The following agencies provided critical funding support for this study: MSTP T32 GM007250 (T.A.E.), 5T32EB7509 (D.S.B.), NCI R01 CA154656 (A.Y.H.), Dana Foundation (A.Y.H.), St. Baldrick&#8217;s Foundation (A.Y.H.), Alex&#8217;s Lemonade Stand Foundation (A.Y.H.), Gabrielle&#8217;s Angel Foundation (A.Y.H.) and Hyundai Hope-on-Wheels Program (A.Y.H.). The authors are thankful for the indispensable help of Jerry Silver and Sarah Busch in learning the Dorsal Column Crush (DCC) injury. The authors are also grateful to Jingquang You, Elisabeth Hare and Hongmei Hu for technical help with genotyping and Ross Anderson for mounting the spinal stabilizing unit.

注意:所有的动物应收容并根据动物护理和使用委员会（IACUC）批准的机构使用。这里描述的所有程序都是由美国凯斯西储大学IACUC批准。 1.转基因动物<ol><li>使用市售的荧光记者小鼠标签轴突（THY-1 YFP高）50和小胶质细胞/单核细胞（CX3CR1 GFP / GFP 51;见材料清单）。这些小鼠应互交，并根据机构动物护理政策维持个别繁殖地。 </li></ol> 2.骨髓嵌合体<ol><li>确定动物是至少8周龄用作受体动物。 </li><li>将宿主动物在圆形照射控股笼设计，按住鼠标在一个位置，以保证均匀，全身照射。 </li><li>设置定时器，一个铯（Cs-137）辐射到管理一个全身辐射处800-1000拉德根据制造商的说明将动物的年龄（例子这里示出的是980拉德）。 </li><li>回到动物的笼子里休息至少4小时的。 </li><li>选择合适的基因型的供体动物（参见图2A），为骨髓祖细胞的来源。 注:将合适的供体是动物表达不同谱系特异性荧光报告从主机来识别不同的细胞群，或相同基因型作为对照之一。 </li><li>填充1培养皿，用70％的酒精，以及第二盘用10ml罗斯威尔公园纪念研究所（RPMI）培养基和一个40微米的细胞滤网浸泡在该媒体上。填写一个15毫升的锥形管RPMI媒体。置于冰上的菜肴和管。 </li><li>根据机构指引CO牺牲捐助动物2窒息。清洁皮肤和毛皮浸泡，用70％的乙醇在整个动物，直到所有的毛皮是湿的。 </li><li>取出SK在从动物的通过切割围绕所述中间部分和拉动皮肤下来在后腿充分暴露腿部肌肉下半部。 </li><li>取出胫骨前路过度在膝盖关节脱臼的骨头，然后用无菌镊子和剪刀，剥离和切肌肉远离胫骨的两端。放置在培养皿用酒精骨长达30秒，然后转移到含有介质的培养皿内的细胞过滤器。 </li><li>脱臼的髋关​​节和剥离附着于股骨肌肉，置于股骨简要醇，然后将其放置在盘内的同一个细胞过滤。 </li><li>在组织培养罩，使用无菌剪刀切断骨的两端，并插入一个21号针头在1ml注射器进入骨的末端。冲洗骨髓到15毫升锥形与媒体。重复该程序的其余骨骼。 </li><li>使用柱塞从3毫升注射器的后面，粉碎骨细胞中结束过滤器上的陪替氏培养皿中提取更多的细胞。 </li><li>放置过滤器上的50ml锥形管中的顶部和传输介质与吸气细胞从15毫升锥形管中。使用柱塞的回粉碎骨髓，以获得单细胞悬浮液。洗的15毫升锥形管中，骨碎片和细胞过滤用30ml媒体到50ml锥形管中。 </li><li>离心细胞5分钟，在500×g离心以沉淀细胞。 </li><li>裂解红血细胞用2ml铵 - 氯 - 钾（ACK）的裂解缓冲液2分钟。骤冷裂解缓冲液用10毫升含有10％胎牛血清的培养基。离心机在500×g离心5-10分钟以沉淀。 </li><li>在盐水在10ml RPMI培养基中一次洗涤细胞两次，在500×g离心离心5-10分钟以沉淀每次洗涤。 </li><li>重悬的细胞以3×10 6个细胞在200μl盐水中的最终浓度。 </li><li>传输3×10 6骨髓经尾静脉注射到细胞内部收益率adiated受体小鼠。 </li><li>将酸化水（pH值3.0）的接收者笼子，让老鼠恢复。 注:失败将移植手术后在10-14天内死亡的小鼠。 </li><li> 8周后，通过检查用流式细胞仪（ 图2B-C）的外周血免疫细胞验证骨髓重建。 </li></ol> 3.背柱破碎损伤注:选择适当的嵌合体动物或基于所需的实验手术的双重转基因动物。动物与任何标记居民或骨髓细胞在前面步骤中创建。 <ol><li>准备和高压灭菌的手术工具，包括咬骨钳，杜蒙＃4镊子，虹膜剪刀，镊子持组织，针驱动器和伤口剪辑。 </li><li>制备的药物施用到老鼠的疼痛控制。 Bupernex和麻卡因通常用于控制疼痛。使用适当的药物治疗需要一个次批准的机构动物护理和使用委员会（IACUC）。 </li><li>诱导麻醉，用4％的异氟醚并用1-2％异氟烷维持实现呼吸速率为每分钟60-100次呼吸。适用于眼部润滑剂，以动物的眼睛，以防止干燥麻醉下，确认没有回应步行捏。