作者要感谢 Yuliya Naumchuk (芝加哥大学) 和 Agne žiniauskaitė (Experimentica Ltd.) 的优秀技术和录像支持。Dr. 卡洛特尼斯卡娅的研究项目是由 Dr. 的约翰 P 和苔蕾丝马尔卡希在美国芝加哥大学的眼科教授的支持。

所有的动物都是按照帕特声明, 为使用动物在眼科和视力研究和欧共体指令 86/609/EEC 动物实验, 使用的协议批准和监测的动物实验委员会芬兰。
1. 激光诱导小鼠 CNV 模型5
<ol><li>从宏观上检查动物的眼睛是否有异常。</li>
	<li>称量鼠标。</li>
	<li>计算和准备适当数量的麻醉剂使用, 根据动物的重量,例如medetomidine (1 毫克/千克), 氯胺酮 (75 毫克/千克) 和蒸馏水 (0.9% NaCl 溶液) 的比例为 1:1. 5:2. 5, 或氯胺酮 (40-75 毫克/千克),嗪 (5 毫克/千克), 蒸馏水 (0.9% 氯化钠溶液) 的比率为 1:2. 5:1;对于20克老鼠, 注入0.1 毫升的混合物。</li>
	<li>注射麻醉腹腔。</li>
	<li>把老鼠放回笼子里, 等到动物被麻醉。确认鼠标没有踏板反射就能正确麻醉。</li>
	<li>确保使用激光安全个人防护设备。</li>
	<li>打开一个狭缝灯和 532 nm 二极管激光器。</li>
	<li>将鼠标从笼子里取出, 放在暖气垫上。</li>
	<li>应用一滴卡进行瞳孔扩张。等待3-5 分钟的全 (3 毫米) 瞳孔扩张。</li>
	<li>将鼠标放在狭缝灯的舞台上。</li>
	<li>将一滴眼科液体凝胶放在片上, 扁平角膜。</li>
	<li>用中心的视神经头定位鼠标的眼睛。</li>
	<li>将激光功率设置为100兆瓦, 持续时间为100毫秒, 光斑尺寸为50µm。</li>
	<li>将激光束聚焦于视网膜色素上皮 (RPE)。</li>
	<li>通过避免视网膜血管理想地在 4, 8, 和12点周围的视神经, 分别做三激光射击入一只眼睛。在所有激光照射后检查眼底, 因为没有视网膜出血。对侧眼作为非激光的控制。</li>
	<li>丢弃片并将鼠标放回加热垫上。</li>
	<li>用一滴 PEG 凝胶滴在两只眼睛上。</li>
</ol>2. SD-OCT 6,7
<ol><li>将鼠标放置到啮齿动物的对齐阶段, 并固定头部。</li>
	<li>将 SD-OCT 系统 (例如, Bioptigen/莱卡 Envisu R2200) 的镜头对准, 以使用 X 和 Y 级控制器对活体进行成像。</li>
	<li>执行 sd oct 扫描以验证赫膜的断裂: 一旦 sd oct 扫描整个眼睛, 在激光站点上手动移动参考线。赫膜的断裂应在激光区域中清晰可见 (请参见图 1)。</li>
</ol>3. 荧光素血管造影7,8,9
<ol><li>取出鼠标与持有人, 并将其放在 FA 系统 (例如, 海德堡 Spectralis HRA2)。</li>
	<li>聚焦在眼睛眼底的激光烧伤区域, 用红外线反射模式与视神经的头部在观察窗口中间。</li>
	<li>注射0.1 毫升的5% 荧光素钠盐为20克鼠皮下或腹腔。</li>
	<li>关注脉络膜水平。</li>
	<li>从脉络膜的焦点水平进行图像。</li>
	<li>重新聚焦在视网膜水平上, 并采取图像。</li>
	<li>等待三十年代, 重复步骤 3.4-3.6。</li>
	<li>将鼠标从支架上取出, 并将其放在加热垫上。</li>
	<li>反向麻醉的2拮抗剂的 medetomidine, 唑 (0.5 毫克/千克, ip), 或等待动物恢复从麻醉。</li>
	<li>在后续的天数5、10和14中, 在麻醉动物中重复在体内 SD OCT 和 FA 成像。</li>
</ol>4. CNV 分级
<ol><li>赫在0天激光后立即采取的 OCT 图像和脉络膜 FA 图像的损伤等级如下: 0-赫的膜未损坏 1-赫膜的成功损坏</li>
	<li>通过比较一系列视网膜 FA 图像中荧光素信号的动态观察, 发现有渗漏的激光斑点 CNV 的存在, 如下所示: 0-视网膜的正常出现 0.5-渗漏的微弱的染色 1.0-漏的 CNV 区域 注: 使用 oct 成像, 以进一步确认 CNV 或可疑 FA 中存在的 intraretinal 流体在 OCT 图像将建议 CNV 评分。</li>
</ol>5. 视网膜厚度测量
<ol><li>使用自动分割软件进行视网膜厚度测量。确保视网膜总厚度被认为是从神经纤维层到 rpe (健康测量点) 的所有层的厚度, 或者是连接 rpe (激光站点) 周围的假想线 (也请参见图 7)。</li>
</ol>

