这项工作得到了部分由 NIH NS059622, NS073636, 国防部 CDMRP W81XWH-12-1-0562, 优异审查奖 I01 BX002356 从美国退伍军人事务部, 克雷格 H 尼尔森基金会 296749, 印第安纳脊髓和脑损伤研究基金会 (ISCBIRF) 印第安纳州卫生署 (019919), 和玛丽罗斯霍曼乔治捐赠基金。

所有外科和动物处理程序均按照《实验室动物护理和使用指南》 (国家研究委员会) 和《印第安纳大学医学院机构动物保育和使用指南》的规定进行。委员会.
1. 手术准备
<ol>
	<li>对包括脊椎稳定剂在内的所有手术工具进行消毒。用70% 乙醇清洁手术桌和周围的所有区域。为准备 non-survival 外科手术, 在37° c 加热垫的顶部放置一个干净的手术垫。</li>
	<li>使用六周的老大 (SD) 大鼠进行这项研究。用腹腔注射氯胺酮 (87.7 毫克/千克) 和嗪 (12.3 毫克/千克) 混合物称量和麻醉大鼠。当动物停止对脚趾捏刺激的反应时, 确认适当的麻醉阶段。皮下注射 0.01-0. 05 毫克/千克丁丙诺啡和5毫克/千克卡洛芬在手术前。</li>
	<li>在2区域刮毛鼠: 颈部脊柱的背部和颈部区域在乳房的一侧。用控告手术磨砂和70% 酒精湿巾擦拭皮肤区域。在手术中用眼药膏防止干眼。将动物放在干净的外科垫上仰卧位。</li>
</ol>
2. 颈外静脉插管
<ol>
	<li>找到颈外静脉, 在锁骨附近找到脉搏点, 用一把小弹簧剪刀切开, 在现场做一个垂直切口, 这是3解剖点的交叉: 右下颌骨的尾支, 更大的结核肱骨和柄 (图 1A)。用弹簧剪刀和细钳隔离容器。将远端与1无菌手术缝合线 (图 1C)20绑在一起。</li>
	<li>准备一个1毫升注射器充满生理盐水和连接一个专门的导管由21口径针 (图 1B)。在容器上用一对微型剪刀做一个小切口, 然后将导管滑入血管。<ol>
		<li>通过绑上近端和远端 (图 1C) 来保护针。特制导管是由21口径的针头制成的。研磨的尖端平坦和焊缝与2毫米的一条尖端切断从另一个21口径针。这可以防止导管在外面打滑。 
		注意:少量的血液流入针头表明, 针头已成功进入血管。</li>
	</ol>
	</li>
</ol>
3. 脊柱稳定和 C5-C7 椎板切除术
<ol>
	<li>将动物置于俯卧的位置。沿中线切割皮肤, No. 15 手术刀刀片在所需的脊柱水平。从两侧解剖 5th到 7th颈椎 (C5-C7) 的肌肉层, 以公开侧面切面 (图 2A)21。 
	注意:找到第二个胸椎 (T2), 找到肩胛骨之间的穗。从 T2 椎体向上计数, 找到 C7 椎骨21,22,23,24。</li>