使用加热垫维持温度和监视使用直肠探针的温度。 </li><li>剃小鼠背部用电动剃须刀在靶向脊髓水平，然后擦拭区域三次，碘伏接着用两次70％的酒精。放置在无菌的悬垂性的动物，并覆盖与另一个无菌披盖带切断在适当的位置，以可视化的手术部位的孔的动物。保持无菌窗帘整个手术的所有工具。 </li><li>切割沿中线在所需脊髓水平与虹膜剪将皮肤暴露肌肉上的动物的背面。 </li><li>下一个迪斯阿拉斯显微镜，切背部肌肉，包括斜方肌，沿着他们连接到背棘突中线背阔肌的韧带，并露出脊柱。 </li><li>除去椎骨的背侧和横突之间的旋转体的肌肉以显示特定椎骨的椎板要去除（T11在这种情况下）。由最后非浮动肋的插入到该过程标识T11。 </li><li>将咬骨钳的提示到上面和下面要删除的过程中，空间略有提升，以确保他们安全地在周围的椎板地方，但不要太深伤害脊椎。关闭咬骨钳于一体的运动，以去除薄层。 </li><li>根据需要通过使用骨钳以除去剩余椎骨的小零件放大的椎板。 </li><li>测量从一个＃4钳提示1毫米持反对他们一把尺子，用永久性记号笔标记。衡量一个1毫米的分离赌注通过保持对尺子钳子吐温两个尖齿和滑动的橡胶镊子环就位在​​镊子的轴固定的尖端至1毫米的最大宽度。 </li><li>创建一个小孔通过硬脑膜插入30号针头在用于镊子的尖齿的两个插入点来访问脊髓。 </li><li>插入钳子尖齿在通过孔到1毫米上标记的镊子的侧面的深度的90°角硬脑膜，确保该标记是硬脑膜以上。捏关闭钳，保持10秒。释放，并重复镊子挤压两次，共3成立。从脊髓中取出镊子。 </li><li>浸泡一块吸收性明胶海绵压缩在生理盐水和地点在损伤部位。 </li><li>使用一个运行针，缝合肌肉之经椎板和浸泡明胶海绵与＃4合成的非吸收缝合线的尼龙。 </li><li>剪辑使用5毫米伤口CLI到位皮肤ps的。 </li><li>注入10微升麻卡因（1.0毫克/千克）在切口周围部位490微升盐水皮下和5μlBupernex（0.1毫克/千克）肌内入臀肌。 </li><li>让动物恢复在一个温暖的垫和监测在运动后肢任何困难或瘫痪的迹象。不要让动物无人看管或其他动物的存在，直到从麻醉中完全恢复。不要使用可观察到的显示运动障碍的任何动物。 注:虽然这个美眉病变可以沿着脊髓的任何级别执行，务实，T10-L4构成成像较少的技术挑战相对于呼吸和稳定使用卡的身体运动。夹具放置必须围绕肋骨进行修改，以图像的胸椎水平。 </li></ol> 4.活体成像<ol><li>准备和高压灭菌的手术工具，包括杜蒙＃4镊子，虹膜剪刀，镊子抱着纸巾，针ð河流和伤口剪辑。 </li><li>异氟醚作为麻醉前的鼠标。诱导麻醉，在4-5％，并在1-2％，以维持每分钟60-100次呼吸呼吸速率和缺乏响应于脚捏的维护。按住鼠标加热，监测时不能成像呼吸频率和持续监测动物的温度与直肠探头。 </li><li>清洁伤口周围剪辑和聚维酮碘，然后将皮肤醇，并根据制造商的说明除去伤口剪辑。一旦卷绕夹子被除去，如果需要的话去除毛发用奈尔并用聚维酮碘和70％乙醇再次清洗的部位。 </li><li>再打开，用剪刀将皮肤。从肌肉中取出任何剩余的缝线和拉肌肉分开用钳子，如果需要用剪刀裁剪。 </li><li>在解剖显微镜下，创建口袋脊髓通过切割用剪刀的肌肉在横突的侧面上的椎骨一个椎骨水平的上方和下方层压钳nectomy。 </li><li>将围绕夹子每个椎体并拧紧。根据需要留出空间显微镜物镜到达脊髓调整。确保脊髓平行于成像平台，并且维持组织的稳定性以进行成像。如果呼吸工件时看到的，重新调整相应夹具，以尽量减少在成像场运动。 </li><li>用封口膜封住夹子。 </li><li>从脊髓用钳子取出吸收性明胶海绵压缩，精心挑选过许多作品成为可能，也除去了脑膜尽可能多的疤痕组织。盖暴露脊髓液，新的海绵，如果需要的。 注意:如果发生任何流血从周围的肌肉，无论是坚定的用海绵或根据需要烧灼。但是，请注意不要用烧灼触摸脊髓。烧灼时捂住脊髓或者一块湿润的纸巾或明胶海绵压缩的。 </li><li>创建WEL升由第一密封的皮肤和组织与Vetbond，然后用牙科用丙烯酸系脊髓保持器的两个夹具和动物的侧面之间建立一个很好地保持浸没流体。等到丙烯酸固化。 </li><li>填写好人工脑脊髓液（脑脊液），并再次检查周围的任何手术部位出血。 </li><li>任选在这一点上，静脉注射荧光染料容器通过尾静脉注射在成像期间以突出的血管。 注:荧光葡聚糖高于70 kDa的经常使用，尽管量子点和荧光标记的外源凝集素也可作为有用的容器染料。 50微升15万WM TRITC葡聚糖通常是静脉注射。 </li><li>以获得生理有关的免疫细胞运动性，鼠标的温度必须保持在37℃。将鼠标移动到温度控制的环境中，在显微镜下进行成像和监测动物的正确ANES通过呼吸速率和脚捏thesia水平。在成像过程中经常监测动物，以确定麻醉深度，旨在达到60呼吸率 - 每分钟100次呼吸。 </li><li>找到通过显微镜目镜的成像部位。 </li><li>调整的双光子激光扫描显微镜拍到的z栈图像每隔30-60秒，以获得动态成像数据。 </li><li>一旦成像会话完成从加热箱取出的动物。 </li><li>松开脊髓夹，并从夹子取下鼠标。从表皮的同时去除夹子拉丙烯酸得好远。 </li><li>如果鼠标再次成像，放置盐水浸泡明胶海绵压缩在脊髓。关闭肌肉和皮肤上的手术部位。不要让动物无人看管或其他动物的存在，而从麻醉中恢复。如果动物表演后恢复神经功能缺损的迹象，动物应安乐死。否则，安乐死从麻醉中按照IACUC批准的协议安乐死的小鼠在CO 2室中出现之前。 </li><li>按照制造商的指示进行分析使用图像分析软件的荧光图像。 </li></ol>