<ol>
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作者 Symantas Ragauskas, 博士是 Experimentica Ltd. 的一名雇员 (研究科学家) 和股东, 提供合同研究服务, 采用了本文所用的临床前 CNV 模型。
作者 Eva Kielczewski 是一个员工 (研究应用工程师, oct) 的徕卡系统, 生产的 SD oct 在本文中使用。
作者约瑟夫万斯是一个雇员 (NA oct 销售总监) 的徕卡微系统, 生产的 SD oct 体系在本文中使用。约瑟夫. 万斯也是 Spective 的总裁兼董事总经理。
作者西蒙卡洛特尼斯卡娅博士是 Experimentica Ltd. 的顾问首席科学官和股东, 这是一家提供合同研究服务的临床前合同研究机构, incl. 了本文所用的临床前 CNV 模型。西蒙卡洛特尼斯卡娅, 博士也是 & p 科学, LLC, 生命科学咨询公司的首席执行官, 并担任 Dr. 约翰 p 和苔蕾丝 e. 马尔卡希在 Stritch 医学院芝加哥大学的眼科教授。这项安排的条款已根据其利益冲突政策, 由美国芝加哥大学学院审查和批准。
作者 Giedrius Kalesnykas, 博士是 Experimentica Ltd. 的一名雇员 (CEO) 和股东, 提供合同研究服务, 采用本文所用的前 CNV 模型。

多模态成像为 CNV 病理评价提供了有价值的工具。在这里, 我们提出了一个成像协议, 包括 FA, SD OCT, 自动分割的快速, 重现性, 可靠的评价 CNV 病理。经激光管理后, 赫膜断裂。此外, 在这个阶段使用 SD OCT 也可以立即可视化可能的 intraretinal 和视网膜出血, 这可能混淆结果的解释。根据 FA 图像的荧光素信号对视网膜渗漏进行分级。使用 SD OCT 提供了更详细的描述 CNV 病理。此外, 纵向 SD OCT 分析在不同的时间点在整个随访期强调的时间差异的病理, 将仍然难以捉摸, 如果依赖于 FA 单独。
用自动分割法测量视网膜总厚度。人工测量 CNV 诱导的视网膜厚度。对脉络膜 flatmount 的组织学评价进行了验证, 用图像分析软件图像 J 对新生血管面积进行了测量。
可视轴的正确透明度对于所提出的协议的成功执行至关重要。角膜干燥和白内障的形成是诊断的主要因素。因此, 一旦鼠标被麻醉, 眼睛应该不断水合与人工泪液或凝胶保持适当的角膜水化。建议的协议应在10分钟内优先进行麻醉诱导。延长麻醉时间可能导致白内障的形成, 并防止在体内成像。
所描述的协议仅限于基于视网膜水平血管渗漏的 CNV 进展的观察分级。可以使用海德堡 Spectralis 软件增加对视网膜渗漏的定量评估, 允许对泄漏区域进行划定, 并提供有关区域的定量数据。此外, 苏莱曼和同事 (2015) 最近提出了一个计算 CNV 体积从在体内获取 OCT 图像使用椭球方法10。椭圆形模型最有可能引入偏倚, 以估计病变体积的 CNV 在大多数情况下有一个不规则的形状。然而, 从 OCT 图像的共焦分析和建议的椭球量化的体积测量之间的高度相关性, 提供了证据证明该方法是定量评估 CNV 卷的重要工具10.
最后, 我们认为, 不同的在体内成像模式的组合, 以及自动分割和组织学分析, 提供了可重复和可靠的评估 CNV 病理学临床前研究。该方法对于概念治疗干预研究的证明尤其有用。

第一次成功的尝试模仿人类 CNV 在啮齿目动物的病理证实了近三年前与氪激光在长埃文斯鼠1。此后, 氪激光被用来打破赫的细胞膜在最流行的鼠标应变, C57BL/6J2,3,4。用 FA 和组织学染色验证了 CNV 诱导的成功率。快速发展的无创成像模式, 如 OCT, 促进了啮齿动物的临床前模型领域的增长。在同一眼的多个时间点, 监测视网膜形态变化的能力大大降低了动物的使用, 提高了实验研究的效率。组织学评价 CNV 病变是相当直接的, 并要求标签的异常血管生长周围的激光管理, 图像采集, 和面积/体积估计使用图像分析软件。相比之下,在体内成像模式引入了对 CNV 病理及其解释的更复杂的分析。
在这里, 我们提出了一个简单的和相对快速的方法来评分的诱导, 进展, 和回归的 CNV 使用 FA, SD OCT, 和自动分割方法的小鼠激光诱导 CNV 模型。

