starkey, M. L., Bartus, K., Barritt, A. W., Bradbury, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroitinase+ABC+promotes+compensatory+sprouting+of+the+intact+corticospinal+tract+and+recovery+of+forelimb+function+following+unilateral+pyramidotomy+in+adult+mice.">Chondroitinase ABC promotes compensatory sprouting of the intact corticospinal tract and recovery of forelimb function following unilateral pyramidotomy in adult mice.</a> Eur J Neurosci. 36, 3665-3678 (2012).</li><li>Brosamle, C., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+of+origin,+course,+and+termination+patterns+of+the+ventral,+uncrossed+component+of+the+mature+rat+corticospinal+tract.">Cells of origin, course, and termination patterns of the ventral, uncrossed component of the mature rat corticospinal tract.</a> The Journal of comparative neurology. 386, 293-303 (1997).</li><li>Brosamle, C., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ipsilateral,+ventral+corticospinal+tract+of+the+adult+rat:+ultrastructure,+myelination+and+synaptic+connections.">Ipsilateral, ventral corticospinal tract of the adult rat: ultrastructure, myelination and synaptic connections.</a> Journal of neuroctology. 29, 499-507 (2000).</li><li>Bareyre, F. 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=The+injured+spinal+cord+spontaneously+forms+a+new+intraspinal+circuit+in+adult+rats.">The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats.</a> Nat Neurosci. 7, 269-277 (2004).</li><li>Chen, Q., Smith, G. M., Shine, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+activation+is+required+for+NT-3-induced+axonal+plasticity+in+chronic+spinal+cord+injury.">Immune activation is required for NT-3-induced axonal plasticity in chronic spinal cord injury.</a> Exp Neurol. 209, 497-509 (2008).</li><li>Z'Graggen, W. J., 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=Compensatory+sprouting+and+impulse+rerouting+after+unilateral+pyramidal+tract+lesion+in+neonatal+rats.">Compensatory sprouting and impulse rerouting after unilateral pyramidal tract lesion in neonatal rats.</a> J Neurosci. 20, 6561-6569 (2000).</li><li>Rosenzweig, E. S., 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=Extensive+spontaneous+plasticity+of+corticospinal+projections+after+primate+spinal+cord+injury.">Extensive spontaneous plasticity of corticospinal projections after primate spinal cord injury.</a> Nat Neurosci. 13, 1505-1510 (2010).</li><li>Z'Graggen, W. J., Metz, G. A., Kartje, G. L., Thallmair, M., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+recovery+and+enhanced+corticofugal+plasticity+after+unilateral+pyramidal+tract+lesion+and+blockade+of+myelin-associated+neurite+growth+inhibitors+in+adult+rats.">Functional recovery and enhanced corticofugal plasticity after unilateral pyramidal tract lesion and blockade of myelin-associated neurite growth inhibitors in adult rats.</a> J Neurosci. 18, 4744-4757 (1998).</li><li>Kartje-Tillotson, G., O'Donoghue, D. L., Dauzvardis, M. F., Castro, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyramidotomy+abolishes+the+abnormal+movements+evoked+by+intracortical+microstimulation+in+adult+rats+that+sustained+neonatal+cortical+lesions.">Pyramidotomy abolishes the abnormal movements evoked by intracortical microstimulation in adult rats that sustained neonatal cortical lesions.</a> Brain Res. 415, 172-177 (1987).</li><li>Thallmair, 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=Neurite+growth+inhibitors+restrict+plasticity+and+functional+recovery+following+corticospinal+tract+lesions.">Neurite growth inhibitors restrict plasticity and functional recovery following corticospinal tract lesions.</a> Nature Neuroscience. 1, 124-131 (1998).</li><li>Akbik, F. V., Bhagat, S. M., Patel, P. R., Cafferty, W. B., Strittmatter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+plasticity+of+adult+brain+is+titrated+by+Nogo+Receptor+1.">Anatomical plasticity of adult brain is titrated by Nogo Receptor 1.</a> Neuron. 77, 859-866 (2013).</li><li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacol. 39, 777-787 (2000).</li><li>Steward, O., Sharp, K., Yee, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+re-assessment+of+the+effects+of+intracortical+delivery+of+inosine+on+transmidline+growth+of+corticospinal+tract+axons+after+unilateral+lesions+of+the+medullary+pyramid.">A re-assessment of the effects of intracortical delivery of inosine on transmidline growth of corticospinal tract axons after unilateral lesions of the medullary pyramid.</a> Experimental neurology. 233, 662-673 (2012).