The authors have nothing to disclose.

在其原生组织区室的不同类型的细胞中的实时随后的病理过程中成像的相互作用产生了极大的兴趣。内的密集网络的中枢神经系统，细胞 - 细胞接触，并与邻近的细胞信号传导对于正常功能和理解的CNS病理必不可少的。在这里，我们在脊髓中描述了使用2-光子激光扫描显微术的观测细胞运动的机械病灶内。除了手术和组织制剂的质量，组织的机械稳定性是最重要的成功时间推移显微镜，从呼吸运动伪影的脊髓特别的隔离。稳定性可通过对应于该动物的心脏速率或呼吸解剖显微镜下寻找脊髓运动进行评估。稳定应进行评估双方在执行了手术，也立刻开始前成像。如果灵魂升并不稳定，调整应使脊髓夹具的放置和密封性。加之骨髓嵌合体的方法的细胞类型特异性荧光报道小鼠模型允许单核细胞与小胶质细胞和轴突的识别。先前的研究已经表明，血液来源的单核细胞和小胶质细胞不负责使用此处43中描述的方法学创伤后继发性轴索损伤。葡聚糖共轭容器染料允许船只识别既提供地标，并确定血管的完整性破坏。合适的荧光报道小鼠模型的选择是至关重要的，以允许要被成像的正确识别所需的细胞类型。 荧光小鼠模型是适合于研究正进行开发也是以实验的成功是至关重要的。 CX3CR1 + / GFP的两个吞噬人群之间的区别</sup&gt;细胞在中枢神经系统历来是困难的。如这里所示，一个常见的​​免疫学技术，照射嵌合体，可用于从骨髓衍生的巨噬细胞区分无线电抗性，中枢神经系统驻留小胶质细胞。照射过程具有破坏血脑屏障，并改变细胞的表型的潜力，所以使用该模型的应该仔细考虑。这些变化的程度不同，与辐射剂量以及恢复期的持续时间，以及它们对不同的炎症模型的影响还没有得到充分的研究。关于CNS血脑屏障照射的效果可以通过屏蔽照射期间的头被最小化，并且这已被证明是降低细胞浸润到脊髓在照射后，即使在脊髓不直接屏蔽52。在这里，我们已经观察到细胞浸润到CNS在稳态作为嵌合体产生的结果的数目而相比之下，麻木是微不足道呃进入病变细胞。其他替代模型考虑的因素包括药物引起的嵌合体52或联体小鼠模型53。 这里，我们已经提出了一个具体的，简单的和可重现的小损伤模型，结果在一个损伤脊髓的背侧白质，很容易使用双光子显微镜图像。这种方法还提供了一个可量化的病变以测定轴突顶梢枯死的，在所述文献中已加上的互补固定的组织分析16,18,43,48,54背侧列的程度。在该模型中，动物仅显示最小缺陷，不需要特别小心损伤后。利用这里所描述的背柱破碎损伤和成像技术未来的研究可以是有力的筛选工具，以评估治疗对脊髓损伤的疗效。这些处理可以包括小分子抑制剂，药物，细胞产品，组织移植物和组合治疗秒。巨噬细胞，小胶质细胞和神经元之间的相互作用也可能起到在脊髓其他疾病模型，包括多发性硬化，肿瘤，脑膜炎和肌萎缩性侧索硬化的作用，并且该技术可在这些疾病的研究是有用的，以及。

