笔者想承认法甲法兰西驳L&#39;épilepsie（MA到-L），国家研究所德拉桑特等德拉RECHERCHE MEDICALE格兰特AVENIR R12087FS（FJ到），从蒙彼利埃大学教育资助（为FJ）和格兰特距离联邦倒拉RECHERCHE河畔乐Cerveau（以NM）。我们承认Chrystel的乐峰在IPAM在蒙彼利埃的体内成像核心平台设施的技术援助。我们也感谢玛丽Vernov（威尔康乃尔医学院）校对稿件。

小鼠被允许食物，水随意访问，并维持12小时光照-黑暗周期。所有涉及实验动物的程序符合国家和欧洲的法律和由法国教育部教育和科学研究（CEEA-LR-00651-01）的批准。共有6个转基因5xFAD和4个同窝野生型（WT）对照组小鼠被用于此过程。 1.手术前准备<ol><li>腹腔（IP）注射甲氧基X04（10毫克/千克）手术标记Aβ存款11前48小时。 </li><li>制备无菌人工脑脊液（ACSF）（120mM氯化钠，26.2毫碳酸氢钠 ，2.5mM的氯化钾，1mM的的NaH 2 PO 4，1.3毫氯化镁 ，10mM葡萄糖;气泡以95％ 的 O 2/5％CO 2的前加入2.5毫摩尔的CaCl 2）。 </li><li>消毒手术工具（剪刀，镊子，刀片和钻头），用高压灭菌或无菌之前热珠灭菌手术。 </li><li>请用70％的乙醇和覆盖手术台和显微镜板用干净的吸水布。 </li><li>注入氯胺酮/赛拉嗪（75毫克/公斤和10毫克/公斤，分别）和镇痛药酮洛芬（2.5毫克/千克）的IP。 </li><li>请与爪子或尾巴捏麻醉适当的水平。 </li><li>应用无菌眼药膏眼睛和刮皮毛（鼻耳），同时避免了胡须。因为它是接着用水，以防止皮肤刺激仔细漂洗脱毛乳膏可以用作长。刮剩下的皮毛用刀片。消毒用碘伏防腐剂溶液皮肤。 </li><li>将鼠标的双目显微镜下和从耳朵到鼻使皮肤切口。侧身拉出皮肤暴露颅骨。切开骨膜并反复冲洗用无菌学联头骨停止可能的出血。 </li></ol> 2.血管系统标签和减薄的头骨窗口准备（40分钟） <ol><li>取出眼膏用棉签。应用局部眼用麻醉（地卡因1％）的下降，轻轻地压在皮肤周围的眼窝实现凸出。 </li><li>在眶后窦以下通过亚德尼和同事12中描述的方法;注入用葡聚糖（70 kDa）的缀合异硫氰酸荧光素（FITC）（皮下注射胰岛素针100毫克/千克或50微升50毫克/毫升溶液）。应用无菌眼药膏的眼睛。 </li><li>保证皮肤回缩，头骨是围绕选定成像区清洁和干燥。申请围绕头部的窗氰基丙烯酸胶少量贴装器件（包括层叠剃须刀刀片，参见图1），和胶周围施加温和的压力的目标区域几秒钟10。 </li><li>皮肤的端部拉向头部的窗口的边缘安装器件，确保的区域是干。固定皮肤和兽医级氰基丙烯酸胶少量的窗口的边缘，并等待直至干燥（ 图1）。 </li><li>固定动物在使用头戴式设备阶段。用无菌脑脊液冲洗，并确保对皮肤和头戴式设备之间的井是完全密封的。包裹鼠标生存毯子，以保持正常体温。 </li><li>如前所述10学联解决方案执行颅骨变薄。注意，头皮未完全除去。定期更改学联清除骨屑，避免组织过热。任何剩余的骨骼碎片可以用简短的空气泡芙被删除。学联解决方案还通过衰减钻探产生的振动。 注:小鼠头骨厚度取决于动物的年龄和感兴趣区域80 150之间，以微米而变化。 </li><li>使用牙钻（以中速和0.7mm的毛刺）开始在区域1变薄颅骨表面。0毫米直径利用垂直平行于头骨有规律的运动，并除去大部分的骨松质（第40 - 70微米）。记得更换学联每隔20-30秒，并限制不间断钻3-4秒。需要注意的是骨缝线附近是高度血管。学联应用明胶海绵预浸停止最终出血。 </li><li>用锋利的一次性眼科显微叶片继续与颅骨变薄，小心不要施加过大的压力。最后区域是大约0.5毫米，直径与厚度为20 - 35微米。 注:由于骨再生和瘢痕形成，有必要在每个成像会话的窗口进一步变薄。 </li></ol> 3.双光子显微镜（45分钟） <ol><li>如果有必要，用19毫克/千克氯胺酮注射以维持足够的麻醉。 </li><li>在双光子激光显微镜下传送鼠标（固定到头戴阶段）。使用20X水浸泡OBJ为1.0的数值孔径ective，在使用外延荧光灯的光场的中心定位的变薄颅窗口。确保目标始终沉浸在学联。 </li><li>与锁模脉冲激光（钛宝石680-1040nm）启动激光扫描。在750纳米设定激发波长分别检测甲氧基XO 4的发射的荧光和FITC-葡聚糖的蓝色和绿色通道。 注:本显微镜具有在506 nm和555 nm的两个分束器。蓝色的光被配备有470±12纳米的过滤器的NDD检测器捕获。绿色光被配备有525±25纳米的过滤器具有高灵敏度的GaAsP检测器捕获。从物镜射出的最大激光功率不应超过10毫瓦，以避免激光诱导的组织损伤。 </li><li>获取低倍率栈（500微米×500微米，512×512像素; 2微米步骤）在0.7X变焦数值创造了3D地图的ROI的精确重新定位在稍后的时间点。 </li><li>获得高4数字放大图像的镶嵌有2倍数字变焦（200微米×200微米，512×512像素; 1微米步骤）。使用图像处理软件在200微米的步骤将窗口移动到捕获所有地区。堆栈的深度通常为250微米，从表面软脑膜镶嵌的每幅图像中开始。 </li><li>从显微镜取出鼠标之前，拍照，或对影像领域的未来重新定位的软脑膜血管的手绘2D地图。 </li></ol> 4.回收和再成像<ol><li>通过施加牵引力而下方保持头骨移除头部安装设备。如果有任何胶水仍然连接到颅骨或皮肤，用细镊子仔细地刮它。缝合头皮和将鼠标在加热的笼返回给家笼之前并监控从麻醉中恢复。应用局部抗生素乳膏于缝线和注入酮洛芬（2.5毫克/千克）我P上的以下2天。 </li><li>重复步骤重复成像会议1.1至2.5。如果需要的话，剃骨用显微叶片，以确保图像的最佳质量。 注意:当成像会话之间的时间间隔是超过一个月与牙钻附加变薄可能是必要的。 </li><li>根据从以前的会话（步骤3.6）所采取的软脑膜血管的2D地图使用啶光显微镜下鼠标位置。 </li><li>启动双光子激光扫描和调整显微镜阶段在使用在步骤3.4中得到的3D地图的微米尺度来实现对准。如步骤3.5所述进行详细的成像。 </li></ol> 5.收购后三维重建与图像分析<ol><li>获得脉管的三维重建和使用3D图像分析软件如IMARIS淀粉样蛋白沉积（版本使用8.0和7.7.2）。 </li&gt; <li>使用图像处理菜单和对比度变化的子菜单上的正常化层插件，以获得在图像栈的整个深度的两个通道中的理想的对比度。 </li><li> 在所有时间点同时打开来自同一区域的图像和总是处理它们并联 ，以比较不同时间点。 </li><li>使用长丝示踪模块跟踪船只。 <ol><li>点击图标长丝创建一个新的细丝，跳过自动追踪并选择自动路径法。 </li><li>是确定所选信道对应的血管图像，并使用指针的选择功能，点击即在保持变速+对照键，以确定一个起点穿过成像会话保守脉管分叉。使用指针的选择功能，同时按住shift键来选择丝的终点单击。 注:obtai的比较斯内德骨骼会显示出血管的结构变化。 </li></ol></li><li>通过平面分析进行平面追踪新的斑块。应用面模块与蓝色通道用于跟踪的Aβ斑块的生长。点击图标面来创建一个新的表面，并与自动曲面创建进行。使用阈值法来设置和减去背景信号。统计数据菜单提供相对于个别淀粉样蛋白沉积的表面数据。 </li></ol>

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作者有没有竞争经济利益。

体内双光子显微镜开放式颅骨技术提供大量成像领域13,14无限成像会议的优势。然而，该技术也是在感兴趣区域14产生炎症，经常不相容或影响神经血管读数15。相反，减薄头骨颅技术不会导致神经炎症，使脑血管结构的可靠的成像和菌斑积累10,14。通过这种技术提出的第二个优点是稀疏的颅骨窗口的成功率可达80％〜90％，对于有经验的操纵者。成功的重大?...