	<li>使用改良的稳定装置稳定大鼠的脊柱。在侧方椎骨两侧做一个狭缝。在裸露的横向加工面下滑动不锈钢臂, 拧紧螺钉以确保稳定性 (图 2B)。</li>
	<li>小心移除 C5-C7 板 (椎板切除术,图 2C)。</li>
	<li>在裸露的硬脑膜上放置一小块盐水浸泡的海绵, 使其保持滋润 (图 2D)。</li>
</ol>
4. 双光子 (2P) 成像窗口的安装
<ol>
	<li>小块的海绵进入肌肉和脊椎之间的缝隙, 还有一条细线的海绵在脊髓和脊椎之间, 然后用纸巾粘胶封住周围的肌肉骨区。等待5分钟完全干燥 (图 2E)。 
	注意:这一步有效地防止未来的出血到窗口和泄漏的浸泡解决方案。</li>
	<li>在微波炉中用 ddH2O 准备4% 琼脂。在琼脂完全溶解后, 等到它回到可触摸的温度。用琼脂溶液填充1毫升无菌注射器, 并将其置于窗口边缘以构建壁 (图 2E)。该解决方案固化迅速, 保持灵活性, 使镜头或目标自由移动。</li>
	<li>当准备好进行成像时, 卸下海绵并将浸入式液体放入窗口中进行2P 成像 (图 2F)。将稳定的动物转移到2光子显微镜的暗室内, 并将2P 成像窗口直接置于透镜下。将镜片仔细地放到成像窗口中。</li>
</ol>
5. 注射第一荧光染料和基线成像
<ol>
	<li>在盐水中制备0.5 毫升的罗丹明 B 异硫氰酸酯-葡聚糖 (4 毫克/毫升平均分子量 ~ 70 kda)。用溶液填充1毫升无菌注射器, 并将注射器连接到先前安装的导管。 
	注意:建议在使用前准备荧光染料溶液。</li>
	<li>通过使注射器非常缓慢地注入第一个染料 (图 3B)。首先, 用目镜确定感兴趣的区域。使用电荷耦合器件 (CCD) 相机获得一个明亮的表面血管模式的图像, 在较低的放大率作为一个地标图像。切换到激光扫描模式, 然后打开2P 成像软件, 用于收集图像和线扫描数据。</li>
	<li>选择合适的2P 激光激发波长、功率和荧光通道 (第一染料的红色通道) 与成像组织中使用的荧光相匹配, 然后执行活体成像 (图 3E)。在整个过程中, 把动物放在加热垫上。</li>
</ol>
6. C7 挫伤损伤使用莉萨设备
<ol>
	<li>根据先前建立的协议25,26, 使用路易斯维尔伤害系统设备 (LISA) 装置执行 C7 中线钝挫伤损伤。<ol>
		<li>简言之, 将动物放在丽莎台上进行校准。</li></ol></li></ol>在选择零点设置和调整组织位移 (在本例中为 0.800 mm) 后, 单击该软件的 "运行实验" 按钮以触发冲击器释放并造成损伤。
		<li>受伤后, 将另一小片盐水浸泡海绵在裸露的硬脑膜上, 以保持其滋润。</li>
	