在中枢神经系统（CNS）的炎性反应，疾病或损伤是知之甚少，特别是其中的组织内的免疫和固有细胞问候的相互作用。脊髓这些细胞相互作用的调查是在活的动物特别感兴趣。唯一方便CNS白质纤维束是在脊髓背列，这使得在其上注重提高可行的实验方法的努力的一个重要领域。这些研究已受到限制，由于在获得和稳定的中枢神经系统用于成像的技术难度。在脊髓实时成像已经在脊髓损伤了说明先前1-13，但是，很少有研究涉及蜂窝移动超出几个小时的初始损伤后。外伤性脊髓损伤是一个复杂的环境中，与神经元，星形胶质细胞，成纤维细胞，NG2祖细胞，免疫细胞包括第一卷纳克小胶质细胞，嗜中性粒细胞，巨噬细胞，T细胞，B细胞和树突状细胞14,15。巨噬细胞是免疫细胞负责吞噬在病变部位，从循环中浸润的所述子集。这些吞噬细胞的作用一直争论不休，有报告表明，这些细胞可以在受伤的组织既破坏和保护的作用。这些角色的范围从增加二次轴突顶梢枯死受伤后行动的吞噬方式16-22，采取对伤口愈合的表型和功能下降的赤字在受伤的动物21,23,24。 以前，试图区分的巨噬细胞从胶质已经主要依靠形态中的健康组织。然而，活化的小胶质细胞和巨噬细胞表达许多相同的标记和损伤后25-29显示区分形态，使这些细胞难以研究浅的不同活动的分离编辑这些因素仅30。这些细胞可通过CD45的使用流式细胞仪33,34的差异表达来分离，虽然这种方法是在体内鉴别这些细胞类型的有用。 CCR2和CX3CR1在小胶质细胞和巨噬细胞利用差异表达也得到了探索，虽然在这些标记为单核细胞表达的动态变化分化成巨噬细胞可复杂精确的分析35,36。小胶质细胞是驻地的免疫细胞在中枢神经系统和胎儿发育过程中产生于卵黄囊祖细胞，巨噬细胞的同时来源于骨髓祖细胞和侮辱31,32后进入中枢神经系统损伤。另一种方法使用形态来区分这两种类型的细胞，是从供体动物的替代骨髓祖细胞中表达来自供体祖细胞衍生的巨噬细胞一可追踪标记，同时保留在CNS驻地，RECIPient衍生的小胶质细胞。这些骨髓嵌合模型通常用在许多其他应用程序37-40。这种方法具有与损伤引起的用于消灭骨髓祖细胞在宿主，从而限制了其在某些应用中使用辐射或细胞毒性药物的血 - 脑屏障的完整性相关联的唯一的警告。最近，小胶质细胞和巨噬细胞在CNS之间的功能区别都开始通过使用流式细胞仪，基因芯片阵列分析和嵌合方法24,26,41-44，这将证明在将来非常有用的可分辨。虽然分离这两种类型的细胞仍然很难，了解其独特的功能，就是在通往涉及中枢神经系统中的小胶质细胞均和巨噬细胞疾病更有针对性的治疗非常重要。 脊髓病灶内细胞相互作用的描述已经主要来自包括细胞培养模型的研究。这为dUE向涉及成像两者脊髓损伤和免疫细胞一起在活体动物的挑战。不同类型和严重程度的脊髓损伤的电流啮齿动物模型包括挫伤模型45,46，针点刺受伤2，裂伤47，并在这里16,18,48描述的背柱破碎损伤。炎症在脑膜增加了与损伤的严重程度和构成窗户或手术用于串行成像的植入特有的挑战。其中的一些挑战包括吞噬细胞的浸润，以及纤维组织的生成在手术部位。其中的一些问题可以通过创建更小的病变和覆盖暴露的脊髓硬膜及脊柱旁的肌肉，以便随后重新开放手术间无免疫原性敷料被克服，因为在这里做研究背柱挤压伤。能力旋转图像只背部分人帘线也限制损伤的选择，因为挫伤模型主要造成损害中央线，损伤45,49后常常不放过背侧列的最背侧的一部分，尤其是在早期的时间点。因此，我们描述了能够产生一个干净的，可重复的，矩形形状的病变是两个内病变和位置相对于病变轴突的容易量化的观察小区移动的有用的简单损伤模型。