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:275px;">Medetomidine (commercial name Domitor)</td>
			<td style="width:123px;">Orion</td>
			<td style="width:344px;">Vnr 01 56 02</td>
			<td style="width:177px;">Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Ketamine</td>
			<td>Intervet</td>
			<td>Vnr 51 14 85</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">0,9% NaCl</td>
			<td>B Braun</td>
			<td>357 0340</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Xylazine (commercial name Rompun vet)</td>
			<td>Bayer</td>
			<td>vnr 14 89 99</td>
			<td>Anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tropicamide</td>
			<td>Santen</td>
			<td>Vnr 04 12 36</td>
			<td>Mydriatic agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Viscotears</td>
			<td>Alcon</td>
			<td>Vnr 44 54 81</td>
			<td>Lubricant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Systane</td>
			<td>Alcon</td>
			<td>&#160;-</td>
			<td>Lubricant</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5% Fluorescein sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>F6377-100G</td>
			<td>Fluoresent agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Atipamezole (commercial name Antisedane)</td>
			<td>Orion</td>
			<td>Vnr 47 19 53</td>
			<td>Anesthesia</td>
		</tr>
	</tbody>
</table>

in vivo multimodal imaging and analysis of mouse laser-induced choroidal neovascularization model

激光诱导脉络膜新生血管 (CNV) 是模拟年龄相关性黄斑变性 (AMD) 的湿型的一个良好建立的模型。在本议定书中, 我们的目标是引导读者不只是通过技术考虑产生激光诱导的损害, 以触发新生血管的过程, 而是侧重于强大的信息, 可以获得从多式联运纵向在体内的整个随访期内的成像。
激光诱导的小鼠 CNV 模型是由二极管激光管理产生的。多模态在体内成像技术被用来监测 CNV 的诱导, 进展和回归。首先, 在激光后立即进行光谱域光学相干层析成像 (SD OCT) 来验证赫膜的断裂。随后使用荧光素血管造影 (FA) 进行的活体成像证实了赫膜从脉络膜水平获得的序列图像的成功损伤。CNV 增殖和回归的纵向后续在天 5, 10 和14以后使用了 SD OCT 和 FA 激光执行。提出了从 FA 图像中 CNV leasions 的简单可靠的分级方法。自动分割的测量总视网膜厚度, 结合手动口径应用测量视网膜厚度的 CNV 地点, 允许无偏见的评价存在水肿。最后, 利用 isolectin GS-IB4 染色对脉络膜 flatmounts 进行了 CNV 的组织学验证。染色是值, isolectin 阳性面积计算 ImageJ。
该协议特别适用于需要高通量样的 CNV 病理筛查的治疗研究, 因为它可以快速、多式、可靠地分类 CNV 病理和视网膜水肿。此外, 高分辨率 SD OCT 可以记录其他病理特征, 如视网膜或 intraretinal 液的积累。但是, 此方法不提供从 SD OCT 图像中自动进行 CNV 卷分析的可能性, 必须手动执行。