脑血管是一个多细胞结构，这是在解剖学和功能上耦合到神经元。船只的动态重构发生在整个大脑发育和中枢神经系统（CNS）1,2的病状的进展期间。它已被广泛接受脑损伤是几种中枢神经系统疾病，包括癫痫，阿耳茨海默氏病（AD），创伤性脑损伤的一个标志和脑炎3,4。因此， 在体内追踪脑血管变化建模中枢神经系统疾病时，从发病进入慢性期变得显著。由于脑血管修改常与神经元损伤或可塑性伴随发生时，神经血管成像是一个关键切入点破译CNS疾病的病理生理机制。 这个协议描述了一个纵向的双光子基于程序来跟踪cerebrovasculature的重构的小鼠模型AD，由于淀粉样蛋白斑块沉积5-7的大，小口径血管脑血管缺陷标志着一个渐进的病理。此过程允许淀粉样蛋白沉积和其位置和生长的跟踪的可视化对于神经血管重塑整个病程。重要的荧光染料的cerebrovasculature和淀粉样蛋白斑在转基因小鼠公元8可视化每个成像会议召开前被注入。通过减薄头骨颅窗口ROI的重复成像会议是非侵入性的和选择的方法在活老鼠的大脑2,5,9,10评估神经血管重塑。 下面的步骤概括了手术方案，图像采集和处理。淀粉样脑血管病（CAA）主要在软脑膜大与穿透性小动脉的早期发展的特点。

<table><tbody><tr><td>methoxy-X04</td><td>tocris</td><td>4920</td><td>use 10 mg/kg</td></tr><tr><td>FITC-Dextran 70 Kda</td><td>sigma</td><td>46945</td><td>use 100 mg/kg</td></tr><tr><td>gelfoam/Bloxang</td><td>Bausch and Lomb</td><td></td><td></td></tr><tr><td>micorsurgical blade</td><td>surgistar</td><td>6900</td><td>must be sharp and not dented</td></tr><tr><td>povidone-iodine</td><td>betadine</td><td></td><td>antisceptic solution</td></tr><tr><td>binocular stereomicroscope</td><td>olympus</td><td>SX10</td><td>optimal image contrast is crucial for this procedure</td></tr><tr><td>2-photon microscope</td><td>zeiss</td><td></td><td>Zeiss LSM 710mp</td></tr><tr><td>fine scissors-toughcut</td><td>Fine science tools</td><td>14058-09</td><td>this scissors are optimized for cutting skin and soft tissue</td></tr></tbody></table>

longitudinal in vivo imaging of the cerebrovasculature: relevance to cns diseases

大脑脉管系统的重塑是脑病理学的一个共同特征。 体内成像技术是检测脑可塑性或损害的发生加班费和相对于神经元活性或血流的基础。 体内双光子显微镜允许在客厅大脑细胞大单位结构和功能可塑性的研究。特别是，减薄-颅骨窗制剂允许的感兴趣区域（ROI）的皮质区域的可视化而不诱导显著脑炎症。皮质的投资回报率的重复成像会议是可行的，许多中枢神经系统疾病的进展过程中提供疾病特点随时间变化的表征。这种技术在访问内250μm的大脑的软脑膜结构依赖于检测通过基因细胞标记和/或活体染料编码的荧光探针。后者（ 例如，荧光葡聚糖）被用于映射LUMIN脑结构的人舱。锗本文所述的协议是使用淀粉样蛋白沉积物的体内标记物，甲氧基O4，以评估阿尔茨海默氏病（AD）进展。我们还描述了用于跟踪血管变化和淀粉样沉积的采集后图像处理。虽然目前专注于AD模型，描述的协议是有关在那里发生的病理变化脑血管病其他中枢神经系统疾病。

本协议描述的可视化cerebrovasculature和淀粉样蛋白沉积加班的方法。荧光染料注射标记的淀粉样蛋白沉积（甲氧基XO 4）11，以填充脑血管管腔（FITC-葡聚糖）1。 3D图像分析软件模块用于创建视图的连续时间点拍摄的恒定场的3D图像。在5XFAD小鼠5表明大部分的Aβ沉积物出现月龄3和4之间，在软组织生长（ 图2）和围绕穿透血管（ 图3）?...