<table border="0" cellpadding="0" cellspacing="0" width="594">
	<tbody>
		<tr height="85">
			<td height="85" style="height:85px;width:125px;">Name</td>
			<td style="width:165px;">Company</td>
			<td style="width:64px;">Catalog Number</td>
			<td style="width:240px;">Comments</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:125px;">CX3CR1 GFP mice</td>
			<td style="width:165px;">Jackson laboratories</td>
			<td style="width:64px;">5582</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:125px;">Thy-1 YFP H mice</td>
			<td style="width:165px;">Jackson laboratories</td>
			<td style="width:64px;">3782</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:125px;">Mouse pie cage</td>
			<td style="width:165px;">Braintree Scientific</td>
			<td style="width:64px;">MPC 2 set</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:125px;">60mm2 petri dish</td>
			<td style="width:165px;">Corning</td>
			<td style="width:64px;">430166</td>
			<td style="width:240px;">For bone marrow isolation</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:125px;">Falcon 40 um cell filter</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:64px;">08-771-1</td>
			<td style="width:240px;">For bone marrow isolation&#160;</td>
		</tr>
		<tr height="121">
			<td height="121" style="height:121px;width:125px;">1x15mL and 1x50mL conical tube</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:64px;"></td>
			<td style="width:240px;">For bone marrow isolation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">16 gauge needle</td>
			<td style="width:165px;">BD</td>
			<td style="width:64px;">305177</td>
			<td style="width:240px;">For bone marrow isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">1ml syringe</td>
			<td style="width:165px;">BD</td>
			<td style="width:64px;">309628</td>
			<td style="width:240px;">For bone marrow isolation</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">ACK lysis buffer</td>
			<td style="width:165px;">Gibco</td>
			<td style="width:64px;">A10492-01</td>
			<td style="width:240px;">For bone marrow isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:125px;">HBSS</td>
			<td style="width:165px;">Gibco</td>
			<td style="width:64px;">1E+07</td>
			<td style="width:240px;">For bone marrow isolation</td>
		</tr>
		<tr height="79">
			<td height="100" rowspan="2" style="height:100px;width:125px;">RPMI media</td>
			<td rowspan="2" style="width:165px;">Sigma</td>
			<td rowspan="2" style="width:64px;">R8758</td>
			<td rowspan="2" style="width:240px;">For bone marrow isolation</td>
		</tr>
		<tr height="21">
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:125px;">F4/80 antibody</td>
			<td style="width:165px;">Ebioscience</td>
			<td style="width:64px;">12-4801 PE</td>
			<td style="width:240px;">For FACS verification of chimeras</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">30-gauge insulin syringe&#160;</td>
			<td style="width:165px;">BD</td>
			<td style="width:64px;">328280</td>
			<td style="width:240px;">For tail vein injection&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Spinal Cord Clamps</td>
			<td style="width:165px;">Narshinge</td>
			<td style="width:64px;">STS-A</td>
			<td style="width:240px;">For imaging&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Ortho-Jet Dental Acrylic powder&#160;</td>
			<td style="width:165px;">Lang Dental</td>
			<td style="width:64px;">REF1320</td>
			<td style="width:240px;">For imaging</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Ortho-Jet Dental Acrylic liquid</td>
			<td style="width:165px;">Lang Dental</td>
			<td style="width:64px;">REF1404</td>
			<td style="width:240px;">For imaging</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Gelfoam gelatin sponges</td>
			<td style="width:165px;">Pfizer</td>
			<td style="width:64px;">9E+06</td>
			<td style="width:240px;">For imaging</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">4-0 Ethilon sutures</td>
			<td style="width:165px;">Ethilon</td>
			<td style="width:64px;">1667G</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Reflex Clips, 7mm</td>
			<td style="width:165px;">Kent Scientific</td>
			<td style="width:64px;">INS750344</td>
			<td style="width:240px;">For surgery. Sterilize before use</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Iris Scissors</td>
			<td style="width:165px;">Fine Science Tools</td>
			<td style="width:64px;">14061-09</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">45/5 Forceps, Dumoxel</td>
			<td style="width:165px;">Fine Science Tools</td>
			<td style="width:64px;">11251-35</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Rongeurs</td>
			<td style="width:165px;">Fine Science Tools</td>
			<td style="width:64px;">16021-14</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">30 gauge needle</td>
			<td style="width:165px;">BD</td>
			<td style="width:64px;">305106</td>
			<td style="width:240px;">For making access holes in dura</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:125px;">Forceps Dumont #4 Biology tips</td>
			<td style="width:165px;">Dumont</td>
			<td style="width:64px;">11242-40</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Needle Drivers</td>
			<td style="width:165px;">Fine Science Tools</td>
			<td style="width:64px;">12002-12</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Michel Wound Clip Forceps</td>
			<td style="width:165px;">Kent Scientific</td>
			<td style="width:64px;">INS700753</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Wound clip remover</td>
			<td style="width:165px;">Fine Science Tools</td>
			<td style="width:64px;">12033-00</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:125px;">O-rings for Forceps</td>
			<td style="width:165px;">Fine Science Tools</td>
			<td style="width:64px;">11200-00</td>
			<td style="width:240px;">For surgery, Often provided with #4 forceps, can also be ordered separately</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:125px;">Cordless Rechargable Animal trimmer</td>
			<td style="width:165px;">Wahl</td>
			<td style="width:64px;">Series 8900</td>
			<td style="width:240px;">for surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Vetbond</td>
			<td style="width:165px;">3M</td>
			<td style="width:64px;">1469SB</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:125px;">Betadine Solution (10% Providine-Iodine Topical Solution)</td>
			<td style="width:165px;">Purdue Products L.P.</td>
			<td style="width:64px;">NDC 67618-150-08</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:125px;">Nair Hair remover lotion</td>
			<td style="width:165px;">Church &#38; Dwight Co., Inc.</td>
			<td style="width:64px;">NRSL-22329-05</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:125px;">Isoflurane</td>
			<td style="width:165px;">Butler Schein</td>
			<td style="width:64px;">NDC 11695-6776-2</td>
			<td style="width:240px;">Drugs &#8211; for surgery</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Marcaine</td>
			<td style="width:165px;">Henry Schein</td>
			<td style="width:64px;">6E+06</td>
			<td style="width:240px;">Drugs &#8211; for surgery 1.0 mg/kg given subcutaneously&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">Bupernex</td>
			<td style="width:165px;">Henry Schein</td>
			<td style="width:64px;">121-7793</td>
			<td style="width:240px;">Drugs &#8211; for surgery buprenorphine 0.1 mg/kg</td>
		</tr>
		<tr height="60">
			<td height="345" rowspan="6" style="height:345px;width:125px;">aCSF</td>
			<td rowspan="6" style="width:165px;">Chemicals from Sigma</td>
			<td style="width:64px;">119 mm NaCl</td>
			<td style="width:240px;">For surgery</td>
		</tr>
		<tr height="86">
			<td height="86" style="height:86px;width:64px;">26.2 mM NaHCO3</td>
			<td style="width:240px;">From Cold Spring Harbor Protocols</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:64px;">2.5 mM KCl</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:64px;">1 mM NaH2PO4</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:64px;">1.3 mM MgCl2</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:64px;">10 mM glucose&#160;</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:125px;">TRITC-dextran 150,000 MW</td>
			<td style="width:165px;">Sigma</td>
			<td style="width:64px;">T1287</td>
			<td style="width:240px;">For intravenous administration to label vasculature</td>
		</tr>
	</tbody>
</table>

