该项目由KWF-STW资助（儿童扩散内在庞廷胶质瘤和高档胶质瘤的索诺波治疗药物交付）。我们感谢伊利亚·斯卡奇科夫和查尔斯·穆格诺特对该系统开发的投入。

所有体内实验均由荷兰伦理委员会（许可证编号AVD114002017841）和荷兰阿姆斯特丹Vrije大学动物福利机构批准。调查人员接受了FUS系统基础知识的培训，以尽量减少动物的不适。
1. 聚焦超声波系统
注:描述的设置是基于市售组件的内部构建的 BBB 中断系统，包括一个 3D 打印定制圆锥体和可拆卸立体声平台。该系统设计为模块化，可根据现有设备和特定用途进行修改。该协议描述了小鼠大脑庞廷区域较大区域的声波化过程。通过调整目标位置，大脑的不同部分可以成为目标。在这项研究中，使用了1兆赫单元素传感器，焦距为75毫米，光圈为60毫米，焦区为1.5 x 1.5 x 5毫米（峰值压力的FWHM）。传感器的焦点平面通过动物的颅骨定位在与耳栏相交的水平平面上。
<ol><li>为啮齿动物的 BBB 开口选择合适的传感器。 注:根据微泡的特性和使用频率，声学设置，特别是机械指数（MI），可能会发生变化13，38。</li>
	<li>将传感器放在 3D 打印的圆锥体中。</li>
	<li>在圆锥体的底端采用声学透明的 mylar 膜，实现光束传播路径的声学耦合，并在圆锥体中填充脱气水。</li>
	<li>将传感器安装在动物上方的机动线性阶段，如 图 1 所示，允许自动垂直定位传感器。</li>
	<li>根据研究的要求设计一个可拆卸的立体定向平台，包括温度调节加热、咬合和耳栏、麻醉和多模式的纤维标记，如 图 1 和图 2所示。立体定向平台的安装由二维线性舞台系统组成，允许在光束下精确自动定位（< 0.1 毫米）动物。</li>
	<li>将传感器连接到 图 1 中显示的声学发射链，该链由传感器、函数发生器和功率放大器组成。</li>
	<li>设计一个图像处理管道来检测多模式的假象标记，使精确的声波瞄准感兴趣的大脑区域，并收集针水听器检测到的腔化数据。</li>
	<li>校准系统并确定转导器对应动物在立体定向平台上的垂直定位的焦点。</li>
</ol>2. 动物制备
注:以下协议为小鼠指定，但可以适应大鼠。对于这些实验，使用雌性水性裸体Foxn1-/-小鼠（6-8周大）。
<ol><li>允许动物在动物设施中适应至少一周，并定期称体重。</li>
	<li>通过皮下注射（s.c.）注射30分钟之前，FUS治疗开始镇痛治疗，管理丁丙诺啡（0.05毫克/千克）。</li>
	<li>用3%异氟兰、2L/min O2 麻醉动物，验证动物是否深度麻醉。在整个过程中保持动物麻醉，并监测呼吸频率和心率，根据需要调整异氟素的浓度。</li>
	<li>涂抹眼药膏，防止眼睛干燥，避免可能的伤害。</li>
	<li>用剃须刀和脱毛霜去除头顶上的头发，然后用水清洗，以去除任何残留物，以避免对皮肤造成刺激。</li>
	<li>对于BLI肿瘤模型的实验，注射150微L的D-露西芬（30毫克/mL）腹膜内（即.p.），用29G胰岛素注射器进行BLI图像指导。</li>
	<li>插入 26-30 G 尾静脉导管，用少量肝素溶液（5 UI/mL）冲洗导管和静脉。用肝素溶液填充导管，以避免血液凝固。 注意:当血液倒流到导管中时，可以看到良好的导管。避免导管中的气泡，以防止浮雕。为了避免过多的注射压力，请确保导管的长度尽可能短。</li>
