我们感谢鼠资源研究中心，美国密苏里大学，哥伦比亚大学，密苏里州，提供GFP鼠; Neuralstem公司提供人类神经干细胞。这项工作是由退伍军人管理局，美国国立卫生研究院（NS09881），加拿大脊髓研究组织，该克雷格H.尼尔森基金会，伯纳德和安妮·斯皮策慈善基金的支持。

所有动物的协议是由弗吉尼亚州圣地亚哥机构动物护理和使用委员会（IACUC）批准。实验动物护理和安全指引，美国国立卫生研究院严格遵循。动物可以免费获得食物和水在整个研究和充分治疗减少疼痛和不适。 1，含生长因子鸡尾酒纤维蛋白组分的制备<ol><li>溶解25毫克大鼠纤维蛋白原在0.5ml PBS中，以得到50毫克/毫升2×原液（见材料表）。纤维蛋白原是难于溶解，并应置于37℃培养箱或水浴和动摇每隔5-10分钟1-2小时。 </li><li>溶解100 UN大鼠凝血酶在2ml的10mM无菌的CaCl 2，得到50 U / ml的2×储液。 </li><li>溶解在PBS中的生长因子以下列浓度，得到100微升储备溶液：1毫克/毫升：BDNF; NT-3（高浓度股票鞘内输注体内 <sup&gt; 19）; 200微克/毫升：GDNF;胰岛素样生长因子; bFGF的;表皮生长因子;血小板衍生生长因子;的aFGF;肝细胞生长因子（除用于细胞培养大约1000倍以上）。 </li><li>溶解25毫克MDL28170（蛋白酶抑制剂）的130毫升DMSO中，得到50mM的DMSO储备溶液，然后稀释50倍的PBS中，得到1mM储备溶液。 </li><li>混合100μl的各生长因子组分和100μlMDL28170（1mM储备溶液），以产生1,000微升生长因子鸡尾酒溶液（ 图1）。 </li><li>混合500μl的血纤维蛋白原2×原液用500μl生长因子鸡尾酒，得到25毫克/毫升纤维蛋白原工作溶液含有生长因子和分装成10或20微升贮存于-70℃。在-70℃下的溶液是稳定的，最多至12个月</li><li>同样，混合500微升的凝血酶倍原液用500μl生长因子鸡尾酒，得到25单位/毫升凝血酶工作液含有生长因子和分装成类似10或20微升卷进行存储一个吨-70℃长达12个月。 </li><li>上接枝的日子，将纤维蛋白原和含凝血酶生长因子鸡尾酒在冰上，直到与细胞混合。 </li></ol> 2，T3脊髓横断<ol><li>用可接受的方法麻醉成年雌性菲舍尔344大鼠或无胸腺裸鼠体内。主题深度麻醉，当反应到尾巴和爪子掐是完全不存在。注：我们通常用150-200克的大鼠菲舍尔或180-220克无胸腺大鼠和氯胺酮麻醉的鸡尾酒（2毫升/千克）（25毫克/毫升），甲苯噻嗪（1.3毫克/毫升）和乙酰丙嗪（0.25毫克/毫升）。 </li><li>适用眼膏，以防止干燥或眼睛的伤害，而动物在麻醉状态下。 </li><li>刮胡子的上胸面积和清洁与优碘皮肤。 </li><li>切使用＃15刀片在皮肤和肌肉，用外科手术牵开器暴露T3椎体，并在T3椎骨执行椎板使用咬骨钳，露出的T3 / 4脊髓。 </li><li>做一个LONgitudinal正中切口在使用＃11刀片大约2毫米长的硬脑膜。 </li><li>将虹膜切除剪硬脑膜下切开右外侧整个脊髓。使在同一侧的另一个切1.5毫米更多cadually。 </li><li>两切段用钝器23号针头连接到中等强度之间的真空吸脊髓组织。使用可视化验证，以确保完全横断腹和横向。这将完成一个侧面半切。 </li><li>为了延长病变为全宽脊髓横断，将镜切口进入左hemicord。试图离开硬脑膜完整，比先切等，嫁接时，两个星期后，保留该公司移植物/纤维蛋白基质中的病变部位。 </li><li>缝合肌肉，应用抗生素粉，主食皮肤。 </li><li>注入乳酸林格氏（3毫升）中，Bannamine（2.5-5毫克/千克）和氨苄青霉素（80-100毫克/千克）手术和脉后，立即（见材料表）ntain大鼠在一个温暖的孵化器，直到体温重新建立。 </li><li>排空膀胱，并每天两次约两个星期注射林格氏和氨苄西林溶液直至建立反射性膀胱排空（ 图1B）。 </li></ol> 3。