Watch this Scientific Journal Video about Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases at JoVE.com

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

该原稿描述的过程使用纵向双光子显微镜体内淀粉样蛋白斑积累期间跟踪cerebrovasculature的重塑。减薄头骨制备使荧光染料的可视化，以评估在阿尔茨海默氏病的小鼠模型中脑血管损伤的进展。

纵<em&gt;在体内</em&gt;的Cerebrovasculature影像:相关性疾病CNS

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

longitudinal-vivo-imaging-cerebrovasculature-relevance-to-cns

Research

JoVE Journal

Medicine

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

7.1K 次观看.  Institute of Functional Genomics. 该原稿描述的过程使用纵向双光子显微镜体内淀粉样蛋白斑积累期间跟踪cerebrovasculature的重塑。减薄头骨制备使荧光染料的可视化，以评估在阿尔茨海默氏病的小鼠模型中脑血管损伤的进展。 

视频: Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Non-invasive Parenchymal, Vascular and Metabolic High-frequency Ultrasound and Photoacoustic Rat Deep Brain Imaging

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central nervous system of small animals. However, transdermal and transcranial neuroimaging is frequently affected by low sensitivity, image aberrations and loss of space resolution, requiring scalp or even skull removal before imaging. To overcome this challenge, a new protocol is presented to gain significant insights in brain hemodynamics by photoacoustic and high-frequency ultrasounds imaging with the animal skin and skull intact. The procedure relies on the passage of ultrasound (US) waves and laser directly through the fissures that are naturally present on the animal cranium. By juxtaposing the imaging transducer device exactly in correspondence to these selected areas where the skull has a reduced thickness or is totally absent, one can acquire high quality deep images and explore internal brain regions that are usually difficult to anatomically or functionally describe without an invasive approach. By applying this experimental procedure, significant data can be collected in both sonic and optoacoustic modalities, enabling to image the parenchymal and the vascular anatomy far below the head surface. Deep brain features such as parenchymal convolutions and fissures separating the lobes were clearly visible. Moreover, the configuration of large and small blood vessels was imaged at several millimeters of depth, and precise information were collected about blood fluxes, vascular stream velocities and the hemoglobin chemical state. This repertoire of data could be crucial in several research contests, ranging from brain vascular disease studies to experimental techniques involving the systemic administration of exogenous chemicals or other objects endowed with imaging contrast enhancement properties. In conclusion, thanks to the presented protocol, the US and PA techniques become an attractive noninvasive performance-competitive means for cortical and internal brain imaging, retaining a significant potential in many neurologic fields.

The present work describes a new protocol to perform non-invasive high-frequency ultrasound and photoacoustic based imaging on rat brain, to efficiently visualize deep subcortical regions and their vascular patterns by directing signals on skull foramina naturally present on animal cranium.

非侵入性的脑实质，血管和代谢高频超声和光声大鼠深脑成像

Photoacoustics and high frequency ultrasound stands out as powerful tools for neurobiological applications enabling high-resolution imaging on the central ...

科研

神经科学

Quantification of Cerebral Vascular Architecture using Two-photon Microscopy in a Mouse Model of HIV-induced Neuroinflammation

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic neuroinflammatory state within the central nervous system (CNS), thought to be driven principally by virally-mediated activation of microglia and brain resident macrophages. HIV-1 infection is also accompanied by changes in cerebrovascular blood flow (CBF), raising the possibility that HIV-associated chronic neuroinflammation may lead to changes in CBF and/or in cerebral vascular architecture. To address this question, we have used a mouse model for HIV-induced neuroinflammation, and we have tested whether long-term exposure to this inflammatory environment may damage brain vasculature and result in rarefaction of capillary networks. In this paper we describe a method to quantify changes in cortical capillary density in a mouse model of neuroinflammatory disease (HIV-1 Tat transgenic mice). This generalizable approach employs in vivo two-photon imaging of cortical capillaries through a thin-skull cortical window, as well as ex vivo two-photon imaging of cortical capillaries in mouse brain sections. These procedures produce images and z-stack files of capillary networks, respectively, which can be then subjected to quantitative analysis in order to assess changes in cerebral vascular architecture.

