作者感谢 Buckwalter 实验室 (特别是托德. 彼得森博士) 提供老鼠模型和执行 dMCAO 和假手术。此外, 我们还要感谢 Invicro 的托马斯 Liguori 为他的技术援助与 VivoQuant 图像分析软件, Tim 道尔博士, 劳拉 Pisani, Frezghi 博士 Habte 从 SCi3 小动物成像设施在斯坦福的意见和协助开发这一成像协议, 和放射化学设施 (特别是钧博士公园) 为他们的帮助与综合 [11C] DPA-713。

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作者声明没有利益冲突。

该协议描述了使用 [11C] DPA-713-PET 对 dMCAO 和假鼠 neuroinflammation 进行量化的方法。TSPO PET 是目前最广泛研究的影像生物标志物, 用于可视化和测量 neuroinflammation。TSPO 表达是上调在脑胶质细胞炎症允许无创检测和量化的 neuroinflammation。此外, 它是一种高度翻译的技术, 使它成为一个宝贵的工具, 在临床和临床前的研究。该协议和代表性的结果突出了使用 [11C] DPA-713 PET 检测和监测中风和其他神经系统疾病在体内的 neuroinflammatory 改变的适用性。
本研究采用3月大的 C57BL/6 雌性小鼠进行 dMCAO 手术。这一模型的选择, 因为它导致一个高度可重现性梗塞局限于体感皮层, 提供了一个永久性局灶缺血的模型与其他中风模型 (如大脑中动脉, 低变异性)闭塞 (MCAO) 灯丝法)14。脑卒中模型的 PET 成像具有在大脑中包含一个内部参考区域的优势, 每个动物使用 ROIs 在对侧半球。由于手术后会产生一些炎症, 因此在研究设计中包括接受假手术的小鼠是很重要的, 即在没有动脉闭塞的情况下, 对脑膜进行开颅和手法治疗。单开颅手术可能会导致潜在的神经组织中断, 并引入病原体, 导致免疫应答独立于中风20。因此, 假手术后的一些炎症是预期的, 应该与 dMCAO 平行评估, 以排除仅因手术而导致信号的可能性。为了避免在 dMCAO 队列分析中包括没有中风的手术引起的炎症, mr 成像必须进行, 以确认成功的中风手术和梗塞的发展。MRI 也提供了一个结构参考框架, 这是必不可少的准确绘制梗死和对侧 ROIs。此外, 还需要准确的图像处理, 包括图像配准和 ROI 定义, 以确保可靠的量化。
当与 C-11 标记 radiotracers 进行 PET 和显影研究时, 必须牢记额外的限制。必须考虑短半衰期 (20.33 分钟) 的 C-11, 其使用一般限于研究机构, 现场回旋接入。必须事先确定适当的放射性运输路线、剂量管理和采集时间点, 并预先准备好实验工作流程的详细计划, 使团队能够快速有效地工作。这项研究的设计和建立已经概述, 以适应4小鼠的成像同时增加数据输出时, 使用 C-11 示踪剂。如果可能的话, 最好在 C-11 示踪器到达成像设备时, 让所有的老鼠空心化, 并在 CT 扫描中进行, 以确保注射前的放射性衰减最小。这一步步协议最好是由至少3位研究人员进行, 以便在重大放射性衰变之前快速插管、剂量测量、示踪剂注射、PET 扫描和脑切片。这需要两个人进行 PET 扫描和注射所有4只老鼠同时进行。在注射前开始 PET 采集的原因是为了保证血液和感兴趣区域的示踪剂分布的药代动力学和动力学得到准确和完全的捕获。许多步骤可能需要进行有力的训练和练习, 以确保实验的顺利运行。特别是, 这个协议依赖于成功的尾静脉插管的 C57BL/6 小鼠, 这可能是困难的, 因为深色的头发呈现在他们的尾巴上, 可能会变得更具挑战性的中风后发生或如果在多个时间点成像相同的老鼠.