激光后立即出现气泡或视网膜出血并不总是可见的。因此, SD OCT 对于验证赫膜的损伤尤为重要。图 1显示了激光管理后在不同时间点的 OCT 成像的示例。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56173/56173fig1.jpg"> 图 1: 眼睛眼底的 OCT (VIP 图像) 显示在白色、绿色和红色圆圈中勾勒出的三激光区域.OCT B 扫描图像之前采取的激光 (基线), 立即激光后, 以验证赫的膜断裂 (0 天, 箭头点的损害网站), 和 5, 10 和14天后, 激光管理。从白色 (第一排图像) 中所勾画的区域可以看出, CNV 在以后的 timepoints 中没有发展。在绿色和红色概述的区域开发了 CNV, 在跟随的天5被发现了。然而, 在10和14天 timepoints, 这些 CNV 病变退化。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
图2和 3显示了使用 FA 的串行成像, 它证实了赫的细胞膜在0天的所有三个斑点中的 C57BL/6jRj 老鼠的成功损伤。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56173/56173fig2.jpg"> 图 2: 连续 FA 成像每年二十年代 (图像1到 18) 在脉络膜水平后立即激光管理.图像中的白色箭头1指向激光站点, 显示荧光素在以后的 timepoints (图18中为白色箭头)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig2large.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/56173/56173fig3.jpg"> 图 3: 连续 FA 成像每年二十年代 (图像1到 18) 在视网膜水平后立即激光管理.CNV 地区, 有荧光素渗漏和评分 1 (漏 CNV) 是指出的白色箭头的图像18。注意在 FA 成像 (图8和11中的白色箭头) 期间, timecourse 的强度, 以及荧光素阳性区的增加。图18中白色轮廓的两个区域被分级为有微弱的 FA 信号 (等级 0.5)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
除了 CNV 病理学的分级外, SD OCT 还可以在病变部位显示更多的信息,例如, 如, 视网膜液的存在, 水肿, 和 CNV 回归。图 4显示了激光诱导的 CNV 在小鼠体内的主要病理特征。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56173/56173fig4.jpg"> 图 4: CNV 病理学的光谱域光学相干断层成像.SD OCT 提供了一个详细的 CNV 病理在视网膜组织, 从这些代表性的图像可以看到疤痕组织, CNV 形成, 和液体的积累。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
黄斑水肿是人体内湿型 AMD 的主要病理标志之一。在激光诱导的 CNV 模型中, 可以使用自动分割来评估视网膜厚度。人工测量选定的激光的地点是需要测量视网膜厚度在 CNV。图 5显示了自动分段后生成的报告的示例。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56173/56173fig5.jpg"> 图 5: 视网膜厚度的定量.自动分割法测量视网膜厚度<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
使用自动分割是一个快速的方法来提供视网膜厚度的概述 (表 1)。图 6A和6B分别显示了来自健康视网膜区域和 CNV 病理的视网膜区域自动分割的典型例子。尽管在区分个别视网膜层方面存在微小的不精确性, 但总的来说, 该软件能可靠地识别色素小鼠的视网膜总厚度。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56173/56173fig6.jpg"> 图 6: 视网膜层的自动分割.自动分割健康的视网膜区域 (A) 和包含 CNV 的视网膜区域 (在图像B中为星号)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了评估激光地区的视网膜厚度, 每个激光区域都是人工测量的: 视网膜总厚度被认为是所有层的厚度, 从神经纤维层到连接 RPE 周围的假想线损坏 (图 7和表 1)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>地区</td>
			<td>5天</td>
			<td>10天</td>
			<td>14天</td>
		</tr>
<tr>
<td>总视网膜厚度, μ m</td>
			<td>218±7。8</td>
			<td>220±7。2</td>
			<td>221±9。8</td>
		</tr>
<tr>
<td>激光面积1</td>
			<td>200</td>
			<td>204</td>
			<td>214</td>
		</tr>
<tr>
<td>激光面积2</td>
			<td>226</td>
			<td>217</td>
			<td>220</td>
		</tr>
<tr>
<td>激光面积3</td>
			<td>222</td>
			<td>223</td>
			<td>227</td>
		</tr>
<tr>
<td colspan="4">数据显示为 mean±SD</td>
		</tr>
</tbody></table>表1。总视网膜厚度和视网膜厚度在 CNV 站点在14天的后续, 由自动分割使用 inVivoDiver 软件 (v 3.0.8) 确定。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56173/56173fig7.jpg"> 图 7: 人工测量激光区视网膜厚度与 CNV 病理.黄线表明, 在激光管理的地方, 视网膜的总厚度从神经纤维层到假想的 RPE 层 (黑线)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
组织学上, CNV 病变的确认使用 isolectin GS-IB4 标记 (图 8A)。图像分析软件图像 J 被用来计算 CNV 病变面积 (图 8B)。
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56173/56173fig8.jpg"> 图 8: 组织学分析.脉络膜 flatmount CNV 病变的组织学染色 (绿色、 A) 可以使用图像 J (B) 中的阈值进行量化。A的刻度线为50μ m。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56173/56173fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model at JoVE.com

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

在体内小鼠激光诱导脉络膜新生血管模型的多模态成像与分析

在这里, 我们介绍了纵向的在体内成像在激光诱导的小鼠脉络膜新生血管形态学变化中的作用。

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

in-vivo-multimodal-imaging-analysis-mouse-laser-induced-choroidal

Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

9.0K 次观看.  Experimentica Ltd.. 在这里, 我们介绍了纵向的在体内成像在激光诱导的小鼠脉络膜新生血管形态学变化中的作用。

视频: In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

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

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

在中央小鼠中枢神经系统的干细胞移植的多模态成像

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

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 ...

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 &#181;m) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

Using this protocol, we were able to image 160 &#181;m thick brain sections from mice infected with the parasite Toxoplasma gondii, which enables visualization and analysis of the spatial relationship between the encysting parasite and the infected neuron.

神经元的3-D成像和分析感染<em&gt;在体内</em&gt;与<em&gt;弓形虫</em

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Laser-Induced Brain Injury in the Motor Cortex of Rats

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.

The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.

大鼠运动皮层中的激光诱发脑损伤

A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

切除小鼠神经神经纤维神经丝的成像与分析

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...