This paper describes a method by which the vascular architecture in the brain can be quantified using in vivo and ex vivo two-photon microscopy.

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

Human Immunodeficiency Virus 1 (HIV-1) infection frequently results in HIV-1 Associated Neurocognitive Disorders (HAND), and is characterized by a chronic ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

横向慢性颅窗口准备启用<em&gt;在体内</em&gt;观察继末节小鼠大脑中动脉闭塞

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

脑血流动力学和的光散射特性的同步评估<em&gt;体内</em&gt;大鼠脑使用多光谱漫反射成像

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

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

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

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

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

在体内挫伤脊髓损伤后影像血管动力学的双色法

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

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

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

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

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

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

免疫与感染

In Vivo Imaging of Cerebrospinal Fluid Transport through the Intact Mouse Skull using Fluorescence Macroscopy

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and magnetic resonance imaging (MRI) have enabled in vivo quantification of CSF flow but they are limited by reduced imaging volumes and low spatial resolution, respectively. Recent work has found that CSF enters the brain parenchyma through a network of perivascular spaces surrounding the pial and penetrating arteries of the rodent cortex. This perivascular entry of CSF is a primary driver of the glymphatic system, a pathway implicated in the clearance of toxic metabolic solutes (e.g., amyloid-&#946;). Here, we illustrate a new macroscopic imaging technique that allows real-time, mesoscopic imaging of fluorescent CSF tracers through the intact skull of live mice. This minimally-invasive method facilitates a multitude of experimental designs and enables single or repeated testing of CSF dynamics. Macroscopes have high spatial and temporal resolution and their large gantry and working distance allow for imaging while performing tasks on behavioral devices. This imaging approach has been validated using two-photon imaging and fluorescence measurements obtained from this technique strongly correlate with ex vivo fluorescence and quantification of radio-labeled tracers. In this protocol, we describe how transcranial macroscopic imaging can be used to evaluate glymphatic transport in live mice, offering an accessible alternative to more costly imaging modalities.

Transcranial optical imaging allows wide-field imaging of cerebrospinal fluid transport in the cortex of live mice through an intact skull.

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Cerebrospinal fluid (CSF) flow in rodents has largely been studied using ex vivo quantification of tracers. Techniques such as two-photon microscopy and ...

Ex Vivo Pressurized Hippocampal Capillary-Parenchymal Arteriole Preparation for Functional Study

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and cardiac arrest are remarkably sexually dimorphic diseases, and both induce cerebral ischemia. However, progress in understanding the vascular cognitive impairment, and then developing sex-specific treatments, has been partly limited by challenges in investigating the brain microcirculation from mouse models in functional studies. Here, we present an approach to examine the capillary-to-arteriole signaling in an ex vivo hippocampal capillary-parenchymal arteriole (HiCaPA) preparation from mouse brain. We describe how to isolate, cannulate, and pressurize the microcirculation to measure arteriolar diameter in response to capillary stimulation. We show which appropriate functional controls can be used to validate the HiCaPA preparation integrity and display typical results, including testing potassium as a neurovascular coupling agent and the effect of the recently characterized inhibitor of the Kir2 inward rectifying potassium channel family, ML133. Further, we compare the responses in preparations obtained from male and female mice. While these data reflect functional investigations, our approach can also be used in molecular biology, immunochemistry, and electrophysiology studies.

The present manuscript details how to isolate hippocampal arterioles and capillaries from the mouse brain and how to pressurize them for pressure myography, immunofluorescence, biochemistry, and molecular studies.

Ex Vivo 加压希马卡毛细管- 功能研究准备

From subtle behavioral alterations to late-stage dementia, vascular cognitive impairment typically develops following cerebral ischemia. Stroke and ...

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy (pCLE) of Hippocampal Capillaries in Awake Mice

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application to elucidate capillary blood flow effects during seizures, driven by mural cells. In vitro and in vivo cortical imaging have shown that capillary constrictions driven by pericytes can result from functional local neural activity, as well as from drug application, in healthy animals. Here, a protocol is presented on how to use pCLE to determine the role of microvascular dynamics in neural degeneration in epilepsy, at any tissue depth (specifically in the hippocampus). We describe a head restraint technique that has been adapted to record pCLE in awake animals, to address potential side-effects of anesthetics on neural activity. Using these methods, electrophysiological and imaging recordings can be conducted over several hours in deep neural structures of the brain.