intravital imaging of axonal interactions with microglia and macrophages in a mouse dorsal column crush injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

的示意图描绘了背柱破碎损伤示于图1A。病灶后，动物被制备并稳定的使用脊椎夹具（ 图1B）活体显微镜。背柱挤压伤后立即拍摄的图像显示了一个清晰可见的矩形病变钳齿插入点是清晰可辨。轴突在损伤部位在整个背柱（ 图1C）的跨度被切断。血管的完整性保持不变，并通过荧光检测没有证据表明血管染料泄漏。以数周执行相同病变串行成像，肌肉和皮肤，可缝合在椎板​​切除术用于随后再暴露。类似病变形态被认为是在稍后的时间点（ 图1D，E）。损伤后5天，钳子插入位点仍然可见，但病变已经开始增加在大小为dUE引起的炎症继发性损伤。在病变的尾端轴突已从损伤的初始位点通过一个被称为"轴突顶梢枯死"过程缩回。在病变的喙端轴突展出沃勒样变性（ 图1D，E）。在5天至伤后22之间，病变的大小已经稳定。同时，CX3CR1 +细胞的大量涌入可以看出浸润和周围在这些时间点的压碎损伤，尽管这些细胞（小胶质细胞与巨噬细胞）的身份不能在制备区分， 如图1。 为了区别粉碎病变微环境中的小胶质细胞和巨噬细胞的行为中，使用一个辐射嵌合体模型（在图2A中概述）。首先，CX3CR1 + / GFP小鼠的骨髓祖细胞被替换的非荧光供体CELLS，因此只GFP + CNS驻地小胶质细胞是由荧光成像可见。骨髓重建的效率可以通过流式细胞仪8周后骨髓移植（ 图2B，C）的检查的外周血进行确认。在图2B中 ，F4 / 80和GFP阳性的巨噬细胞，以蓝色突出显示，在一个CX3CR1 + / GFP→C57BL / 6的嵌合小鼠中检测到，其中一些的GFP阴性，但F4 / 80阳性单核细胞也存在。在图2C中 ，没有双阳性F4 / 80和GFP阳性细胞更换CX3CR1 + / GFP接受者的骨髓与野生型骨髓后留下。受伤之前，小胶质细胞均匀分布在脊髓内，显示休息，支链的形态（ 图2D）。损伤后8天，只有少数小胶质细胞可以在与病灶周围进行检测。这些小胶质细胞显示变形虫的形态（ 图2E）。二，骨在一个非荧光小鼠骨髓祖细胞通过从CX3CR1 + / GFP小鼠的骨髓细胞取代，因此呈现骨髓来源CX3CR1 +单核细胞/巨噬细胞通过GFP荧光可见（ 图2A）。受伤之前，唯一的GFP +细胞检测在很大程度上是血管周围，这已被报道为骨髓来源并不断翻身定期28（ 图2F）。后一个挤压伤，CX3CR1 + / GFP细胞可以沿着围绕病变（ 图2H-J）的血管的内可以看出，与病灶中心填充有浸润CX3CR1 + / GFP的巨噬细胞（ 图2G）。 可长达6小时执行时间的推移背列成像。在图3中 ，我们示出了非嵌合CX3CR1 + / GFP小鼠立即损伤后，其中从循环细胞可看到Ñ ​​移出朝向病变芯血管和损伤部位（ 图3A，B;补充电影1）内移动。利用荧光强度，以确定细胞可以跟踪所采取的巨噬细胞（; 补充电影1图3A，B）中的路径的图像分析。从成像分析得到的统计数据显示巨噬细胞中的病灶的平均运动是3.6微米/分钟（ 图3D）。最后，在仍能病变证明成像的区域内被检测到损伤，轴突，小胶质细胞和巨噬细胞后第22天保持稳定的时间（ 补充电影2）长时间。 <img alt="图1" fo:content-width="7in" src="/files/ftp_upload/52228/52228fig1highres.jpg" width="600" /> 图1:创建和背柱挤压伤的连续影像。 （A）的背柱破碎损伤是由在感兴趣的水平除去椎骨的背侧过程进行。钳子插入的脊髓1mm处的背柱中，然后关闭3次，以产生紫色所示的病变部位。（B）中的动物再定位脊髓夹具，以获得稳定活体成像。（C）的立即受伤后，镊子插入位点（红色椭圆形）和矩形挤压伤体积（灰色框）清晰可辨。在背根神经轴突可以看出（白色箭头），以及对病灶（实心箭头头部）的尾部侧的轴突损伤末端。（D）的损伤后5天，在钳子插入位点（红色椭圆），以及病变（灰色框的范围内）仍然可以很容易地可视化。病变已在规模从最初的侮辱增加。轴突发生沃勒样变性可以看出喙在additi病变（开放箭头头）上以在背根（白色箭头）和受伤的轴突（E）的损伤后22天结束（白色箭头）。轴突，病灶部位仍然可辨认具有相似的尺寸，以在5天后的伤害。注意和受伤部位周围的大量CX3CR1 +细胞。标记的特征是相同的（D）中。