</li><li>Thallmair, 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=Neurite+growth+inhibitors+restrict+plasticity+and+functional+recovery+following+corticospinal+tract+lesions.">Neurite growth inhibitors restrict plasticity and functional recovery following corticospinal tract lesions.</a> Nature neuroscience. 1, 124-131 (1998).</li><li>Weidner, N., Ner, A., Salimi, N., Tuszynski, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+corticospinal+axonal+plasticity+and+functional+recovery+after+adult+central+nervous+system+injury.">Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury.</a> Proc Natl Acad Sci U S A. 98, 3513-3518 (2001).</li><li>Schnell, L., Schneider, R., Kolbeck, R., Barde, Y. A., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3+enhances+sprouting+of+corticospinal+tract+during+development+and+after+adult+spinal+cord+lesion.">Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion.</a> Nature. 367, 170-173 (1994).</li><li>Starkey, M. L., Bleul, C., Maier, I. C., Schwab, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehabilitative+training+following+unilateral+pyramidotomy+in+adult+rats+improves+forelimb+function+in+a+non-task-specific+way.">Rehabilitative training following unilateral pyramidotomy in adult rats improves forelimb function in a non-task-specific way.</a> Experimental neurology. 232, 81-89 (2011).</li><li>Ueno, M., Hayano, Y., Nakagawa, H., Yamashita, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraspinal+rewiring+of+the+corticospinal+tract+requires+target-derived+brain-derived+neurotrophic+factor+and+compensates+lost+function+after+brain+injury.">Intraspinal rewiring of the corticospinal tract requires target-derived brain-derived neurotrophic factor and compensates lost function after brain injury.</a> Brain : a journal of neurology. 135, 1253-1267 (2012).</li><li>Maier, I. C., 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=Constraint-induced+movement+therapy+in+the+adult+rat+after+unilateral+corticospinal+tract+injury.">Constraint-induced movement therapy in the adult rat after unilateral corticospinal tract injury.</a> J Neurosci. 28, 9386-9403 (2008).</li><li>Schallert, T., Fleming, S. M., Leasure, J. L., Tillerson, J. L., Bland, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+plasticity+and+assessment+of+forelimb+sensorimotor+outcome+in+unilateral+rat+models+of+stroke,+cortical+ablation,+parkinsonism+and+spinal+cord+injury.">CNS plasticity and assessment of forelimb sensorimotor outcome in unilateral rat models of stroke, cortical ablation, parkinsonism and spinal cord injury.</a> Neuropharmacology. 39, 777-787 (2000).</li><li>Montoya, C. P., Campbell-Hope, L. J., Pemberton, K. D., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+&amp;#34;staircase+test&amp;#34;:+a+measure+of+independent+forelimb+reaching+and+grasping+abilities+in+rats.">The &amp;#34;staircase test&amp;#34;: a measure of independent forelimb reaching and grasping abilities in rats.</a> Journal of neuroscience methods. 36, 219-228 (1991).</li><li>Soblosky, J. S., Song, J. H., Dinh, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graded+unilateral+cervical+spinal+cord+injury+in+the+rat:+evaluation+of+forelimb+recovery+and+histological+effects.">Graded unilateral cervical spinal cord injury in the rat: evaluation of forelimb recovery and histological effects.</a> Behavioural brain research. 119, 1-13 (2001).</li><li>Soleman, S., Yip, P., Leasure, J. L., Moon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+sensorimotor+impairments+after+endothelin-1+induced+focal+cerebral+ischemia+(stroke)+in+aged+rats.">Sustained sensorimotor impairments after endothelin-1 induced focal cerebral ischemia (stroke) in aged rats.</a> Experimental neurology. 222, 13-24 (2010).</li><li>Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Rat Nervous System. , (1995).</li><li>Benowitz, L. I., Goldberg, D. E., Madsen, J. R., Soni, D., Irwin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inosine+stimulates+extensive+axon+collateral+growth+in+the+rat+corticospinal+tract+after+injury.">Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury.</a> Proc Natl Acad Sci U S A. 96, 13486-13490 (1999).</li><li>Zhou, L., Baumgartner, B. J., Hill-Felberg, S. J., McGowen, L. R., Shine, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophin-3+expressed+in+situ+induces+axonal+plasticity+in+the+adult+injured+spinal+cord.">Neurotrophin-3 expressed in situ induces axonal plasticity in the adult injured spinal cord.</a> J Neurosci. 23, 1424-1431 (2003).</li></ol>