	<li>将动物放在温度调节立体定向平台上，以避免体温过低。 注:体温过低可减少血液循环，可影响微泡和药物动力学的注射/循环。</li>
	<li>使用耳栏和咬伤杆固定和固定立体定向平台上的动物头部。用带子固定身体，将动物的尾巴绑在平台上。</li>
</ol>3. 体内图像引导聚焦超声波
注意:对于此协议，使用了 1 MHz 单元素传感器，其音冲脉冲持续时间为 10 ms，MI 为 0.4，脉冲重复频率为 1.6 Hz，240s 周期为 40 个周期。该协议被优化为微泡稳定含有硫六氟化物（SF6）作为无害气体，其中平均气泡直径为2.5微米，超过90%的气泡小于8微米。
<ol><li>将立体定向平台与安装的动物置于成像模式（如 BLI 或 X 射线）中， 并拍摄动物的图像。</li>
	<li>使用多模式的假象标记与图像处理管道相结合，根据传感器的对焦点标记动物的位置。</li>
	<li>通过在获得的 X 射线图像上放置大脑轮廓或使用 BLI 图像确定肿瘤中心（图 2）来确定目标区域。大脑特定部位的位置在Paxinos大脑图集40 中使用头骨标记布雷格玛和兰姆达作为参考点。例如，庞斯位于 x=-1.0，y=-0.8 和 z=-4.5 从兰姆达。</li>
	<li>用胶带屏蔽动物的鼻孔和嘴部，防止超声波凝胶干扰呼吸。</li>
	<li>在动物的头顶上涂抹超声波凝胶。</li>
	<li>缩回动物颈部的皮肤，用超声波凝胶润滑针水听器，并将针水听器放在眼骨的直接附近。</li>
	<li>使用图像处理管道和对焦点引导传感器到正确的位置。</li>
	<li>将预配置设置应用到所有附加设备上，并针对感兴趣的大脑区域。 注:根据研究问题，肿瘤或脑区域可以作为单个焦点或体积形状，如 图2所示。</li>
	<li>激活制造商描述的微泡。注入一个120微L（5.4微克）微泡的博卢斯。</li>
	<li>用盐水冲洗尾静脉导管，检查导管的开口。</li>
	<li>注入微泡并开始共振。</li>
	<li>用针水听器记录微泡腔。</li>
	<li>声波手术后施用血管内对比剂或药物。剂量、时间和计划取决于研究的目的和药物。 注:埃文斯蓝是一个常见的颜色代理评估BBB开幕41。</li>
	<li>监视动物，直到预先确定的时间点或人道终点之前。</li>
</ol>4. 微泡腔分析
注:这里描述了应用过程，它适用于SF6-磷脂微泡的体内实验，平均直径为2.5微米（80%的气泡低于8微米），在1MHz的频率下，爆音脉冲为10毫秒，最初由麦克丹诺德等人建议。
<ol><li>将录制的 PCD 信号从时间域转换为频率域。</li>
	<li>集成由此产生的光谱功率，在 2号 和 3号 谐波（± 50 kHz）周围进行稳定空腔检测，如 图 3 所示（2 和 3 MHz 的绿色框）。</li>
	<li>集成惯性腔检测的光谱功率，在主频、2号、3号谐波、1号和2号超谐波以及第一个亚谐波（±150 kHz）之间，如图3（红色框）所示。</li>
	<li>将光谱功率集成在主频（1 MHz ± 50 kHz）周围，实现先前获得的两个 PCD 信号的正常化。 注:PCD信号，用于SF6-磷脂微泡在体内实验在1 MHz，不显示超谐波或亚和谐之前惯性气泡设置，如 图3所示。</li>
</ol>