	
	<li>重复步骤4.3 和 re-perform在体内在与先前注入的红色染料 (图 3C和 #38; F) 可见的同一区域上进行成像。</li>
	<li>将动物放回手术台上, 并在 IACUC-IUSM 协议后, 用适当的麻醉使动物镇静。</li>

7. 注射第二荧光染料和损伤后的成像
<ol>
	<li>准备0.5 毫升异硫氰酸荧光素-葡聚糖 (4 毫克/毫升, 平均分子量〜 70 kDa) 在生理盐水中相同的5.1。用1毫升无菌注射器填充溶液, 并与先前安装的导管连接。</li>
	<li>将稳定的动物放回2P 显微镜暗室内, 映像同一个区域与第一染料的红色通道和第二染料的绿色通道 (图3D 和 #38; G)。</li>
	<li>在成像结束时, 从脊柱稳定装置中释放出大鼠, 清洗琼脂壁。</li>
</ol>
8. 动物祭祀
<ol>
	<li>成像后, 牺牲的老鼠 transcardial 灌注协议27。收集脊髓标本, 并修复4% 粉煤灰。</li>
</ol>
9. 离线数据分析: 船舶直径的量化
<ol>
	<li>将图像文件传送到工作站进行离线分析。</li>
	<li>打开 ImageJ 并选择 "文件", 然后选择以前保存的原始数据并打开相关的单个图像文件 (图 4B)。</li>
	<li>通过选择 "分析" 后跟 "设置刻度" (图 4C) 来校准图像。使用图 4A中显示的公式计算以 "像素距离" 为单位的值。2光子光学透镜的标定决定了方程中的缺省值。"opticalZoom" 的值在与单个图像文件 (图 4B) 关联的 Excel 可扩展标记 Languagefile (XML 文件) 中找到。</li>
	<li>绘制垂直于容器的长轴的直线 (图 4D1和 #38; E1), 然后选择 "分析", 后跟 "测量"。容器直径的测量显示在结果窗口中 (图 4D2和 #38; E2)。在容器中重复3次以获得平均值。</li>
</ol>
10. 离线数据分析: 红细胞 (红细胞) 速度的量化
<ol>
	<li>将行扫描文件传输到工作站进行分析。</li>
	<li>启动 ImageJ 软件, 然后选择 "文件", 然后选择以前保存的原始数据, 然后打开具有扩展名 "。</li>
	<li>打开 "图像", 然后选择 "堆栈", 然后是 "图像堆叠"。将所有的文件转换为单个图像堆栈 TIFF 文件。</li>
	<li>启动 Matlab 软件, 点击 "打开", 选择 "LSPIV_parallel" 代码文件。注: 可在 https://sourceforge.net/projects/lspivsupplement/files/18中下载用于 LS-PIV 的 Matlab 代码</li>
	<li>选择以下命令: "运行" 和 #62; "更改文件夹" 和 #62; "动脉"。选择在10.3中生成的图像堆栈 TIFF 文件。</li>
	<li>键入 "Y" 并按 Enter 键。</li>
	<li>将光标分别放在图像的左侧和右侧, 程序开始处理数据。</li>
	<li>在程序结束时, 输入2值以计算最终读取: "pixel_meter 转换值" 和 "扫描时间转换值"。这两者都可以在与行扫描数据相关的 XML 文件中找到。最后的值被表示为每秒毫米单位的速度的平均值和标准偏差 (毫米/s)。</li>
</ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+in+vivo+imaging+in+the+mouse+spinal+cord+using+an+implanted+chamber.">Chronic in vivo imaging in the mouse spinal cord using an implanted chamber.</a> Nat Methods. 9 (3), 297-302 (2012).</li><li>Evans, T. A., Barkauskas, D. S., Myers, J. T., Huang, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+axonal+interactions+with+microglia+and+macrophages+in+a+mouse+dorsal+column+crush+injury.">Intravital imaging of axonal interactions with microglia and macrophages in a mouse dorsal column crush injury.</a> J Vis Exp. (93), e52228 (2014).</li><li>Davalos, D., Akassoglou, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+the+mouse+spinal+cord+using+two-photon+microscopy.">In vivo imaging of the mouse spinal cord using two-photon microscopy.</a> J Vis Exp. (59), e2760 (2012).</li><li>Davalos, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+in+vivo+imaging+of+densely+populated+glia,+axons+and+blood+vessels+in+the+mouse+spinal+cord+using+two-photon+microscopy.">Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy.</a> J Neurosci Methods. 169 (1), 1-7 (2008).</li><li>Shen, Z., Lu, Z., Chhatbar, P. Y., O'Herron, P., Kara, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+artery-specific+fluorescent+dye+for+studying+neurovascular+coupling.">An artery-specific fluorescent dye for studying neurovascular coupling.</a> Nat Methods. 9 (3), 273-276 (2012).</li></ol>

