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><tbody><tr><td>CX3CR1 GFP mice</td><td>Jackson laboratories</td><td>5582</td><td></td></tr><tr><td>Thy-1 YFP H mice</td><td>Jackson laboratories</td><td>3782</td><td></td></tr><tr><td>Mouse pie cage</td><td>Braintree Scientific</td><td>MPC 2 set</td><td></td></tr><tr><td>60 mm&lt;sup&gt;2&lt;/sup&gt; Petri dish</td><td>Corning</td><td>430166</td><td>For bone marrow isolation</td></tr><tr><td>Falcon 40 &amp;mu;m cell filter</td><td>Fisher Scientific</td><td>08-771-1</td><td>For bone marrow isolation</td></tr><tr><td>1 x 15 ml and 1 x 50 ml conical tube</td><td>Fisher Scientific</td><td></td><td>For bone marrow isolation</td></tr><tr><td>21 gauge needle</td><td>BD</td><td>305177</td><td>For bone marrow isolation</td></tr><tr><td>1 ml syringe</td><td>BD</td><td>309628</td><td>For bone marrow isolation</td></tr><tr><td>ACK lysis buffer</td><td>Gibco</td><td>A10492-01</td><td>For bone marrow isolation</td></tr><tr><td>HBSS</td><td>Corning Cellgro</td><td>21-022-CV</td><td>For bone marrow isolation</td></tr><tr><td>RPMI media</td><td>Sigma</td><td>R8758</td><td>For bone marrow isolation</td></tr><tr><td>F4/80 antibody</td><td>Ebioscience</td><td>12-4801 PE</td><td>For FACS verification of chimeras</td></tr><tr><td>30 gauge insulin syringe</td><td>BD</td><td>328280</td><td>For tail vein injection</td></tr><tr><td>Spinal Cord Clamps</td><td>Narshinge</td><td>STS-A</td><td>For imaging</td></tr><tr><td>Ortho-Jet Dental Acrylic powder</td><td>Lang Dental</td><td>REF1320</td><td>For imaging</td></tr><tr><td>Ortho-Jet Dental Acrylic liquid</td><td>Lang Dental</td><td>REF1404</td><td>For imaging</td></tr><tr><td>Gelfoam gelatin sponges</td><td>Pfizer</td><td>9034201</td><td>For imaging</td></tr><tr><td>4-0 Ethilon sutures</td><td>Ethilon</td><td>1667G</td><td>For surgery</td></tr><tr><td>Reflex Clips, 7 mm</td><td>Kent Scientific</td><td>INS750344</td><td>For surgery. Sterilize before use</td></tr><tr><td>Iris Scissors</td><td>Fine Science Tools</td><td>14061-09</td><td>For surgery</td></tr><tr><td>45/5 Forceps, Dumoxel</td><td>Fine Science Tools</td><td>11251-35</td><td>For surgery</td></tr><tr><td>Rongeurs</td><td>Fine Science Tools</td><td>16021-14</td><td>For surgery</td></tr><tr><td>30 gauge needle</td><td>BD</td><td>305106</td><td>For making access holes in dura</td></tr><tr><td>Forceps Dumont #4 Biology tips</td><td>Dumont</td><td>11242-40</td><td>For surgery</td></tr><tr><td>Needle Drivers</td><td>Fine Science Tools</td><td>12002-12</td><td>For surgery</td></tr><tr><td>Michel Wound Clip Forceps</td><td>Kent Scientific</td><td>INS700753</td><td>For surgery</td></tr><tr><td>Wound clip remover</td><td>Fine Science Tools</td><td>12033-00</td><td>For surgery</td></tr><tr><td>O-rings for Forceps</td><td>Fine Science Tools</td><td>11200-00</td><td>For surgery, Often provided with #4 forceps, can also be ordered separately</td></tr><tr><td>Cordless Rechargable Animal trimmer</td><td>Wahl</td><td>Series 8900</td><td>for surgery</td></tr><tr><td>Vetbond</td><td>3M</td><td>1469SB</td><td>For surgery</td></tr><tr><td>Betadine Solution (10% Providine-Iodine Topical Solution)</td><td>Purdue Products L.P.</td><td>NDC 67618-150-08</td><td>For surgery</td></tr><tr><td>Nair Hair remover lotion</td><td>Church &amp;amp; Dwight Co., Inc.</td><td>NRSL-22329-05</td><td>For surgery</td></tr><tr><td>Isoflurane</td><td>Butler Schein</td><td>NDC 11695-6776-2</td><td>Drugs &amp;ndash; for surgery</td></tr><tr><td>Marcaine</td><td>Henry Schein</td><td>NDC 0409-1587-50</td><td>Drugs &amp;ndash; for surgery 1.0 mg/kg given subcutaneously</td></tr><tr><td>Bupernex</td><td>Henry Schein</td><td>121-7793</td><td>Drugs &amp;ndash; for surgery buprenorphine 0.1 mg/kg</td></tr><tr><td>aCSF</td><td>Chemicals from Sigma</td><td>119 mM NaCl&lt;br /&gt;26.2 mM NaHCO&lt;sub&gt;3&lt;/sub&gt;&lt;br /&gt;2.5 mM KCl&lt;br /&gt;1 mM NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;br /&gt;1.3 mM MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;br /&gt;10 mM glucose</td><td>For surgery&lt;br /&gt;From Cold Spring Harbor Protocols</td></tr><tr><td>TRITC-dextran 150,000 MW</td><td>Sigma</td><td>T1287</td><td>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