作者没有什么可透露的。

在这项研究中，我们开发了一个基于成本效益的基于FUS的图像引导系统，用于瞬时BBB中断，以增加药物输送到大脑的帕奇马。该系统主要由市售组件构建，并与 X 射线和 BLI 配合使用。拟议设计的模块化允许在高通量工作流程中使用多种成像模式进行规划和评估。该系统可以与更全面的高分辨率 3D 成像模式（例如高分辨率 MRI 或微型 CT）相结合，而对于大部分研究，2D 成像模式（如 2D X 射线和/或 ...

血脑屏障 （BBB） 是药物输送到大脑帕伦奇马的主要障碍。大多数已经开发的治疗药物不会越过BBB，因为他们的物理化学参数（如嗜脂，分子量，氢键接受者和捐赠者），或不保留，因为他们的亲和力在大脑1，2的外流运输器。能穿过BBB的一小群药物通常是小脂质分子，它们只对数量有限的脑部疾病1、2有效。因此，对于大多数脑部疾病，药理治疗的选择是有限的，需要新的药物输送策略3，4。
治疗超声波是一种新兴技术，可用于不同的神经学应用，如BBB中断（BBBD），神经调节，和消融4，5，6，7。为了通过颅骨实现BBB开口，通过颅骨发射器，聚焦超声波（FUS）与微泡结合。微泡介导的FUS导致大脑中药物的生物利用率增加，第5、8、9。在声波的存在下，微泡开始振荡，引发转细胞化，并破坏BBB内皮细胞之间的紧密交汇点，使较大的分子10的准细胞传输。先前的研究证实了声学发射的强度与生物对BBB开口11、12、13、14的生物影响之间的相关性。FUS与微泡结合已用于临床试验，用于治疗胶质母细胞瘤，使用特莫佐洛米德或脂质多索鲁比辛作为化疗剂，或用于治疗阿尔茨海默氏症和肌萎缩性侧索硬化症5，9，15，16。
由于超声波介质 BBB 的开启为药物治疗提供了全新的可能性，因此需要临床翻译的临床前研究来评估选定药物候选者的治疗反应。这通常需要具有高空间精度的高吞吐量工作流，最好是用于监控具有高可重复性的定向 BBB 开口的集成腔检测。如果可能，这些系统在初始投资和运行成本方面都需要具有成本效益，以便根据研究规模进行扩展。大多数前CFUS系统都与MRI相结合，用于图像指导和治疗规划15、17、18、19。虽然MRI提供了有关肿瘤解剖学和体积的详细信息，但它是一种昂贵的技术，通常由训练有素/熟练的操作员执行。此外，高分辨率MRI可能并不总是可供药物前设施的研究人员使用，并且需要每个动物长时间的扫描时间，因此它不太适合高通量的药理学研究。值得注意的是，对于神经肿瘤学领域的前科研究，特别是渗透肿瘤模型，可视化和瞄准肿瘤的可能性对于治疗成功至关重要。目前，这一要求仅通过 MRI 或用光蛋白转化的肿瘤来实现，从而实现生物发光成像 （BLI） 与光蛋白基板管理相结合的可视化。
MRI制导的FUS系统通常使用水浴来确保超声波传播用于颅面应用，即动物的头部部分淹没在水中，即所谓的"自下而上"系统15、17、18。虽然这些设计在较小的动物研究中通常效果良好，但在动物准备时间、便携性和实际可维护的卫生标准之间是一种妥协。作为核磁共振成像的替代方案，立体定向导航的其他制导方法包括使用啮齿动物解剖图集21、22、23、激光笔辅助目视24、针孔辅助机械扫描装置25或BLI26。这些设计大多是"自上而下"的系统，其中传感器被放置在动物的头顶上，动物处于自然位置。"自上而下"的工作流程包括22、25、26或充满水的圆锥体21、24。在封闭的圆锥体内使用传感器的好处是更紧凑的占地面积、更短的设置时间和直接的净化可能性简化了整个工作流程。