PET 成像的另一个考虑因素包括仔细记录放射性剂量和残余活动测量, 包括确切的测量时间。这对于在扫描时注入剂量的精确衰减校正是必不可少的, 并且用于获得对每个 ROI 的示踪剂摄取量 (即% ID/g) 的精确测量。在扫描时, 要知道每只老鼠的放射性确切量, 以确保准确的图像分析。因此, 最好同步扫描仪计算机上的时钟和剂量校验仪, 以避免在使用 C-11 等短命同位素时出现错误。
精确的 PET 图像量化也可以被扫描仪的精度和设置所限制。因此, 为了确保准确量化的 PET/ct 图像, 重要的是进行质量控制检查的 CT 和 pet 部件的扫描仪。CT 质量控制检查包括 x 射线源调节, 暗/光, 和中心关闭设置校准。这些校准措施和正确的系统噪音, 必须在收购之前按照扫描仪制造商的建议执行。对 PET 扫描仪也应进行校准。这通常包括扫描 "标准/宠物幻影" 扫描, 其中包含已知的放射性浓度。在制定标准时, 最好使用与研究中使用的放射性同位素相同的放射性核素, 这一剂量与老鼠体内的一只小鼠相比, 与动物成像的采集参数相同。在本议定书中, 用放射性稀释的20毫升注射器作为标准, 随后的 PET 成像结果用于根据校准探测器测量的实际剂量计算校正系数。该校正比可应用于实验获得的成像数据, 以确保对 PET 图像感兴趣区域的示踪剂摄取进行准确量化。除了考虑扫描当天出现的任何背景活动外, 这也说明了放射性核素的正电子范围。由于剂量校验仪是这一校正因子生成的一个组成部分, 因此必须根据制造商指南定期校准该设备。
在进行体外显影时, 选择注射后安乐死的最佳时间点是很重要的, 以确保感兴趣区域的高信号到背景。三十分钟后注射是选择 [11C] DPA-713 显影使用在动态 PET 成像中获得的数据-即, 在体内动态口香糖作为指导, 同时也考虑到 C-11 的短半衰期和时间参与切片并在提取后暴露脑组织。考虑到这一点, [11C] DPA-713 显影必须在单独的一组小鼠上进行, 以允许注射更高的 [11C] DPA-713 剂量和30分钟的时间点用于麻醉下灌注和安乐死。在进行体外显影前对3-4 只小鼠进行小体内 PET 试验研究将有助于确定显影的最佳时间点。另外一个考虑的是体内显影是在注射后恢复小鼠, 还是将其麻醉直到安乐死。让他们麻醉模仿扫描的条件, 并确保放射性分布或排泄动力学不改变的恢复。此外, 这可以防止对小鼠的额外压力, 避免恢复和随后的诱导。最后, 一个有用的补充, 在前体协议将是评估的区域损害的脑切片用于显影通过免疫组化染色 (放射性衰变后), 以产生高分辨率图像的梗塞地点和体积。
由于使用基于 C-11 的跟踪器有限制, 因此可以很容易地修改此协议, 以便使用 F-18 (半衰期为109.77 分钟) 的 TSPO 跟踪程序, 这可能更适用于没有现场回旋加速器的位置。此外, 此协议还描述了使用4鼠标映像设置的情况。虽然这种高通量方法在使用 C-11 示踪器时是最佳的, 但对于使用单鼠标成像床的那些协议也可能进行修改。对本议定书所概述的技术进行周密的规划和一致的培训将导致使用 [11C] DPA-713 生成大量数据, 这可以很容易地用于探讨 neuroinflammation 在疾病表现中的作用和神经系统疾病的其他啮齿动物模型的进展。此外, 这项技术可以用来评估在体内对小胶质细胞的免疫调节疗法的反应。