作者没有什么可透露的。

对血管学研究的一个挑战是技术上的限制, 因为传统的技术主要局限于死后样本的血管结构变化。这本小说的活体成像方法, 使动态测量血流量和相关参数 (速度和血管直径) 使用 2 p LSM 在活鼠。它也允许重复检查在同一组的船只在不同时间点后, 挫伤 SCI。以前的2光子显微成像技术无法捕获创伤血管结构由于一个单一的示踪剂泄漏。我们的双色设计使动态血管成像的创伤模型。此外, 这种方法的灵活性提供了一个机会, 以产生一个时间空间剖面的急性血管变化后 SCI。
在我们的 in 体内双色成像方法中有几个关键步骤. 首先, 在延时成像之前, 确保脊髓的物理稳定性是至关重要的, 尤其是减少呼吸运动伪影。我们设计的脊柱钳形, 以提高脊柱的高度, 在稳定的轻微。因此, 与呼吸动物相关的脊髓运动可以大大减少 (图 1D-f, 2B)。建议在每一次成像会议开始前检查脊髓的稳定性。如果脊髓不能达到稳定, 则应调整脊髓钳的对准和松紧。第二, 外周组织 (骨骼、肌肉层和皮肤) 出血到成像窗口可能会受到污染的看法。为顺利成像会议, 海绵和组织胶粘胶应适用于周围组织, 以有效预防。第三, 我们选择的荧光染料的大小与白蛋白 (66 kDa) 相似, 这是主要的高分子量血浆蛋白。在稳态条件下, 染料主要保留在类似白蛋白28的血管内。损伤后, 染料通过破坏的内皮结构, 并泄漏到软组织, 导致显着增加的荧光强度的外围区域的血管 (图 3F-g)。此外, 我们选择外颈静脉插管有两个原因。首先, 它可以提供在任何时候的实验, 可持续的传递路线。其次, 它可以作为未来治疗注射的途径。
虽然我们的体内双色方法能够为创伤性血管成像研究提供一个新的场所, 但必须解决有关此技术的一些告诫。目前, 该技术的目的是评估血管的变化在2时间点 (基线和1后的时间点), 但它是可行的切换到多个时间点, 如果有额外的荧光染料和渠道可用。虽然有几项研究使用植入玻璃窗口为慢性活体成像, 他们没有能提供基线信息在同一艘船在创伤损伤以后19,29,30, 31,32。不像这些研究, 我们的窗口是一个 no-glass 窗口。这是方便的前后成像, 但它可能是一个挑战, 为长期观察重建窗口。我们未来的研究工作是对慢性影像技术的改进。血管系统由多种血管类型 (动脉、静脉、毛细血管) 组成, 在形态学和功能方面各有不同。在成像过程中, 血管类型的分化有助于梳理出明显的血管变化规律。上面的协议依赖于根据形态学和速度来识别容器的观察者;然而, 可以很容易地添加一种动脉特定的染料, 以便在容器类型33之间提供更明确的分类。
这项技术不仅限于评估挫伤和其他创伤模型, 如挤压伤和辐射损伤, 但也研究的重点是破坏 BSCB, 以及血管通透性的变化。除 SCI 外, 它还可用于研究其他神经退行性疾病, 如肌萎缩侧索硬化 (ALS) 和多发性硬化 (MS) 后的血管变化。此外, 它可以可的转基因动物模型研究动态神经血管相互作用。作为一种强有力的筛选工具, 未来的研究可以利用这里描述的成像技术来评估脊髓损伤的治疗效果。
总之,在体内双色方法是一种可靠的、real-time 的、体内方法, 用于评估动态血管变化, 这是对颞空间血管剖面的表征和对治疗的筛选减少继发性损害后 SCI。

外伤性脊髓损伤 (SCI) 是一种常见的损伤, 导致运动、感觉和自主功能受损。根据国家脊髓损伤统计中心 (和) 在 2016年, 大约有28.2万人受影响, 而其中69% 个主要是由于交通事故或瀑布1。这些病人往往需要重症监护;然而, 目前尚无有效的治疗方法。因此, 迫切需要新的有效的科技战略。
SCI 主要分为两个阶段: 原发性损伤和继发性损伤。主要损伤包括在撞击部位引起出血性坏死的物理侮辱2, 其次是一系列继发性损伤事件, 如炎症、细胞凋亡和残余轴突鞘, 逐步导致到形态学和功能缺陷的扩展3,4,5,6。出血是第一个可见的损伤迹象, 表明在 SCI7,8的急性阶段立即发生血管中断。旨在减少早期血管损伤的神经保护策略可以改善患者的康复, 但这需要更好地了解早期后血管事件的病理生理机制。
尽管以往的研究采用各种方法研究脊髓血管, 但仍有明显的局限性。最共同的缺点是只研究验尸样本, 例如, 氢清除9, 显影10, microangiogram8, 血管腐蚀铸造11, 和免疫组织化学12 ,13。虽然激光多普勒血提供无创 real-time 监测脊髓血流14, 它无法区分血管系统和检测血管形态学变化。动态对比增强 mri (DCE-mri) 也是无创的, 但它生成低分辨率的图像, 并需要昂贵的基础结构15。
虽然在体内使用2光子激光扫描显微镜 (2 p-LSM) 进行成像, 但已开发用于研究皮层中的 vasodynamics16、17、18, 但数量有限的研究显示的血管变化后, SCI. 唐 et al. 在半模型19中显示了病变部位边缘的血流变化, 但挫伤损伤后的影像更具挑战性, 原因有二。首先, 传统的玻璃光学窗口在损伤站点不会承受机械冲击和保持功能为成像。其次, 由于出血引起的软组织渗漏, 导致后成像困难。
在这里, 我们提出了一种新的双色成像方法, 允许成像相同的个别船只在前后的时间点。此外, 它提供了一个时间空间剖面的血管动态变化后, 挫伤 SCI。它还有可能在多个后时间点成像。本协议可直接应用于转基因动物研究神经血管的相互作用。

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an in vivo duo-color method for imaging vascular dynamics following contusive spinal cord injury