In Vivo Fiber-Coupled Pre-Clinical Confocal Laser-scanning Endomicroscopy pCLE of Hippocampal Capillaries in Awake Mice

Whereas multiphoton imaging is only effective at limited depths from the tissue&#8217;s surface, it is possible to achieve 3 &#181;m resolution imaging at any depth via pCLE. Here, we present a protocol to conduct pCLE imaging to measure microvascular dynamics in the hippocampus of ictal and wild-type mice.

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜 （pCLE）

The goal of this protocol is to describe fiber-optic-bundle-coupled pre-clinical confocal laser-scanning endomicroscopy (pCLE) in its specific application ...

<meta charset="utf8"></head><body>用于小鼠血管认知障碍研究的针头模型

A Mouse Model for Vascular Cognitive Impairment and Dementia Based on Needle-guided Asymmetric Bilateral Common Carotid Artery Stenosis

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue rising as the population ages, it is critical to establish a robust animal model for the disease. This paper presents a novel method of creating a mouse model of VCID that is based on asymmetric bilateral common carotid artery stenosis, which mimics human chronic cerebral hypoperfusion caused by carotid atherosclerosis.
Briefly, common carotid arteries (CCAs) are ligated to different gauge needles (32 G for the right CCA and 34 G for the left CCA) using 7-0 silk sutures followed by immediate needle removal. The remaining suture rings cause persistent blood flow reduction and long-term cognitive impairment associated with white matter injury, microinfarcts, and reactive gliosis, thus closely mimicking the pathogenesis of VCID. Importantly, in this needle model, the clinical representations do not revert with time, providing reliable long-term cognitive impairment. Moreover, the survival rate 24 weeks post surgery was 81.6%, which is higher compared to the other established models of VCID with a similar level of blood flow reduction.
Additional advantages include low material cost and compatibility with MRI to monitor brain injury in live animals since no metal is implanted. The main challenge in employing the needle model of VCID is the requirement for developing advanced surgical skills since mouse CCAs are less than 0.6 mm in diameter and are very fragile. High-quality visual representation of the surgery will thus help researchers to master this technique and advance our understanding of VCID, potentially leading to the development of novel therapeutic modalities to decrease the devastating cognitive decline associated with VCID.

<meta charset="utf8"></head><body>基于针引导不对称双侧颈总动脉狭窄的血管性认知障碍和痴呆小鼠模型

The needle method of asymmetric bilateral common carotid artery stenosis is proposed to create a mouse model for vascular cognitive impairment and dementia. It results in longer-term outcomes compared to previously established models and is compatible with live MRI. Visual representation demonstrating the procedure provides guidance for mastering the surgery.

基于针引导不对称双侧颈总动脉狭窄的血管性认知障碍和痴呆小鼠模型

Vascular cognitive impairment and dementia (VCID) results from vascular brain injury. Given VCID&#39;s high incidence, which is expected to continue ...

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此视频中的章节

非侵入性的脑实质，血管和代谢高频超声和光声大鼠深脑成像

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

横向慢性颅窗口准备启用

脑血流动力学和的光散射特性的同步评估

在体内挫伤脊髓损伤后影像血管动力学的双色法

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Ex Vivo 加压希马卡毛细管- 功能研究准备

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜（pCLE）

基于针引导不对称双侧颈总动脉狭窄的血管性认知障碍和痴呆小鼠模型

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摘要

探索更多视频

此视频中的章节

非侵入性的脑实质，血管和代谢高频超声和光声大鼠深脑成像

利用双光子显微镜的艾滋病毒引起的神经炎症的小鼠模型脑血管结构的量化

横向慢性颅窗口准备启用

脑血流动力学和的光散射特性的同步评估

在体内挫伤脊髓损伤后影像血管动力学的双色法

精确的大脑映射，在小鼠中执行神经-免疫动力学的重复体内振动成像

使用荧光宏镜检查,通过"完整小鼠头骨"进行脑脊液传输的 Vivo 成像

Ex Vivo 加压希马卡毛细管- 功能研究准备

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜 （pCLE）

基于针引导不对称双侧颈总动脉狭窄的血管性认知障碍和痴呆小鼠模型

清醒小鼠海马毛细血管的体内纤维耦合临床前共聚焦激光扫描内窥镜（pCLE）