作者什么都没有透露。

我们所描述的锥体束损伤模型。皮质脊髓束被切断在脑干的水平。从技术角度来看这个手术需要大量的谨慎性和准确性。脑干容纳呼吸中心和心血管中心中的多个核，如疑核和孤嵌入在金字塔下方的网状结构。如果切口是由过深，这可能导致突然的呼吸困难，这可能是致命的。我们给Dopram刚切，增加呼吸活动，以防止突然停止呼吸之前。如果呼吸停止，值得通风动物为以下五个分钟，因为它可能会恢复氧气。此外，应绝对避免在基底动脉的损害。在最初几天伤后的并发症有：脑干出血的另一个重要的影响作用，相当消瘦，严重的呼吸diffic由于大量的伤害，包括橄榄只是横向的金字塔，由于气管移位和动物的严重的方向和平衡问题ulties。这些并发症可以被看作端点和动物可能会受到人道杀害。在第一周期间密切监测手术后是必需的。补充食物可以以抗衡体重减轻给出。气道炎症（刺耳呼吸）在一到两天正常解析。一般情况下，年轻的动物更容易操作的是他们的脖子少深。此外，他们更弹性，较少有呼吸并发症。 它需要一些练习来达到CST完整的病变有锥体束。同样，在两项研究由管家等 16，其中规定了由Benowitz 等 28复制前的实验，最初报道的高死亡率，而他们的一些病变是不完整的。行为缺陷也将是可变的。我们建议一个新的外科医生进行很多实践锥体束手术进行任何全面的实验来测试候选人治疗前。如何防止不完全的病变追溯切用注射器针头，确保在本协议描述的皮质纤维接近基底动脉被切断。其他选项是如Zhou 等人，2003年29描述了一个倾斜的玻璃吸移液管吸移病灶部位。的细钨线，也可用于切割区域22。另一种为锥体束是削减颈椎C3背部CST单方面，然后切断与一个腹办法CST 18 ipsilesional C2腹侧部。但可以说，一个完整的病变是没有必要的，如果我们的目标是研究对侧CST发芽;如果大多数金字塔被损毁，并从未病变侧的发芽响应发生这可以是足够了。 的锥体束病变模型的一个目的是要烧蚀的皮质脊髓束的一个侧面，并使用神经可塑性诱导疗法刺激完整的非受伤纤维的可塑性和发芽。为了测试神经保护治疗这种模式是不适合和其他损伤模型，如挫伤受伤应该被使用。 pyramidotomies的成果进行评估最常使用的行为测试和解剖。水平阶梯测试，缸饲养试验和蒙托亚楼梯测试是最常用的行为评估。在文献中描述的其它试验是颗粒到达测试，爬绳试验，步态分析和胶带测试3,17-19。然而，大多数行为测试的结果往往只监测最多42天postlesion 3,4,11,17,18,20-22。从过去的经验，啮齿动物恢复大部分功能，在稍后的时间点未经任何处理的干预。我们已经表明塔吨的水平阶梯测试和蒙托亚楼梯颗粒达到试验检测残疾高达稍后的时间点。它们可以用于测量功能恢复因治疗。病变完整性被PKC伽玛染色（其标签在背柱CST纤维）脊髓延髓在宫颈级别或病变部位的横截面铬花青染色评判。生物素化的葡聚糖胺可注入的对侧皮层，以评估完整纤维穿过中线的发芽到去神经脊髓区域。 总之，锥体束模式是很好的评估，如果手术成功掌握了治疗药物中枢神经系统损伤的神经可塑性诱导能力。它评估的完整纤维的可塑性。下一个步骤将是使用一个更临床相关的模型如挫伤损伤评估，以克服更大injuri药物的能力ES影响多个大片。