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

在体内脊髓的鼠标线使用双光子显微镜成像

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

科研

神经科学

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

玻璃植入脊髓的Windows在成年小鼠实验性自身免疫性脑脊髓炎

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

医学

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

急性脑外伤小鼠其次是纵向双光子成像

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

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 ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

一个程序，植入脊柱商会纵<em&gt;在体内</em&gt;鼠标脊髓成像

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

在体内挫伤脊髓损伤后影像血管动力学的双色法

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Precise Brain Mapping to Perform Repetitive In Vivo Imaging of Neuro-Immune Dynamics in Mice

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. Although studying these cells in isolation in vitro or together ex vivo provides useful physiological information; salient features of neural cell physiology will be missed in such contexts. Therefore, there is a need for studying neural cells in their native in vivo environment. The protocol detailed here describes repetitive in vivo two-photon imaging of neural cells in the rodent cortex as a tool to visualize and study specific cells over extended periods of time from hours to months. We describe in detail the use of the grossly stable brain vasculature as a coarse map or fluorescently labeled dendrites as a fine map of select brain regions of interest. Using these maps as a visual key, we show how neural cells can be precisely relocated for subsequent repetitive in vivo imaging. Using examples of in vivo imaging of fluorescently-labeled microglia, neurons, and NG2+ cells, this protocol demonstrates the ability of this technique to allow repetitive visualization of cellular dynamics in the same brain location over extended time periods, that can further aid in understanding the structural and functional responses of these cells in normal physiology or following pathological insults. Where necessary, this approach can be coupled to functional imaging of neural cells, e.g., with calcium imaging. This approach is especially a powerful technique to visualize the physical interaction between different cell types of the CNS in vivo when genetic mouse models or specific dyes with distinct fluorescent tags to label the cells of interest are available.

This protocol describes a chronic cranial window implantation technique that can be used for longitudinal imaging of neuro-glio-vascular structures, interactions, and function in both healthy and diseased conditions. It serves as a complementary alternative to the transcranial imaging approach that, while often preferred, possesses some critical limitations.

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

The central nervous system (CNS) is regulated by a complex interplay of neuronal, glial, stromal, and vascular cells that facilitate its proper function. ...

免疫与感染

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

清醒小鼠小胶质细胞动力学和神经元活动的同时成像

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Intravital Imaging of Fluorescent Protein Expression in Mice with a Closed-Skull Traumatic Brain Injury and Cranial Window Using a Two-Photon Microscope

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell types of an animal's brain, upon exposure to exogenous stimuli. Here, the administration of a closed-skull traumatic brain injury (TBI) and simultaneous implantation of a cranial window for subsequent longitudinal intravital imaging in mice is shown. Mice are intracranially injected with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) under a neuronal specific promoter. After 2 to 4 weeks, the mice are subjected to a repetitive TBI using a weight drop device over the AAV injection location. Within the same surgical session, the mice are implanted with a metal headpost and then a glass cranial window over the TBI impacting site. The expression and cellular localization of EGFP is examined using a two-photon microscope in the same brain region exposed to trauma over the course of months.

This study demonstrates delivery of a repetitive traumatic brain injury to mice and simultaneous implantation of a cranial window for subsequent intravital imaging of a neuron-expressed EGFP using two-photon microscopy.

使用双光子显微镜对闭合颅骨创伤性脑损伤和颅窗小鼠荧光蛋白表达进行活体成像

The goal of this protocol is to demonstrate how to longitudinally visualize the expression and localization of a protein of interest within specific cell ...