所有比例尺= 200微米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52228/52228fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="7in" src="/files/ftp_upload/52228/52228fig2highres.jpg" width="600" /> 图2:嵌合小鼠的构建以跟踪CX3CR1 + / GFP的表达在背柱破碎损伤的CNS驻地小胶质细胞或骨髓来源的巨噬细胞。 （A）嵌合体小鼠生成的示意图有两种CX3CR1 + / GFP表达英里croglia或巨噬细胞。首先，THY-1 YFPħ小鼠照射和骨髓复原与骨髓细胞从CX3CR1 + / GFP小鼠中分离，产生的嵌合小鼠的巨噬细胞是GFP +。第二，THY-1 YFP的H / CX3CR1 + / GFP小鼠照射和骨髓复原与骨髓细胞从非荧光小鼠中分离，产生的嵌合小鼠的小胶质细胞是GFP +。（B）的流式细胞术数据的实施例与F4 / 80 +和CX3CR1的GFP +细胞在血液从嵌合动物与CX3CR1 + / GFP骨髓供体移植到被照射的C57BL / 6的收件人。流从CX3CR1阳性供体是阳性的GFP和F4 / 80的细胞都显示在蓝色突出显示的区域。（℃）实施例术与F4 / 80 +和CX3CR1的GFP +细胞的数据中的嵌合动物与C57BL6供体骨髓移植进的IR辐射CX3CR1 + / GFP收件人。注意缺乏双阳性CX3CR1 + / GFP和F4 / 80的细胞内的蓝色突出显示的区域。（D）的从第一嵌合方案的小鼠的活体双光子显微镜快照，示出了背内的GFP +胶质细胞与支形态柱（箭头）。这些细胞被穿插轴突（黄色）在背根和背柱之间。比例尺= 100微米。（E）相同的鼠标在活体成像（B）8天后，背柱损伤透着一些CX3CR1 +小胶质细胞与缺乏流程（箭头）大变形虫状细胞体。比例尺= 100微米。（F）从第二嵌合方案鼠标快照（A）中，示出很少的GFP +巨噬细胞，中枢神经系统实质内。比例尺= 100微米。（G）中的相同的小鼠中（F）的活体成像背柱伤8天后揭示人和周围的病灶的巨噬细胞的ARGE涌入。比例尺= 100微米。（H），CX3CR1 +血来源的细胞与在未受伤的动物中血管接触较高放大倍数图像。比例尺= 30微米。（I）中 CX3CR1的细胞与所述容器中，从容器内部看触点A重建。比例尺= 30微米。（J），与所述容器接触阿重建CX3CR1的细胞，从容器的外部观察。比例尺= 20微米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52228/52228fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" fo:content-width="7in" src="/files/ftp_upload/52228/52228fig2highres.jpg" width="600" /> 图3:。背柱破碎后，立即在一个非嵌合CX3CR1 + / GFP小鼠代表细胞跟踪数据伤害。在110（A）的背柱破碎损伤后立即CX3CR1 +小胶质细胞和补充电影1巨噬细胞损伤周围神经轴突（黄色）的快照。（B）的路径（灰色）中（A）所采取的CX3CR1 +细胞分活体成像会话。（C）的轨道中（B）一时间编码表示。磁道是基于他们的时间有色整个110分钟电影内，如图中的时间条。在CX3CR1 +细胞的整体运动的（D）的直方图。所有比例尺为30微米。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52228/52228fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 补充电影1:代表活电影背柱破碎损伤的双重杂合子后你的-1 YFP / + <st荣&gt; / CX3CR1 GFP / +鼠标。随即一个背柱破碎损伤在T11级后进行活体成像脊柱。右图 ​​显示的CX3CR1 +小胶质细胞和巨噬细胞的运动。细胞运动的路径显示为左侧面板上的白色轨迹。这次受伤是位于左上角。 CX3CR1 +细胞可以看出移动到沿着这些轨道的损伤部位。轴突显示为黄色。比例尺= 40微米。总时间:110分钟。播放速度:300X。 补充电影2:代表活电影在22天背柱破碎损伤中的双重杂合子你的-1 YFP / + / CX3CR1 GFP / +鼠标后，这里展示的是具有代表性的电影最初的背柱破碎后第22天，在鼠标拍摄受伤的脊髓水平T11。该注射URY位于该帧的右上角。轴突（黄色）升序吻侧示于右下角。背静脉和血管标以TRITC葡聚糖（红色）。 CX3CR1 +细胞内病变动以较慢的速度相比，这些损伤后立即使用。比例尺= 50微米。总时间:90分钟。播放速度:600X。