皮质脊髓束（CST）是一个主要的机动车道内，在人类的脊髓。损伤后脊髓损伤造成相当大的损失灵巧此道。 CST是细电机运动在人类，如数字移动和远端肌肉的控制尤为重要。还发现在啮齿类虽然脊髓内其解剖学位置是不同的，人类的和CST损伤较少禁用1-4。 一种在体内 CST损伤模型涉及单侧锥体束，其中在腹侧脑干1棱锥被切断延髓到十字交叉（ 图1）。金字塔是在延髓的腹侧清晰的解剖特征，通过它的CST运行它decussates之前。在大鼠中，大多数纤维（约95％）交互对生在髓质的尾端和脊髓的背内侧部分此后找到。然而，approximat伊利10％decussated纤维在背外侧列中运行。纤维的剩余〜5％留不交叉同侧和旅行的腹内侧部分5,6。锥体束后脊髓失去其从对侧CST直接输入（ 图1）。 该锥体束模式是针对未病变CST的完好纤维的反应测试，以治疗特别好。它允许可塑性横跨中线的完整纤维在脊髓，其支配神经支配区和发芽的评估。皮质脊髓束已经观察到在脊髓损伤模型相当发芽并介导尤其前肢恢复7-10。一个可能的治疗目的是加强这方面的前肢恢复。一疗法的功效可与行为评估，所述未病变道的解剖追踪的技术和终端电生理实验-3,11,12-进行测试。&lt;/ P&gt; 相比，脊椎损伤的大多数其他临床前模型的优点在于，它特别影响一个道相对于其他脊椎损伤往往影响多个束。在一个明确的，肤浅的解剖位置只有一个道被损伤。另一个优点是，锥体束是一个可再现的损伤模型。它是一个合适的在体内的资源为研究人员测试潜在神经可塑性诱导疗法，在受伤或影响电机系统13,14病症之后恢复运动功能，其目的的效果。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Carprofen</td>
			<td>Norbrook</td>
			<td>Vm No; 02000/4229</td>
			<td>give 5mg/kg twice daily</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Harvard Instruments</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>B506</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Scissors- Tough Cut</td>
			<td>Fine Science Tools</td>
			<td>14058-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11019-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Long-toothed Alm retractors</td>
			<td>Fine Science Tools</td>
			<td>17009-07</td>
			<td></td>
		</tr>
		<tr>
			<td>cautery system kit</td>
			<td>Harvard Instruments</td>
			<td>726067</td>
			<td></td>
		</tr>
		<tr>
			<td>fine Dumont forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Steel burrs</td>
			<td>Fine Science Tools</td>
			<td>19007-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dopram V-drops</td>
			<td>Pfizer</td>
			<td></td>
			<td>apply 1 drop to tongue</td>
		</tr>
		<tr>
			<td>Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>gelfoam</td>
			<td>Equimedical</td>
			<td>EQU705001</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 Vicryl sutures</td>
			<td>Ethicon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>operating microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compact Anaesthesia System Isoflurane K/F Single Gas</td>
			<td>VetTech Solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scales</td>
			<td>Ohaus</td>
			<td>NVT3201</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Steel Scalpel blades No. 10</td>
			<td>Swann-Morton</td>
			<td align="right">201</td>
			<td></td>
		</tr>
		<tr>
			<td>25g needles</td>
			<td>Terumo</td>
			<td>NN-2525R</td>
			<td></td>
		</tr>
		<tr>
			<td>syringes (1ml and 5ml)</td>
			<td>Terumo</td>
			<td>SS+01T1 / SS*05SE1</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline (Sodium Chloride 0.9%)</td>
			<td>Fresenius Kabi</td>
			<td>Pl 08828/0178</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton buds</td>
			<td>Johnson and Johnson</td>
			<td align="right">5000207582502</td>
			<td>sterilize before use</td>
		</tr>
	</tbody>
</table>

unilateral pyramidotomy of the corticospinal tract in rats for assessment of neuroplasticity-inducing therapies

皮质脊髓束（CST），可以完全在啮齿动物脑干的延髓金字塔单方面切断。 CST就是一个马达道具有用于在人中远端肌肉控制重视，并在较小程度上，在啮齿类动物。一个金字塔导致脊髓主要是脊髓导致短暂的电机残疾前肢的对侧的CST神经支配的损失单方面削减和持续灵巧的损失。皮质脊髓束的同侧预测是次要的。我们修订我们的手术方法，以增加损伤完整性的机会。我们描述术后护理。在蒙托亚楼梯颗粒达到测试与这里显示的水平梯试验赤字检测多达伤后8周。只检测瞬时在气缸饲养试验赤字。因此，气缸测试可能只适合于检测短期恢复。我们展示如何，电生理和解剖，可以评估病变和塑料的变化。我们还描述了如何分析跨中线萌到贫困地区从无恙CST纤维。它是具有挑战性的，以获得完整的&gt; 90％的病变始终由于靠近基底动脉在延髓和生存率可低。另一种手术方法和行为测试在本协议中的描述。的锥体束模型是评估神经可塑性诱导治疗，其中损伤后增加完整纤维的发芽的良好工具。