中风是第五大死因, 也是美国1残疾的主要原因。缺血性中风是绝大多数这些病例 (87%), 发生在血液流向大脑的局部中断 (例如,由血块或脂肪堆积)。随后减少了对受影响地区的氧气和养分供应, 并启动了复杂的病理级联, 导致脑卒中核心 (梗塞) 内的神经元死亡, 除了周围地区。Neuroinflammation 是导致这种损害的途径中的一个关键组成部分, 包括居民脑免疫细胞和浸润性外周免疫细胞 (中性粒细胞、T 细胞、B 细胞和单核巨噬细胞), 认为这有助于破坏性级联2,3。活化小胶质细胞和巨噬菌是这 neuroinflammatory 反应的中心, 报告的有害和有益的影响后, 缺血性中风2。因此, 必须评估这些细胞在中风后的体内贡献。
PET 是一种强大的3维分子成像技术, 通过使用标记为正电子 (β +) 放射放射性核素的特定分子 (如11C、 13N), 可以在体内可视化生物过程, 15O 和18F。这种非侵入性方法比体外方法 (如免疫组化) 有许多优点, 因为它允许实时获取分子信息, 在活着的完整的主题, 并允许纵向调查。PET 成像的 TSPO, 一个标记的活化小胶质细胞和外周髓系体细胞, 提供了一种手段, 以量化和跟踪体内的先天免疫细胞反应, 并可用于评估中风后炎症和反应治疗干预。TSPO, 以前称为外周型苯二氮受体, 是一个 18 kDa 蛋白, 被认为是在胆固醇运输和合成神经甾体4的作用。此外, 证据表明, TSPO 参与了 neuroinflammation 和神经元存活5,6, 报告说, 在许多神经系统疾病包括中风7的表达增加,痴呆症8, 帕金森病9和多发性硬化症10。TSPO 位于外线粒体膜上, 在周围有高度表达, 特别是在类固醇相关组织 (如腺体) 中, 以及在心脏、肾脏和肺部10中可见的中层水平。然而在健康的大脑中, TSPO 水平低, 主要受限于胶质细胞6,11。在脑卒中观察到的神经元损伤后, 中枢神经系统 (CNS) TSPO 水平显著增加。这种观察到的上调 TSPO 可以被利用在体内图像 neuroinflammation, 表达水平提供了准确的炎症严重性指标。因此, 该方法的目的是准确量化 neuroinflammation 在小鼠脑缺血性中风模型中的作用, TSPO PET。
针对 neuroinflammation 的 PET 成像, 研制了多种 TSPO 示踪剂。在这里, TSPO-PET 成像被描述使用 [11C] DPA-71312, 一个有希望的第二代 TSPO 示踪器, 它显示了增强信号的噪声和较低的非特定绑定比历史上使用 [11C] PK11195 13.以此方法为例, 选择了 dMCAO 小鼠中风模型14。该模型包括颞侧开颅术和远端大脑中动脉的永久性结扎, 导致体感皮层局灶性缺血。这有利于临床前中风的研究, 由于缺血性损伤的高重现性和低死亡率相关的模型。到目前为止, TSPO PET 成像研究尚未在 dMCAO 啮齿动物模型中报道。然而, 以往的 PET 成像研究使用大脑中动脉闭塞 (MCAO) 模型, 一个更严重和可变中风模型, 在小鼠和大鼠, 报告 TSPO 表达增加从3天和峰值7天后中风15, 16,17,18。因此, 我们进行了6天后 dMCAO 的 PET 成像与高 TSPO 表达一致。[11C]在同侧 (梗塞) 和对侧半球评估脑 DPA-713 吸收。TSPO-PET 结合结构 MRI, 可精确划定梗死和对侧区域的兴趣 (ROIs)。在这里, 我们描述了基于 atlas 和 MRI 驱动的 ROI 方法来计算 [11C] DPA-713 的吸收。放射性在脾脏中的摄取量也被评估以调查组间炎症的周围水平。这种方法有可能为脑卒中和其他神经系统疾病的时空动力学和特定 neuroimmune 交互作用提供重要的洞察力。