脊髓损伤 (SCI) 在损伤部位引起明显的血管破坏。血管病理立即发生在 SCI 之后, 并持续贯穿于急性损伤阶段。事实上, 内皮细胞似乎是第一个死亡后, 挫伤 SCI。早期血管事件, 包括血-脊髓屏障 (BSCB) 的通透性增高, 引起性水肿, 并导致由复杂损伤机制造成的二次损伤。因此, 靶向血管破坏可能是减少继发性损伤的主要策略, 从而导致组织学和功能损伤。以往的研究大多是在尸检样本上进行, 无法捕捉血管网络的动态变化。在本研究中, 我们开发了一个体内双色双光子成像法来监测挫伤 SCI 后的急性血管动态变化。这种方法可以检测血流, 血管直径和其他血管病变在同一大鼠和后的不同地点。总的来说, 这种方法为研究血管动力学提供了一个绝佳的场所。

该方法能够监测体内在个别血管和外伤后脊髓血管的动态变化。首先, 通过颈外静脉安装导管, 为后续的荧光染料注射提供通道 (图 1A-C,图 3)。在第二步中, 使用专用设备来稳定暴露的 C5-C7 (图 1D-f,图 2AB)。这一稳定步骤可以消除呼吸伪影, 并提供稳定的成像。继椎板切除术 (图 2C) 后, 下一步是在 C5-C7 上安装2P 成像窗口 (图 2D-f)。在脊柱成像窗口周围减少周围组织出血是成功的血管成像的关键。下面的步骤是通过上述导管将罗丹明-葡聚糖荧光染料 (红色) 注入地标, 并将血管网络映射为基线 (图 3A-b, E)。在 C7 中线挫伤损伤与适度严重性后, FITC 葡聚糖 (绿色) 在所需的后时间点 (图 3A和 #38; D) 中引入。双色法的妙处在于, 当第一种染料因损伤而泄漏到软组织时, 仍然可以使用第二染料来检测血管结构 (图 3G)。
在成像过程中, 建议将动物放在加热垫上, 在麻醉诱导后保持身体核心温度。
采用双色法, 可以测量和计算单个血管的直径和红细胞流速 (红细胞速度)。对于直径, 你可以使用 ImageJ 测量容器在其最大直径3重复校准后 (图 4)。对于速度, 用 matlab 程序 (matlab R2013a) 测量线扫描图像以计算红细胞速度 (图 5)18。根据形态学、血流速度和血管直径, 血管可分为2类: 动脉和静脉 (见表 1)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56565/56565fig1.jpg"> 
图 1.颈静脉插管和脊柱稳定。 (a)用于定位颈外静脉的示意图。(B) 由21口径针头制成的专用导管。该尖端是地面平坦和焊接的一块2毫米尖端从另一个21口径的针头切断。(C)导管的示意图。远端先结扎, 其次为近端导管稳定, 最后用针与血管 (血管结扎, 蓝色箭头) 固定在一起。(D)修改后的脊柱稳定器的图像。显示在椎板切除术前和挫伤 SCI (F)之后的 C5-C7 窗口。<a href="//ecsource.jove.com/files/ftp_upload/56565/56565fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56565/56565fig2.jpg"> 
图 2.光学窗口安装 step-by 步骤示意图。 (A)步骤 1: 通过沿中线切割皮肤和肌肉来暴露脊椎。(B) 步骤 2: 脊柱稳定。(C)步骤 3: 椎板切除术。(D)步骤 4: 通过放置一块盐水浸泡的海绵保持脊髓的水分。(E)步骤 5: 用无菌海绵和 vetbond 密封间隙。干燥后, 在窗口边缘建立一层琼脂壁。(F)步骤 6: 当准备好进行成像时, 卸下海绵并将浸入式流体置于2P 成像窗口中。<a href="//ecsource.jove.com/files/ftp_upload/56565/56565fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56565/56565fig3.jpg"> 
图 3.在体内双色方法过程 step-by 步骤. 整个过程由5步骤(A)组成。在步骤1和步骤2之后, 一对具有近似 70 KDa 大小的葡聚糖示踪剂, 在 (B和C) 和挫伤 SCI (D)之后, 依次注入一组用于标记脊髓血管的。(E)-(G)代表2P 图像在步骤3到步骤5中显示脊髓血管。白色箭头指向第一波红色染料泄漏区域 (F和G), 绿松箭头显示第二波绿色染料泄漏(g)。缩放栏 = 50 µm.<a href="//ecsource.jove.com/files/ftp_upload/56565/56565fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56565/56565fig4.jpg"> 
图 4.脊髓血管直径的采集和定量。 在准备之后, 单个图像文件是在2P 显微镜下获取的, 同时还有校准值的 XML 文件(B)。(A)等式显示基于光学变焦值的 "每微米像素" 的计算。在 ImageJ (C)中校准后, 容器直径在(D1)和(E1)损伤之前的纵向轴上测量3点。(D2)和(E2)显示测量值。(F)基线和30分钟后的船只直径的定量。缩放条 = 50 µm. 数据显示为平均值± SD, ** 和 #60; 0.0001, 双尾配对 t 检验. <a href="//ecsource.jove.com/files/ftp_upload/56565/56565fig4large.jpg" target="_blank">请点击这里查看更大版本的这个数字。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56565/56565fig5.jpg"> 
图 5.脊髓血管速度的采集和定量。 在2P 显微镜下获取线扫描图像文件, 计算单船速度。(a)一个选定的容器和方法的例子来评估血管红细胞的速度。(B)行扫描图像的动脉示例以及计算速度的相应绘图文件, 以及静脉(C)的示例。<a href="//ecsource.jove.com/files/ftp_upload/56565/56565fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>动脉</td>
			<td>静脉</td>
		</tr>
		<tr>
			<td>形态</td>
			<td>直、光滑、厚容器壁</td>
			<td>树枝, 粗糙的边缘</td>
		</tr>
		<tr>
			<td>血流速度</td>
			<td>快速</td>
			<td>缓慢但变化</td>
		</tr>
		<tr>
			<td>直径</td>
			<td>30-80 µm</td>
			<td>100-250 µm</td>
		</tr>
	</tbody>
</table>
表 1: 识别船只类型的标准