存活率。 在美国进行的一项代表性的研究，我们有16个出20名女性李斯特连帽大鼠（200〜250克）生存率。最常见的并发症是呼吸，因为气管位移或损坏在脑干的呼吸中枢的困难只是背侧到金字塔（核孤，暧昧和parabrachilis）。类似的结果已经在文献中报道16。 行为的结果。 的行为测试一系列已被用来评估锥体束在啮齿动物后感觉运动结果：在水平梯试验中，单粒料到达测试，缸饲养试验和蒙托亚楼梯测试是最常用的行为评估。在文献中描述的其它试验是颗粒到达测试，爬绳试验，步态分析和胶带测试3,17-19。大多数这些显示损伤后的功能性运动障碍1周。目前，在文献中描述的多数研究只能执行行为测试42天4,11,13,20,21，或者用于更短的时间3,18,22。我们已经进行行为测试用于在延长的时间周期和所看到的基本上完全恢复的马达功能后8周锥体束对某些行为测试的，除了蒙托亚楼梯测试和水平阶梯测试。这可能是有几个原因。首先，受伤CST喙病灶上可能会形成在脑干神经元充当间接中继到运动神经元在脊髓突触。其次，未受伤（健侧）CST可以发芽，形成突触在脑干或脊髓神经元作为继电器运动神经元。第三，其他备用电机大片，如网状脊髓，前庭或红核脊髓束可能会接管一些功能。因此，持久吨reatment效果难以检测与许多行为测试在锥体束模式。一些行为测试在这个模型然而可能的伤害，这是关系到一个处理后的早期指示加速恢复。 缸饲养试验 3,4,23 基于缸饲养试验对应的主导前爪的CST受伤。将大鼠置于一个有机玻璃圆筒和饲养行为是观察期为3分钟。伤前有轻微偏爱使用一种前爪，占主导地位的前爪，期间垂直探测（ 图3A）。伤病影响的主导方和使用对侧前爪下降到28％1周伤后（重复测量ANOVA P &lt;0.05;事后分析发现动物有在第1周，2和3，P &lt;0.05显著赤字， Fisher的LSD）。 4周伤后的恢复动物的ð使用受影响的前肢用于后轮的45％，和6周后，这是57％（n = 16的平均值±示出标准误差）。使用气缸试验锥体束后42天同样，斯达克等人（4）评估了小鼠和报告，在使用受影响的前爪在整个测试期间一个显著下降。 蒙托亚楼梯测试 24 只需要训练前本次测试之前伤害适应环境。要在测试环节动物增加的动力是食品限制在每鼠15克食物的前一天晚上。食物剥夺大鼠被放置在蒙托亚楼梯15分钟。在每一侧，有7个步骤与井，各含有3粒料楼梯。每侧检索粒料的总数被记​​录。检索颗粒与对侧/前爪受影响的人数从61％伤前显著下降至3％3天伤后（ 图3B）。检索粒料的能力仍然在第4周伤后显著障与检索粒料的20％。老鼠表现出显著赤字高达8星期只有29％吃在那个时间点的颗粒（重复测量ANOVA P &lt;0.05， 事后分析发现动物对对侧方显著赤字可言周，P &lt;0.05，Fisher的LSD）。所述ipsilesional前爪受到影响长达1周伤后;然而，这种复苏在第二周（重复测量ANOVA P &lt;0.05， 事后分析发现动物有3节和第1周显著赤字的ipsilesional前爪，P &lt;0.05，Fisher的LSD）。所述ipsilesional前爪的初始缺陷可能由皮质脊髓束通过切口金字塔运行，随后发芽和恢复的同侧部的损毁进行说明。 横梯形图测试25 只需要训练前进行此测试。只走三次在1米长的梯子，用不规则的横档间距。影片将在后面的慢镜头和脚滑倒，并为每个爪子失误总数分析量化。在图3C的曲线图表示未命中和受影响的和受影响较小侧（前爪+后爪），以百分比来采取步骤的总数的卡瓦的总数。老鼠有误差分别1周伤后用10％或患侧11％，受到的影响较小侧显著增加。这个赤字是持久的对侧/患侧多达8个星期10％的错误做，但是，2周解析ipsilesional /受影响较小的一侧（重复测量方差分析， 事后分析发现动物有显著赤字可言周在对侧方和显著赤字在1周和3对ipsilesional边p &lt;0.05，Fisher的LSD）。 电生理检测 在一个终端电的建立将动物麻醉腹膜内用尿烷。在整个过程中，它保持在37℃±1℃。其腹侧颈部区域，它的前肢和胸部区域被剃光和消毒用碘酒擦洗。如先前所述和桡神经暴露在上臂由腹侧方法切开皮肤和胸大肌金字塔暴露。桡神经的末端被切断，神经被放置在两个银线钩电极在矿物油浴中。任一金字塔，刺激与同心双极电极在不同深度具有5个脉冲以300Hz和增加刺激幅度。录音总是从禁用臂（ 图4）的桡神经进行。 没有受伤阿尼姆ALS有在延迟12至20毫秒之间在径向神经强烈反应时对侧金字塔进行刺激（ 图4B）。同侧的刺激只会导致偶尔的激活（ 图4C）。动物，其中有以前收到pyramidotomies，在对侧的手臂记录显示，在桡神经无反应时，损伤呼吸道的病变（ 图4B&#39;）以上的刺激。然而，CST纤维在金字塔的刺激同侧电极导致了桡神经更强活化相比未受伤的动物在第12周伤后（ 图4C&#39;）的记录。 解剖的结果。 