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			<td height="21" style="height:21px;width:215px;">Inveon PET/CT scanner</td>
			<td style="width:89px;">Siemens</td>
			<td style="width:119px;">Version 4.2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">MRI scanner</td>
			<td style="width:89px;">Varian</td>
			<td style="width:119px;"></td>
			<td>7 Telsa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">ParaVision software</td>
			<td style="width:89px;">Bruker</td>
			<td>Version 6.0.1</td>
			<td>MRI operating software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">VivoQuant software</td>
			<td style="width:89px;">InVicro</td>
			<td>Version 2.5</td>
			<td>Image analysis software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Inveon Research Workspace software</td>
			<td style="width:89px;">Siemens</td>
			<td>Version 4.2</td>
			<td>Scanner operating software. Includes microQView, the post-processing managing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Dose calibrator</td>
			<td style="width:89px;">Capintech</td>
			<td style="width:119px;"></td>
			<td>CRC-15 PET</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Typhoon phosphor imager 9410</td>
			<td style="width:89px;">GE Healthcare</td>
			<td style="width:119px;">8149-30-9410</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Butterfly catheters</td>
			<td style="width:89px;">SAI Infusion Technologies</td>
			<td style="width:119px;">BFL-24</td>
			<td>27.5 G needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">1 mL syringes</td>
			<td style="width:89px;">BD</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Insulin syringes</td>
			<td style="width:89px;">BD</td>
			<td align="right" style="width:119px;">329461</td>
			<td>0.5 mL insulin syringes with needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">20 mL syringe&#160;</td>
			<td style="width:89px;">VWR</td>
			<td style="width:119px;">BD302831</td>
			<td>BD Syringe Slip Tip Graduated</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Tissue glue</td>
			<td style="width:89px;">Santa Cruz Animal Health</td>
			<td style="width:119px;">sc-361931</td>
			<td>3 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Heat lamp</td>
			<td style="width:89px;">Fluker</td>
			<td align="right" style="width:119px;">27002</td>
			<td>5.5&#34; reptile heat lamp with clamp and switch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">0.9% sterile saline</td>
			<td style="width:89px;">Pfizer</td>
			<td style="width:119px;">00409-4888-10</td>
			<td>0.9% sodium chloride for injection, 10 mL</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Eye lubricant</td>
			<td style="width:89px;">Watson Rugby</td>
			<td style="width:119px;">PV926977</td>
			<td>Artificial Tears Lubricant Eye Ointment, 1/8 oz</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Chux absorbent sheets</td>
			<td style="width:89px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">1420662</td>
			<td>Disposable absorbent padding</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:215px;">Iris scissors</td>
			<td style="width:89px;">World Precision Instruments</td>
			<td style="width:119px;">503708-12</td>
			<td>11.5cm, Straight, 12-pack</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Surgical tape</td>
			<td style="width:89px;">3M Durapore</td>
			<td style="width:119px;">1538-0</td>
			<td>1/2&#34;X10 yard roll, silk, hypoallergenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Mouse PET bed</td>
			<td style="width:89px;">In house</td>
			<td style="width:119px;"></td>
			<td>4 mouse PET bed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Lighter</td>
			<td style="width:89px;">Bic</td>
			<td style="width:119px;">UDP2WMDC</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Isoflurane</td>
			<td style="width:89px;">Henry Schein</td>
			<td style="width:119px;">NDC 11695-6776-2</td>
			<td>Isothesia, inhalation anesthetic, 250 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Oxygen</td>
			<td style="width:89px;">Praxiar</td>
			<td style="width:119px;">UN1072</td>
			<td>Compressed gas</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Autoradiography cassette</td>
			<td style="width:89px;">Cole Palmer</td>
			<td style="width:119px;">EW-21700-34</td>
			<td>Aluminum, 8&#34; x 10&#34;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Autoradiography film</td>
			<td style="width:89px;">GE Life Sciences</td>
			<td style="width:119px;">28-9564-78</td>
			<td>Storage Phosphor Screen BAS-IP SR 2025 E Super Resolution, 20 &#215; 25 cm, screen only</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Microtome blades</td>
			<td style="width:89px;">ThermoFisher Scientific</td>
			<td style="width:119px;">30-508-35</td>
			<td>MB35 Premier Disposable, 34&#176; cutting angle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Microtome</td>
			<td style="width:89px;">Microm</td>
			<td style="width:119px;"></td>
			<td>HM 550</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Microscope slides</td>
			<td style="width:89px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-15</td>
			<td>Superfrost&#8482; Plus Microscope Slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">OCT liquid</td>
			<td style="width:89px;">VWR</td>
			<td style="width:119px;">25608-930</td>
			<td>Formulation of water-soluble glycols and resins for cryostat sectioning at temperatures of -10&#176;C (14&#176;F) and below</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Freezing molds</td>
			<td style="width:89px;">Poly sciences</td>
			<td style="width:119px;">18646A-1</td>
			<td>Disposable paraffin molds</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Saran wrap</td>
			<td style="width:89px;">Saran</td>
			<td align="right" style="width:119px;">25700001300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Disinfectant</td>
			<td style="width:89px;">Virkon S</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