的新鲜分离胚胎一天准备14脊髓神经干细胞<ol><li>获得近交转基因绿色荧光蛋白F344大鼠（F344-的Tg（UBC-EGFP）F455Rrrc）从大鼠资源研究中心，美国密苏里大学。 </li><li>注射40微克脱-甘氨酸10，[D-丙氨酸6]-促黄体生成激素释放激素乙基酰胺（LHRH）2后，在PBS中稀释，在200的浓度微克/毫升腹膜内（IP）的每成熟雌性大鼠（8周或更老） -3小时光照诱导的胚胎收集的同步。 </li><li>交配成熟的绿色荧光蛋白纯合或杂雄性大鼠过夜注射后4天。 </li><li>收集胚胎在几天13.5-14.5交媾（PC）在冷的HBSS缓冲液，检查下一个“NightSea”手电筒和过滤眼镜（BLS1 - 蓝星手电筒和型号VG1屏障过滤器）GFP表达或荧光显微镜。 </li><li>从每个GFP阳性胎儿解剖脊髓出无菌，取出覆脑膜及附加脊神经节与虹膜切除术剪刀和细珠宝商镊子。 </li><li>收集解剖脊髓中，加入2ml冷的HBSS缓冲液在15毫升cornical管并保持在冰上。 </li><li>按照参考第20到分离解剖脊髓： <ol><li>简单地说，加2毫升0.25％胰蛋白酶-EDTA含有解剖脊髓管（线数可以一起消化，1线可以产生足够的细胞为一个主题的移植物）和消化，在37℃下10-12分钟。 </li><li>停止胰蛋白酶反应通过加入含有10％FBS的管和离心组织于2500 rpm离心2分钟的10ml DMEM。 </li><li>悬浮组织在2毫升Neurobasal培养基合作ntaining 2％B27，以及使用越来越小的火抛光的巴斯德吸管磨碎的脊髓组织。 </li><li>离心将细胞以2,500 rpm离心2分钟，再悬浮于2ml的Neurobasal培养基中含有B27，并用40μm的细胞过滤器过滤器过滤细胞。 </li></ol></li><li>计数细胞，用血细胞计数和分裂细胞同样分为两个Eppendorf管中。 </li><li>离心机的细胞，彻底消除上清液（1-2分钟都必须撤出所有上清液用低真空小费），所以按剩余上清细胞再悬浮后，生长因子鸡尾酒将不会被摊薄。 </li><li>重悬细胞的每一半中含有的生长因子，分别以250,000细胞/微升的浓度（细胞沉淀计数大约⅓最终体积）的纤维蛋白原或凝血酶的工作溶液，并放置在冰上事先移植（ 图1A）。请注意，当与细胞混合纤维蛋白原可能自发地胶前s的凝血酶在病变/移植部位相结合;避免过早胶凝，立刻体内细胞移植前混合细胞中纤维蛋白原。 </li></ol> 4，移植<ol><li>两个星期后脊髓横断Reexpose的损毁T3 / 4脊髓。 </li><li> 5微升纤维蛋白原细胞和5微升凝血酶的细胞可以直接注射到九分，通过密封的疤痕组织和硬脑膜完整病灶部位，而无需重新打开病灶部位。 </li><li>使用27号针头以创建9个孔的疤痕组织和完整硬脑膜1-2周后的病变的表面上：在中心3（一个在中间点和两个支管，并间隔0.5毫米分开），并在这两个延髓3和在尾部接口（大约0.5mm或延髓尾端向病灶中心）3。 </li><li>然后纤维蛋白原细胞溶液体积的一半注入病灶部位和四分之一体积的三个核心点到3点吻端接口和AQ的uarter量为3分尾接口。 </li><li>然后，立即注入throbmbin细胞进入相同的斑点。血纤维蛋白原的细胞和病灶部位内凝血酶细胞的混合物会形成纤维蛋白凝胶，以保持细胞到损伤位点和生长因子鸡尾酒将支持细胞的生存。细胞使用的是连接到一个PicoSpritzer（通用阀门公司）拉玻璃移液管注入。 </li><li>移植培养的人神经干细胞21在与上述相同的程序，利用从人的试剂制成的纤维蛋白和生长因子鸡尾酒。 </li></ol>

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我们什么都没有透露。

一个用于NSC移植在脊髓损伤的主要障碍是生存于病灶中心不佳。在病灶部位的任何间隙或腔体有可能减少或削弱棘上轴突和下方的伤害分离的脊髓节段之间的功能神经元中继的形成。另外，移植的神经干细胞存活不佳可能影响其与宿主组织整合，并因此减少移植神经元与神经元的主机的连通性。可能的机制来解释细胞的损失包括急性脊髓损伤产生的细胞毒性环境中，主机脊髓继发变性，受伤的区?...