动物根据动物附表1（科学程序）法1986年之前两周安乐死10周术后，未受伤的CST被曝与生物素葡聚糖胺（BDA）注 ​​射到运动皮层26同侧残疾人臂（ 图1）。这使得未受伤的纤维，它越过中线和供应对侧脊髓（ 图5C，D）的定量。未受伤的动物只有几个纤维横过中线，而这增加了响应于伤害。横颈脊髓切片用蛋白激酶C（PKC）伽马评估失神经支配的损伤CST（ 图5E）16的比例。可替代地，对病变的完整性可通过注入BDA示​​踪剂进入电动机皮质上的锥体束损伤ipsilesional侧进行评估。在对侧背内侧CST缺乏标签和同侧腹内侧CST提供单侧完全性横断证据。 <img alt="图1" fo:conte<html> NT-WIDTH =“4英寸”SRC =“/文件/ ftp_upload / 51843 / 51843fig1highres.jpg”WIDTH =“400”/&gt; 图1：示意图显示，在大鼠和科技委在金字塔单边干切断的皮质脊髓束顶部图像：皮质神经细胞起源于皮质层5锥体细胞。生物素化的葡聚糖胺（以绿色示出）之前两个星期的实验结束注入对侧皮层。中间的图像：该束上形成的髓质腹面并在spinomedullary结大多数轴突的交互对生的金字塔。单侧锥体束在尾髓质（显示为红色）进行。较低的图像：大多数脊髓的背内侧和背外侧部分向下运行的皮质脊髓束。少数保持同侧和运行ventromedially。 1843fig2highres.jpg“WIDTH =”500“/&gt; 图2：大鼠脑腹面观 。金字塔是与众不同的，因为他们提出，平行凸（黄框）。金字塔的边界是由基底动脉（BAS）通过中线和横向的凸中途基底动脉和动脉paraolivary（POL）之间的碱，（C）中可见内侧限定。开颅手术进行了基底动脉和横向延伸到paraolivary动脉（在C红色框）。一个金字塔垂直切到基底动脉（红线在C中，切口上示出在A和B试样）。因为CST开始交互对生，并因椎动脉被掀起中线放置太靠近尾端的椎动脉（垂直）病灶将是不完整的。 （C）图片由乔治Paxinos 27改编自“金鼠神经系统”。注：此图显示了图像的左手侧的病变：这是动物的钻机HT锥体束，因为它是仰卧。但是请注意，在视频显示病变的动物的左锥体束。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig3highres.jpg" width="500" /> 图3：在缸饲养试验代表性的成果，蒙托亚楼梯颗粒达到测试和水平梯形测试没有通过损伤后所有测试检测到电机赤字。然而，没有赤字可以与在第4周伤后气缸饲养试验来检测，而持续的赤字可以与蒙托亚楼梯测试和水平梯试验8周伤后（每组n =，平均值和标准误差16来检测示出）。星号表示P &lt;0.05相对于受伤前的基线（Fisher的LSD测试）。 <a href="https://www.jove.com/files/ftp_upload/51843/51843fig3highres.jpg" target="_blank">请点击</a>这里查看这个数字的放大版本。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig4highres.jpg" width="500" /> 图4：电评估皮质脊髓束和前锥体束后（A）示出的建立用于终端的实验。我们刺激金字塔与5脉冲喙损毁和十字交叉的部位不同深度的一列火车。红色系的损伤道。我们从上侧对侧桡神经记录。（B）和（C）表示实施例的记录与ipsilesional刺激或对侧刺激前或12周后锥体束。 Ipsilesional刺激诱 ​​发多种复合动作电位（在跟踪尖峰）的桡神经损伤（B）之前。这些是由损伤废除和活动不returñ在12周（B&#39;）。对侧刺激唤起了罕见的单一化合物动作电位伤前（C）。 12周伤活动后明显增加（C&#39;），这可能是由于完整的轴突横贯性脊髓内的中线和塑料变化发芽。 <a href="https://www.jove.com/files/ftp_upload/51843/51843fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51843/51843fig5highres.jpg" width="500" /> 图5：锥体束后解剖学评价（A，B，C，D）的生物素化葡聚糖胺被用于通过其注入相应于未病变CST运动皮层跟踪皮质脊髓束（见绿于图1）。背内侧，对CS的腹内侧部件T分别清晰可见（A，背内侧部分放大中的B）。（C）和（D）是纤维横跨中线发芽到失神经侧灰质响应于治疗的从（A）的放大倍数。为了评估宫颈级别4（12周伤后的组织）的病变横截面的完整性被切断，并用抗PKC伽玛（E）。科技委上的图像的左手侧是完好无损，可以看出在整个中线鼓出的受损背CST的结构崩解，大概是因为进入去神经侧。