pet imaging of neuroinflammation using [11c]dpa-713 in a mouse model of ischemic stroke

Neuroinflammation 是缺血性中风后病理级联的中心。非侵入性分子成像方法有可能对脑卒中某些 neuroimmune 相互作用的时间动力学和角色提供重要的洞察力。具体地说, 正电子发射断层扫描 (PET) 成像的转运体蛋白 18 kDa (TSPO), 一个标记的活化小胶质细胞和外周髓系体细胞, 提供了检测和跟踪 neuroinflammation在体内的手段。在这里, 我们提出了一个方法, 以准确量化 neuroinflammation 使用 [11C] N, n-二乙基-2-[2-(4-甲氧基苯基)-57-dimethylpyrazolo [15-a] 嘧啶-3-基] 乙酰胺 ([11C] DPA-713), 一个有希望的第二代 TSPO-PET放射性, 在大脑中动脉闭塞 (dMCAO) 与假手术小鼠相比。MRI 进行2天后 dMCAO 手术, 以确认中风和定义梗死的位置和体积。PET/计算机断层扫描 (CT) 成像术后6天 dMCAO, 以捕捉峰值增加的 TSPO 水平后中风。对 PET 图像进行定量测定, 以评估 dMCAO 和假鼠大脑和脾脏中 [11C] DPA-713 的摄取量, 以评估中枢和周围炎症水平。体内[11C]DPA-713 脑吸收是通过体内显影证实的。

小鼠进行 MRI 检查成功的中风, 并通过扫描4小鼠同时进行 [11C] DPA-713 PET。PET、CT 和 MR 图像在手动绘制脑 ROIs 和执行半自动分裂脑图谱分析之前进行了联合注册, 以调查同侧和对侧区域的示踪物摄取情况 (图 2)。
PET/CT 图像和时间活动曲线 (口香糖-放射性活动作为时间的函数) 显示增加 [11C] DPA-713 吸收在同侧半球 (图 3a)。定量的动态 PET 脑图像, 使用总结数据从50-60 分钟, 显示显着增加的示踪剂摄取 (% ID/g) 在同侧半球 (梗死) 相比, 在 dMCAO, 但不是在假鼠使用人工绘制ROI 方法 (图 3B)。dMCAO 和假鼠在同侧半球也观察到了增加的摄取量。使用 atlas 方法观察到同侧半球之间没有显著差异, 可能是由于 atlas ROIs 大于梗塞的大小 (通常限于体感皮层), 因此稀释了信号。然而, 所有 ROIs 对 dMCAO 的总摄取量比假的增加, 这与以前使用 MCAO 模型小鼠的报告一致, 表明在梗塞19以外地区的 TSPO 表达增加。dMCAO 与假鼠同时使用两种方法增加同侧/对侧比值;然而, 由于 ROI 方法的差异较大, 这种差异仅在大脑皮层使用大脑图谱方法时才显著。这可以通过增加每组老鼠的数量来克服。定量的 [11C] DPA-713 的摄取在脾脏显示没有显著差异组 (图 4)。
脑 dMCAO 小鼠 PET 成像结果经活体高分辨率数字显影证实 (图 5)。增加 [11C] DPA-713 的摄取在梗死组织中观察到, 在周围健康的脑组织中可忽略的信号。这些图像的定量显示与对侧比从1.4 到2.09 的 dMCAO 小鼠。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57243/57243fig1.jpg"> 图 1:PET 扫描仪和工作区设置.所有工作区都覆盖在保护性吸收填料, 以创造一个无菌环境。(a)校准后, 一张 3 d-打印的小鼠床, 同时配有4只小鼠的成像设备, 用于所有4只麻醉小鼠的扫描仪和鼻锥。(B)预先准备了 PET 成像所需的设备, 包括含盐水的27.5 克导管、眼润滑剂、乙醇拭子、热灯、外科胶带、组织胶水、0.5 毫升注射器、剪刀和打火机。(C)放射性注射时, 将盐水冲洗注射器和剪刀放在扫描仪背面。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57243/57243fig1large.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/57243/57243fig2.jpg"> 图 2:同侧/对侧 ROI 和右/左分裂半球脑图谱的 PET 图像分析过程.利用人工绘制的 ROIs 和半自动化3D 分裂脑图谱方法, 利用图像分析软件确定了同侧和对侧区域的示踪物摄取量 (ROIs)。自动 3D PET/CT 注册后进行人工注册的大脑 MRI 在相应的鼠标头骨定义的 CT 图像。3D ROI 工具用于手动绘制同侧 (红色) 和对侧 (绿色) ROIs 使用 MRI 梗塞作为参考。对于分裂大脑的方法, 3D 左/右分裂小鼠大脑图谱被加载和安装在头骨中的 CT 图像定义。脑 ROIs 用于定量在这个3D 鼠标大脑图谱包括左皮层 (深灰色), 左海马体 (矢车菊蓝色), 左纹状体 (深粉红色), 右皮层 (番茄红色), 右海马 (绿色), 右 Striatium (青色)。在每一个区域获得 [11C] DPA-713 的吸收, 并随后转换为%ID/g 通过正常化的衰减校正剂量时, 每只老鼠扫描。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57243/57243fig2large.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/57243/57243fig3.jpg"> 图 3: 代表体内[11C] DPA-713 DMCAO 和假鼠的脑吸收。(A)动态 PET/CT 图像和口香糖显示增加 [11C] DPA-713 在小鼠的同侧皮层接受 DMCAO (n = 3) 和轻微增加假 (n = 3) 操作小鼠, 与 DMCAO 小鼠表现显着更大的与脑梗塞和对侧侧的比例注射剂量的对比 (%ID/g)。(B) PET 量化 (50-60 分钟总结) 显示, 使用 ROI 方法和在皮质 (Ctx) 使用分裂脑图谱方法显著增加了同侧 roi 的吸收。海马 (HC) 或纹状体 (Str) 无明显差异。使用两种分析方法均能提高同侧比对侧比, 但 Ctx 使用脑图谱方法在统计学上有显著意义。* (p < 0.05), ** (p < 0.001)<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57243/57243fig3large.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/57243/57243fig4v2.jpg"> 图 4:代表体内[11C] DPA-713 dMCAO 和假鼠的脾吸收。(A) [11C] DPA-713 动态 PET/CT 图像显示脾脏 ROIs 在 dMCAO (n = 3) 和假 (n = 3) 小鼠。(B)定量结果表明, dMCAO 和假鼠在脾脏摄取方面没有显著的结果。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57243/57243fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57243/57243fig5v2.jpg"> 图 5: 代表显影结果.数字显影图像显示增加 [11C] DPA-713 吸收在同侧半球相比。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57243/57243fig5v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke at JoVE.com