声场与微泡的相互作用是压力依赖的，范围从低振幅振荡（称为稳定气泡）到瞬态气泡崩塌（称为惯性气泡）27，28。有一个既定的共识，超声波BBBD需要一个声压远远高于稳定的气穴阈值，以实现成功的BBBD，但低于惯性气泡阈值，这通常与血管/神经损伤29。最常见的监测和控制形式是分析（背）散发出声信号使用被动腔检测（PCD），如麦克丹诺德等人建议的12。PCD 依赖于微泡发射信号的 Fourier 光谱分析，其中稳定气泡特征（谐波、亚和声波和超谐波）和惯性气泡标记（宽带响应）的强度和外观可以实时测量。
由于微泡配方的多散射性（振荡幅度强烈取决于气泡直径）、品牌间气泡壳特性的差异以及声学振荡（这在很大程度上取决于频率和压力30、31、32），用于精确压力控制的"一刀切"PCD 分析变得复杂。因此，提出了许多不同的PCD检测方案，这些协议已适应所有这些参数的特定组合，并已用于各种应用方案（从体外实验小动物协议到PCD用于临床使用），用于强力气穴检测，甚至用于压力11、14、30、31、32、33、34、35的追溯性反馈控制。本研究范围内采用的PCD协议直接来自麦克丹诺德等人12，并监测谐波发射是否存在稳定的空腔和宽带噪声，用于惯性气穴检测。
我们已经开发了一个图像引导神经导航FUS系统，用于BBB的短暂打开，以增加药物输送到大脑的帕奇马。该系统基于市售组件，可根据动物设施中可用的成像技术轻松适应多种不同的成像方式。由于我们需要高通量工作流程，我们选择使用 X 射线和 BLI 进行图像指导和治疗规划。用光蛋白（如红素酶）转化的肿瘤细胞适合BLI成像20。光蛋白基底施用后，肿瘤细胞可在体内监测，肿瘤生长和位置可确定20、36。BLI是一种低成本的成像方式，它能够随时间跟随肿瘤生长，具有快速的扫描时间，它与MRI36，37测量的肿瘤生长密切相关。我们选择用一个装满水的锥体代替水浴池，以灵活地移动安装啮齿动物的平台。设计基于一个可拆卸平台，配备集成了（I）小动物立体定向平台（II）与X射线和光学图像兼容性（III）快速可拆卸麻醉面膜和（IV）集成温度调节动物加热系统。麻醉后，动物被安装在平台上的精确位置，在整个过程中，它仍然存在。因此，整个平台通过整个干预工作流程的所有站点，同时保持准确和可重复的定位和持续麻醉。控制软件允许自动检测假象标记，并自动将所有类型的图像和图像模式（即微CT、X 射线、BLI 和荧光成像）注册到立体定向平台的参考框架中。在自动校准程序的帮助下，超声波传感器的焦距在内部被精确认识，从而实现了介入规划、声学传输和后续成像分析的自动融合。如图1和图2所示，该设置提供了高度的灵活性，设计专用的实验工作流程，并允许在不同的站点交错处理动物，从而促进高通量实验。我们已使用这项技术成功地在小鼠异种移植的高档胶质瘤，如弥漫中线胶质瘤。