Watch this Scientific Journal Video about Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury at JoVE.com

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

轴突的相互作用与小胶质细胞和巨噬细胞在小鼠背柱破碎损伤活体成像

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

intravital-imaging-axonal-interactions-with-microglia-macrophages

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.4K 次观看.  Case Western Reserve University. Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury. 

视频: Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

多鼠神经解剖学磁共振成像

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

科研

神经科学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Advanced Diffusion Imaging in The Hippocampus of Rats with Mild Traumatic Brain Injury

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous variability and heterogeneity (age, gender, type of trauma, other possible pathologies, etc.), animal models play a key role in unraveling factors that are limitations in clinical research. They provide a standardized and controlled setting to investigate the biological mechanisms of injury and repair following TBI. However, not all animal models mimic the diffuse and subtle nature of mTBI effectively. For example, the commonly used controlled cortical impact (CCI) and lateral fluid percussion injury (LFPI) models make use of a craniotomy to expose the brain and induce widespread focal trauma, which are not commonly seen in mTBI. Therefore, these experimental models are not valid to mimic mTBI. Thus, an appropriate model should be used to investigate mTBI. The Marmarou weight drop model for rats induces similar microstructural alterations and cognitive impairments as seen in patients who sustain mild trauma; therefore, this model was selected for this protocol. Conventional computed tomography and magnetic resonance imaging (MRI) scans commonly show no damage following a mild injury, because mTBI induces often only subtle and diffuse injuries. With diffusion weighted MRI, it is possible to investigate microstructural properties of brain tissue, which can provide more insight into the microscopic alterations following mild trauma. Therefore, the goal of this study is to obtain quantitative information of a selected region-of-interest (i.e., hippocampus) to follow up disease progression after obtaining a mild and diffuse brain injury.

The overall goal of this procedure is to obtain quantitative microstructural information of the hippocampus in a rat with mild traumatic brain injury. This is done using an advanced diffusion-weighted magnetic resonance imaging protocol and region-of-interest based analysis of parametric diffusion maps.

轻度创伤性脑损伤大鼠海马学的高级扩散成像

Mild traumatic brain injury (mTBI) is the most common type of acquired brain injury. Since patients with traumatic brain injury show a tremendous ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

多尾根结鱼巨噬细胞的快速隔离

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...