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

正电子发射层析成像 (PET) 转运体蛋白 18 kDa (TSPO) 提供了一种非侵入性的手段来可视化 neuroinflammation 在脑疾病的发展和进展中的动态作用。本协议描述 TSPO-PET 和前体显影在小鼠缺血性中风模型中检测 neuroinflammation。

Neuroinflammation 的 PET 成像 [11C] DPA-713 在小鼠缺血性中风模型中的应用

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...

pet-imaging-neuroinflammation-using-11cdpa-713-mouse-model-ischemic

Nanyang Technological University

Research

JoVE Journal

Medicine

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

12.5K 次观看.  Stanford University. 正电子发射层析成像 (PET) 转运体蛋白 18 kDa (TSPO) 提供了一种非侵入性的手段来可视化 neuroinflammation 在脑疾病的发展和进展中的动态作用。本协议描述 TSPO-PET 和前体显影在小鼠缺血性中风模型中检测 neuroinflammation。

视频: PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

腔内中东大脑中动脉闭塞（MCAO）缺血性中风模型在小鼠体内的激光多普勒血流指导

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

科研

医学

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

多光子成像在口腔鳞状细胞癌原位小鼠模型的肿瘤细胞的侵袭

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

内皮素-1诱导大脑中动脉闭塞模型缺血性中风激光多普勒血流仪在大鼠指导

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

MRI和PET心肌梗死小鼠模型

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

蛛网膜下腔出血的小鼠模型

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脑Microslices的兴奋性刺激作为<em&gt;在体外</em&gt;中风模型

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

栓塞性脑梗塞（缺血）的大鼠缺血性中风与血同源凝块

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

新生儿缺氧缺血性脑病的小猪模型

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

同时PET / MRI成像在小鼠脑缺氧缺血

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

玫瑰红Photothrombosis共聚焦光学成像<em&gt;在体内</em&gt;:单支脑卒中模型

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...