<table><tbody><tr><td>1 mL luer-lock syringe</td><td>Becton Dickinson</td><td>309628</td><td>Plastipak</td></tr><tr><td>19 G needle</td><td>Terumo Agani</td><td>8AN1938R1</td><td></td></tr><tr><td>23 G needle</td><td>Terumo Agani</td><td>8AN2316R1</td><td></td></tr><tr><td>3M Transpore surgical tape</td><td>Science applied to life</td><td>7000032707</td><td>or similar</td></tr><tr><td>Arbitrary waveform generator</td><td>Siglent</td><td>n.a.</td><td>SDG1025, 25 MHz, 125 Msa/s</td></tr><tr><td>Automated stereotact</td><td>in-house built</td><td>n.a.</td><td>Stereotact with all elements were in-house built</td></tr><tr><td>Bruker In-Vivo Xtreme</td><td>Bruker</td><td>n.a.</td><td>Includes software</td></tr><tr><td>Buffered NaCl solution</td><td>B. Braun Melsungen AG</td><td>220/12257974/110</td><td></td></tr><tr><td>Buprenorfine hydrochloride</td><td>Indivior UK limitd</td><td>n.a.</td><td>0.324 mg</td></tr><tr><td>Cage enrichment: paper-pulp smart home</td><td>Bio services</td><td>n.a.</td><td></td></tr><tr><td>Carbon filter</td><td>Bickford</td><td>NC0111395</td><td>Omnicon f/air</td></tr><tr><td>Ceramic spoon</td><td>n.a</td><td>n.a.</td><td></td></tr><tr><td>Cotton swabs</td><td>n.a.</td><td>n.a.</td><td></td></tr><tr><td>D-luciferin, potassium salt</td><td>Gold Biotechnology</td><td>LUCK-1</td><td></td></tr><tr><td>Ethanol</td><td>VUmc pharmacy</td><td>n.a.</td><td>70%</td></tr><tr><td>Evans Blue</td><td>Sigma Aldrich</td><td>E2129</td><td></td></tr><tr><td>Fresenius NaCl 0.9%</td><td>Fresenius Kabi</td><td>n.a.</td><td>NaCl 0.9 %, 1000 mL</td></tr><tr><td>Histoacryl</td><td>Braun Surgical</td><td>n.a.</td><td>Histoacryl 0.5 mL</td></tr><tr><td>Hydrophone</td><td>Precision Acoustics</td><td>n.a.</td><td></td></tr><tr><td>Insulin syringe</td><td>Becton Dickinson</td><td>324825/324826</td><td>0.5 mL and 0.3 mL</td></tr><tr><td>Isoflurane</td><td>TEVA Pharmachemie BV</td><td>8711218013196</td><td>250 mL</td></tr><tr><td>Ketamine</td><td>Alfasan</td><td>n.a.</td><td>10 %, 10 mL</td></tr><tr><td>Mouse food: Teklad global 18% protein rodent diet</td><td>Envigo</td><td>2918-11416M</td><td></td></tr><tr><td>Neoflon catheter</td><td>Becton Dickinson</td><td>391349</td><td>26 GA 0.6 x 19 mm</td></tr><tr><td>Oscilloscope</td><td>Keysight technologies</td><td>n.a.</td><td>InfiniiVision DSOX024A</td></tr><tr><td>Plastic tubes</td><td>Greiner bio-one</td><td>210261</td><td>50 mL</td></tr><tr><td>Power amplifier</td><td>Electronics &amp;amp; Innovation Ltd</td><td>210L</td><td>Model 210L</td></tr><tr><td>Preamplifier DC Coupler</td><td>Precision Acoustics</td><td>n..</td><td>Serial number: DCPS94</td></tr><tr><td>Scissors</td><td>Sigma Aldrich</td><td>S3146-1EA</td><td>or similar</td></tr><tr><td>Sedazine</td><td>AST Farma</td><td>n.a.</td><td>2%</td></tr><tr><td>SonoVue microbubbles</td><td>Bracco</td><td>n.a.</td><td>8 &amp;micro;l/ml</td></tr><tr><td>Sterile water</td><td>Fresenius Kabi</td><td>n.a.</td><td>1000 mL</td></tr><tr><td>Syringe</td><td>n.a.</td><td>n.a.</td><td>various syringes can be used</td></tr><tr><td>Temgesic</td><td>Indivior UK limitd</td><td>n.a.</td><td>0.3 mg/ml</td></tr><tr><td>Transducer</td><td>Precision Acoustics</td><td>n.a.</td><td>1 MHz</td></tr><tr><td>Tweezers</td><td>Sigma Aldrich</td><td>F4142-1EA</td><td>or similar</td></tr><tr><td>Ultrasound gel</td><td>Parker Laboratories Inc.</td><td>01-02</td><td>Aquasonic 100</td></tr><tr><td>Vidisic gel</td><td>Bausch + Lomb</td><td>n.a.</td><td>10 g</td></tr></tbody></table>