脊髓损伤（SCI）往往损害不仅白质束携带的信号和从脑，而且中央灰质，引起的interneurons和运动神经元的节段性损失。脊髓损伤的后果是两个马达和病变以下感觉功能的丧失。不幸的是，成人中枢神经系统（CNS）不自发地再生，从而产生永久功能缺损1。因此，重建受伤的成年脊髓和改进运动，感觉和植物神经功能是脊髓损伤研究的一个重要目标。神经干细胞（NS​​Cs），无论是从胚胎或成年中枢神经系统中直接分离，是引人注目的候选细胞，以取代失去的神经元和神经胶质细胞。此外，这些细胞具有形成新的功能性继电器跨越病变部位2,3恢复轴突传导的潜力。 迄今为止，还没有解剖的详细阐释，electrophysiological和神经元中继形成了神经干细胞移植的重度脊髓损伤后行为的影响。有几个原因：第一，移植的神经干细胞或胎儿中枢神经系统组织中存活不佳时，嫁接到大病灶腔。以往的研究表明大量的早期细胞脱落，留下大空囊状病灶腔4,5。在一些研究中移植的细胞随后会分裂，并填写病灶腔4,5，但几天到几周的延迟之后这可能会发生，以及随后病灶部位填充可能不完整或一致的。其次，高效的跟踪系统，可提供对细胞存活，分化/成熟，并移植神经干细胞生长的声音数据缺乏。大多数早期的研究采用顺行和逆行标记来跟踪从移植2,3轴突的预测。然而，这些技术只是部分，往往从移植细胞所产生的标记不清轴突预测及示踪方法都是主观吨引起的染料泄漏以外植入的细胞的工件。其他组的人使用特定的神经元标记物来标记人类胎儿神经干细胞到受伤的啮齿类动物脊髓5,6移 ​​植后轴突的预测。然而，在这些研究中， 移植并没有一贯良好的生存。最近，病毒递送的GFP报告基因的用于标记培养的神经干细胞7,8。但是，GFP的表达常常不一致，并且可以下调7。最近，利用转基因供体小鼠或大鼠稳定表达报告基因，绿色荧光蛋白，或人胎盘碱性磷酸酶，已显着提高体内 9,11移植的神经干细胞/祖细胞的跟踪。第三，一些研究表明，无论从胚胎或成年中枢神经系统导出体外培养的大鼠神经干细胞完全分化成神经胶质谱系当移植到完整的或受伤的成年脊髓心病的环境ð7,12,13，尽管事实是这些神经干细胞能够分化成神经元和神经胶质细胞在体外的，这表明局部环境可能要求干细胞的命运。可选地，培养的神经干细胞，尤其是那些从成年CNS衍生的，可具有固有defaulty属性分化为体内 13胶质谱系。 因为上面所讨论的限制，我们小组最近开发出一种新的协议，以提高胚胎NSC跟踪，生存，并在严重受伤的成年脊髓分化/成熟。简单地说，我们开始与表达绿色荧光蛋白报告基因在体内移植后14支撑GFP表达一个稳定的转基因菲舍尔344大鼠自交系。接下来，我们使用新鲜分离的神经干细胞从胚胎第14天菲舍尔344脊髓，发展阶段，其保存以产生神经元和神经胶质细胞的潜力。最后，我们的嵌入式新鲜分离的神经干细胞成含增长纤维蛋白基质因素15-17保留了细胞和均匀的大病灶腔内散发，旨在支持移植细胞的存活，分化和整合。移植物置入的T3完全横断，脊髓损伤后两星期网站。这些移植细胞始终充满完全横断网站和分化成神经元丰富的扩展大量的轴突到宿主脊髓长距离18。类似的结果用培养的人神经干细胞移植到免疫缺陷大鼠18获得的。