Watch this Scientific Journal Video about Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies at JoVE.com

Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

皮质脊髓束的主要感觉运动束之一，可以单方面地损伤在啮齿类脑干为了测试神经可塑性诱导疗法的中枢神经系统。此手术（“锥体束”）和术后评估本协议描述。

皮质脊髓束的大鼠单侧锥体束的神经可塑性诱导疗法的评估

The corticospinal tract (CST) can be completely severed unilaterally in the medullary pyramids of the rodent brainstem. The CST is a motor tract that has ...

unilateral-pyramidotomy-corticospinal-tract-rats-for-assessment

Research

JoVE Journal

Neuroscience

15.7K 次观看.  King's College London. 皮质脊髓束的主要感觉运动束之一，可以单方面地损伤在啮齿类脑干为了测试神经可塑性诱导疗法的中枢神经系统。此手术（“锥体束”）和术后评估本协议描述。 

视频: Unilateral Pyramidotomy of the Corticospinal Tract in Rats for Assessment of Neuroplasticity-inducing Therapies

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

在大鼠的药物自我管理的评估超声发声

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

科研

神经科学

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for tissue formation during embryonic development and tissue homeostasis during adulthood 1. Currently, great efforts are invested towards controlling the switch of somatic stem cells from expansion to differentiation because this is thought to be fundamental for developing novel strategies for regenerative medicine 1,2. However, a major challenge in the study and use of somatic stem cell is that their expansion has been proven very difficult to control.
Here we describe a system that allows the control of neural stem/progenitor cell (altogether referred to as NSC) expansion in the mouse embryonic cortex or the adult hippocampus by manipulating the expression of the cdk4/cyclinD1 complex, a major regulator of the G1 phase of the cell cycle and somatic stem cell differentiation 3,4. Specifically, two different approaches are described by which the cdk4/cyclinD1 complex is overexpressed in NSC in vivo. By the first approach, overexpression of the cell cycle regulators is obtained by injecting plasmids encoding for cdk4/cyclinD1 in the lumen of the mouse telencephalon followed by in utero electroporation to deliver them to NSC of the lateral cortex, thus, triggering episomal expression of the transgenes 5-8. By the second approach, highly concentrated HIV-derived viruses are stereotaxically injected in the dentate gyrus of the adult mouse hippocampus, thus, triggering constitutive expression of the cell cycle regulators after integration of the viral construct in the genome of infected cells 9. Both approaches, whose basic principles were already described by other video protocols 10-14, were here optimized to i) reduce tissue damage, ii) target wide portions of very specific brain regions, iii) obtain high numbers of manipulated cells within each region, and iv) trigger high expression levels of the transgenes within each cell. Transient overexpression of the transgenes using the two approaches is obtained by different means i.e. by natural dilution of the electroporated plasmids due to cell division or tamoxifen administration in Cre-expressing NSC infected with viruses carrying cdk4/cyclinD1 flanked by loxP sites, respectively 9,15.
These methods provide a very powerful platform to acutely and tissue-specifically manipulate the expression of any gene in the mouse brain. In particular, by manipulating the expression of the cdk4/cyclinD1 complex, our system allows the temporal control of NSC expansion and their switch to differentiation, thus, ultimately increasing the number of neurons generated in the mammalian brain. Our approach may be critically important for basic research and using somatic stem cells for therapy of the mammalian central nervous system while providing a better understanding of i) stem cell contribution to tissue formation during development, ii) tissue homeostasis during adulthood, iii) the role of adult neurogenesis in cognitive functions, and perhaps, iv) better using somatic stem cells in models of neurodegenerative diseases.

Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection

Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.

扩展胚胎及成体神经干细胞<em&gt;在子宫内</em电穿孔或病毒立体定向注射

Somatic stem cells can divide to generate additional stem cells (expansion) or more differentiated cell types (differentiation), which is fundamental for ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

一个鼠标，两种文化：隔离和成人神经的培养干细胞从单个小鼠的两个神经源性区

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

间刺激间隔对啮齿类动物时间处理评估的力量

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Bilateral Assessment of the Corticospinal Pathways of the Ankle Muscles Using Navigated Transcranial Magnetic Stimulation

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans and can be assessed using transcranial magnetic stimulation (TMS). Given the role of distal leg muscles in upright postural and dynamic tasks, such as walking, a growing research interest in the assessment and modulation of the corticospinal tracts relative to the function of these muscles has emerged in the last decade. However, methodological parameters used in previous work have varied across studies making the interpretation of results from cross-sectional and longitudinal studies less robust. Therefore, use of a standardized TMS protocol specific to the assessment of leg muscles&#39; corticomotor response (CMR) will allow for direct comparison of results across studies and cohorts. The objective of this paper is to present a protocol that provides the flexibility to simultaneously assess the bilateral CMR of two main ankle antagonistic muscles, the tibialis anterior and soleus, using single pulse TMS with a neuronavigation system. The present protocol is applicable while the examined muscle is either fully relaxed or isometrically contracted at a defined percentage of maximum isometric voluntary contraction. Using each subject&#39;s structural MRI with the neuronavigation system ensures accurate and precise positioning of the coil over the leg cortical representations during assessment. Given the inconsistency in CMR derived measures, this protocol also describes a standardized calculation of these measures using automated algorithms. Though this protocol is not conducted during upright postural or dynamic tasks, it&#160;can be used to assess bilaterally any pair of leg muscles, either antagonistic or synergistic, in both neurologically intact and impaired subjects.

The present protocol describes the simultaneous, bilateral assessment of the corticomotor response of the tibialis anterior and soleus during rest and tonic voluntary activation using a single pulse transcranial magnetic stimulation and neuronavigation system.

导航经颅磁刺激踝关节脊椎通路的双边评价

Distal leg muscles receive neural input from motor cortical areas via the corticospinal tract, which is one of the main motor descending pathway in humans ...

Determining the Functional Status of the Corticospinal Tract Within One Week of Stroke

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential for recovery based on clinical assessments alone. The functional integrity of the corticospinal tract is an important prognostic biomarker for recovery of UL function, particularly for those with severe initial UL impairment. This article presents a protocol for evaluating corticospinal tract function within 1 week of stroke. This protocol can be used to select and stratify patients in trials of interventions designed to improve UL motor recovery and outcomes after stroke. The protocol also forms part of the PREP2 algorithm, which predicts UL function for individual patients 3 months poststroke. The algorithm sequentially combines a UL strength assessment, age, transcranial magnetic stimulation, and stroke severity, within a few days of the stroke. The benefits of using PREP2 in clinical practice are described elsewhere. This article focuses on the use of a UL strength assessment and transcranial magnetic stimulation to evaluate corticospinal tract function.

This protocol is for evaluating corticospinal tract function within 1 week of stroke. It can be used to select and stratify patients in trials of interventions designed to improve upper limb motor recovery and outcomes and in clinical practice for predicting upper limb functional outcomes 3 months after stroke.

确定中风一周内皮质脊柱的功能状态

High interindividual variability in the recovery of upper limb (UL) function after stroke means it is difficult to predict an individual&#39;s potential ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

基于旋转加速大鼠漫射性无羟基脑损伤的诱导

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...