Watch this Scientific Journal Video about An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury at JoVE.com

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

在体内挫伤脊髓损伤后影像血管动力学的双色法

我们介绍了一个在体内成像方法使用两种不同的荧光染料跟踪动态脊髓血管变化后, 挫伤脊髓损伤成人大大鼠。

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

an-vivo-duo-color-method-for-imaging-vascular-dynamics-following

Research

JoVE Journal

Neuroscience

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

9.3K 次观看.  Indiana University School of Medicine. 我们介绍了一个在体内成像方法使用两种不同的荧光染料跟踪动态脊髓血管变化后, 挫伤脊髓损伤成人大大鼠。

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

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 ...

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 ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

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 ...

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) ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

神经干细胞向大鼠和小鼠大脑的动脉内传递:对脑缺血的应用

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Induction of Complete Transection-Type Spinal Cord Injury in Mice

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, such as cell transplantation methods, are being researched to overcome the debilitating disabilities arising from SCI. Most pre-clinical animal trials are conducted in rodent models of SCI. While rat models of SCI have been widely used, mouse models have received less attention, even though mouse models can have significant advantages over rat models. The small size of mice equates to lower animal maintenance costs than for rats, and the availability of numerous transgenic mouse models is advantageous for many types of studies. Inducing repeatable and precise injury in the animals is the primary challenge for SCI research, which in small rodents requires high-precision surgery. The transection-type injury model has been a commonly used injury model over the last decade for transplantation-based therapeutic research, however a standardized method for inducing a complete transection-type injury in mice does not exist. We have developed a surgical protocol for inducing a complete transection type injury in C57BL/6 mice at thoracic vertebral level 10 (T10). The procedure uses a small tip drill instead of rongeurs to precisely remove the lamina, after which a thin blade with rounded cutting edge is used to induce the spinal cord transection. This method leads to reproducible transection-type injury in small rodents with minimal collateral muscle and bone damage and therefore minimizes confounding factors, specifically where behavioral functional outcomes are analyzed.

This protocol describes how to create a precise laminectomy for induction of stable transection-type spinal cord injury in the mouse model, with minimal collateral damage for spinal cord injury research.

小鼠完全转型脊髓损伤的诱导

Spinal cord injury (SCI) largely leads to irreversible and permanent loss of function, most commonly as a result of trauma. Several treatment options, ...