<table border="1">
 <tbody>
 <tr>
 <td>Reagents</td>
 <td>Company</td>
 <td>Catalogue</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Fibrinogen(rat)</td>
 <td>Sigma</td>
 <td>F6755-25MG</td>
 <td>2 hr at 37 &deg;C to dissovle 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Thrombin (rat)</td>
 <td>Sigma</td>
 <td>T5772-100UN</td>
 <td>Dissovle in 10 mM CaCl2 
 Stock Concentration: 50 U/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>bFGF (human)</td>
 <td>Sigma</td>
 <td>F0291 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>EGF (murine)</td>
 <td>Sigma</td>
 <td>E1257 (0.1 mg)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>BDNF(human)</td>
 <td>Peprotech</td>
 <td>452-02 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>NT-3 (human)</td>
 <td>Peprotech</td>
 <td>452-03 (1 mg)</td>
 <td>Stock Concentration: 1000 ng/&mu;l 
 Final Concentration: 50 ng/&mu;l</td>
 </tr>
 <tr>
 <td>GDNF (rat)</td>
 <td>Sigma</td>
 <td>G1401 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>IGF-1(mouse)</td>
 <td>Sigma</td>
 <td>I8779 (50 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>aFGF (human)</td>
 <td>Sigma</td>
 <td>F5542 (25 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l </td>
 </tr>
 <tr>
 <td>PDGF-AA (human)</td>
 <td>Sigma</td>
 <td>P3076 (10 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>HGF (human)</td>
 <td>Sigma</td>
 <td>H9661 (5 &mu;g)</td>
 <td>Stock Concentration: 200 ng/&mu;l 
 Final Concentration: 10 ng/&mu;l</td>
 </tr>
 <tr>
 <td>MDL28170</td>
 <td>Sigma</td>
 <td>M6690 (25 mg)</td>
 <td>Stock in DMSO 
 Stock Concentration: 1 mM 
 Final Concentration: 50 &mu;M</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Millipore</td>
 <td>BSS-1005-B</td>
 <td>Stock Concentration: 1X 
 Final Concentration: 1X</td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine</td>
 <td>Putney</td>
 <td>26637-411-01</td>
 <td>40-80 mg/kg 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 25 mg/ml</td>
 </tr>
 <tr>
 <td>Xylazine</td>
 <td>Lloyd</td>
 <td>0410,</td>
 <td>2.5-8 mg/ml 
 Stock Concentration: 100 mg/ml 
 Final Concentration: 1.3 mg/ml</td>
 </tr>
 <tr>
 <td>Acepromazine</td>
 <td>Butler</td>
 <td>003845</td>
 <td>0.5-4 mg/ml 
 Stock Concentration: 10 mg/ml 
 Final Concentration: 0.25 mg/ml</td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Healthpets</td>
 <td>BET16OZ</td>
 <td></td>
 </tr>
 <tr>
 <td>Ringers</td>
 <td>Abbott</td>
 <td>04860-04-10</td>
 <td>2-3 ml/inj</td>
 </tr>
 <tr>
 <td>Banamine</td>
 <td>Schering-Plough</td>
 <td>0061-0851-03</td>
 <td>2.5 -5 mg/kg 
 Stock Concentration: 50 mg/ml 
 Final Concentration: 0.5 mg/ml</td>
 </tr>
 <tr>
 <td>Ampicillin</td>
 <td>Sandoz</td>
 <td>0781-3404-85</td>
 <td>80-100 mg/kg 
 Final Concentration: 50 mg/ml</td>
 </tr>
 <tr>
 <td>LH-RH</td>
 <td>Sigma</td>
 <td>L4513</td>
 <td>200 &mu;g/kg 
 Final Concentration: 200 &mu;g/ml</td>
 </tr>
 <tr>
 <td>HBSS</td>
 <td>Gibco</td>
 <td>14175-096</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Gibco</td>
 <td>25200056 </td>
 <td>Stock Concentration: 0.25 % 
 Final Concentration: 0.125 %</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>11995073 </td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Gibco</td>
 <td>16000044 </td>
 <td>Stock Concentration: 100 % 
 Final Concentration: 10 %</td>
 </tr>
 <tr>
 <td>Neurobasal Medium</td>
 <td>Gibco</td>
 <td>21103049 </td>
 <td></td>
 </tr>
 <tr>
 <td>B27</td>
 <td>Gibco</td>
 <td>17504044</td>
 <td>Stock Concentration: 100x 
 Final Concentration: 1x</td>
 </tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Note, use human reagents for grafts of human NSCs</td>
 </tbody>
</table>

promotion of survival and differentiation of neural stem cells with fibrin and growth factor cocktails after severe spinal cord injury

神经干细胞（NS​​Cs）能自我更新和分化成神经元和神经胶质细胞。移植的神经干细胞可以脊髓损伤（SCI）后替代丢失的神经元和神经胶质细胞，并能形成功能性继电器重新连接的上方和下方的病变脊髓节段。以前的研究接枝神经干细胞已经被不完全移植物存活的脊髓损伤腔内的限制。另外，接枝细胞存活，分化和扩展过程跟踪没有被优化。最后，在以往的研究，培养的大鼠神经干细胞通常报告分化成神经胶质细胞在移植到损伤脊髓，而不是神经元，除非命运被驱赶到一个特定的细胞类型。为了解决这些问题，我们开发了新的方法，以提高神经干细胞的存活，整合和分化，甚至严重的脊髓损伤部位。神经干细胞是新鲜从表达绿色佛罗里达州一个稳定的转基因菲舍尔344大鼠行胚胎第14天脊髓（E14）隔离uorescent蛋白（GFP），并嵌入到含有生长因子的纤维蛋白基质;该制剂的目的是保留移植细胞在病灶腔和支持细胞的生存。神经干细胞在纤维蛋白/生长因子鸡尾酒植入两周后胸3级（T3）完全性脊髓横断，从而避免炎症的高峰期。导致移植物完全充满病灶腔和分化为两个神经元，轴突延伸到宿主脊髓过相当长的距离，和神经胶质细胞。培养的人神经干细胞表达绿色荧光蛋白移植物导致类似的结果。因此，方法被定义为提高神经干细胞移植，存活和体内研究结果的分析。

的GFP免疫组织化学标记表明接枝的大鼠神经干细胞重新悬浮于磷酸盐缓冲盐水（PBS）（缺乏纤维蛋白基质和生长因子），在T3切断现场不良存活，只有附着在病灶/主机余量，留下大部分病灶部位的空的无移植的细胞（ 图2A）。神经干细胞的存活改善时共接枝与单独的纤维蛋白基质，但接枝并没有完全填满一个大腔病变（数据未显示）。因此，我们嵌入到神经干细胞含有生长因子鸡尾?...

Watch this Scientific Journal Video about Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury at JoVE.com

Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

含有生长因子的纤维蛋白基质被用来留住嫁接神经干细胞分化为完全脊髓横断网站。移植细胞完全充满病灶腔和分化为多种神经细胞类型，包括神经元轴突延伸到宿主脊髓长距离。

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 ...

promotion-survival-differentiation-neural-stem-cells-with-fibrin

Research

JoVE Journal

Neuroscience

12.3K 次观看.  Veterans Administration Medical Center, San Diego. 含有生长因子的纤维蛋白基质被用来留住嫁接神经干细胞分化为完全脊髓横断网站。移植细胞完全充满病灶腔和分化为多种神经细胞类型，包括神经元轴突延伸到宿主脊髓长距离。 

视频: Promotion of Survival and Differentiation of Neural Stem Cells with Fibrin and Growth Factor Cocktails after Severe Spinal Cord Injury

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

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

科研

神经科学

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These animals also resist ischemic injury to the brain in vivo 2,3 and oxygen-glucose deprivation in vitro 4,5. These unique qualities provided the impetus to isolate AGS neurons to examine inherent neuronal characteristics that could account for the capacity of AGS neurons to resist injury and cell death caused by ischemia and extremely cold temperatures. Identifying proteins or gene targets that allow for the distinctive properties of these cells could aid in the discovery of effective therapies for a number of ischemic indications and for the study of cold tolerance. Adult AGS hippocampus contains neural stem cells that continue to proliferate, allowing for easy expansion of these stem cells in culture. We describe here methods by which researchers can utilize these stem cells and differentiated neurons for any number of purposes. By closely following these steps the AGS neural stem cells can be expanded through two passages or more and then differentiated to a culture high in TUJ1-positive neurons (~50%) without utilizing toxic chemicals to minimize the number of dividing cells. Ischemia induces neurogenesis 6 and neurogenesis which proceeds via MEK/ERK and PI3K/Akt survival signaling pathways contributes to ischemia resistance in vivo7 and in vitro 8 (Kelleher-Anderson, Drew et al., in preparation). Further characterization of these unique neural cells can advance on many fronts, using some or all of these methods.

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

成人北极黄鼠海马神经干细胞的生长和分化

Arctic ground squirrels (Urocitellus parryii, AGS) are unique in their ability to hibernate with a core body temperature near or below freezing 1. These ...

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

小鼠神经干细胞移植手术与器官的诱向性鼠肝炎病毒感染小鼠的脊髓

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

侧向液压冲击：创伤性脑损伤小鼠模型

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

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

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

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

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

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

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

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

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

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

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

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

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

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

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

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

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

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

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 ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

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

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

脊髓损伤 wistar 大鼠跑步机的作业训练

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