a high-throughput image-guided stereotactic neuronavigation and focused ultrasound system for blood-brain barrier opening in rodents

血脑屏障 （BBB） 是治疗各种脑部疾病的主要障碍。内皮细胞，通过紧密的结点连接，形成生理屏障，阻止大分子（>500 Da）进入脑组织。微泡介导聚焦超声波 （FUS） 可用于诱导局部 BBB 开口，使较大的药物进入大脑帕奇马。
除了用于临床翻译的大型临床设备外，药物候选者治疗反应评估的临床前研究还需要专门的小型动物超声波设置，以便有针对性地打开 BBB。最好是，这些系统允许高吞吐量的工作流，既具有高空间精度，也可进行集成气穴监测，同时在初始投资和运行成本方面仍然具有成本效益。
在这里，我们展示了一个生物发光和X射线引导立体定向小型动物FUS系统，该系统基于市售组件，满足上述要求。特别强调高度自动化，以促进在大量药物前药物评估研究中通常遇到的挑战。这些挑战的例子是需要标准化，以确保数据可重复性，减少组内变异性，减少样本量，从而符合道德要求，减少不必要的工作量。拟议的BBB系统已在BBB开放范围内得到验证，方便了对患者衍生的胶质母细胞瘤多形式和弥漫中线胶质瘤模型的药物输送试验。

描述的FUS系统（图1和图2）和相关工作流程已用于100多只动物，并产生了健康小鼠和肿瘤携带小鼠的可重复数据。根据微泡泡泡注注峰值时记录的空腔和谐波的光谱密度，可以使用《议定书》第 4 步中解释的 Fourier 分析计算每个频率的光谱功率。基于声学协议（1 MHz，10ms脉冲持续时间），MI为0.4，结合微泡，在第二和第三谐波的标准化?...

Watch this Scientific Journal Video about A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents at JoVE.com

A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

血脑屏障 （BBB） 可以通过微泡介导的聚焦超声波 （FUS） 暂时中断。在这里，我们描述了一个逐步协议的高通量BBB打开体内使用模块化FUS系统，为非超声专家访问。

高通量图像引导立体定向神经导航和聚焦超声系统，用于在啮齿动物中打开血脑屏障

The blood-brain barrier (BBB) has been a major hurdle for the treatment of various brain diseases. Endothelial cells, connected by tight junctions, form a ...

a-high-throughput-image-guided-stereotactic-neuronavigation-focused

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

4.7K 次观看.  Cancer Center Amsterdam. 血脑屏障 （BBB） 可以通过微泡介导的聚焦超声波 （FUS） 暂时中断。在这里，我们描述了一个逐步协议的高通量BBB打开体内使用模块化FUS系统，为非超声专家访问。

视频: A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI引导下的破坏血脑屏障在大鼠模型经颅聚焦超声

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

科研

医学

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

神经科学

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

一个多认知功能测试，并记录在啮齿动物的神经元活动的完全自动化和高度灵活的系统

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

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 ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

自动图像引导贴片夹在脑切片神经元研究中的应用

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Real-Time Intravital Multiphoton Microscopy to Visualize Focused Ultrasound and Microbubble Treatments to Increase Blood-Brain Barrier Permeability

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles has emerged as an effective method to transiently and locally increase the permeability of the BBB, facilitating para- and transcellular transport of drugs across the BBB. Imaging the vasculature during ultrasound-microbubble treatment will provide valuable and novel insights on the mechanisms and dynamics of ultrasound-microbubble treatments in the brain.
Here, we present an experimental procedure for intravital multiphoton microscopy using a cranial window aligned with a ring transducer and a 20x objective lens. This set-up enables high spatial and temporal resolution imaging of the brain during ultrasound-microbubble treatments. Optical access to the brain is obtained via an open-skull cranial window. Briefly, a 3-4 mm diameter piece of the skull is removed, and the exposed area of the brain is sealed with a glass coverslip. A 0.82 MHz ring transducer, which is attached to a second glass coverslip, is mounted on top. Agarose (1% w/v) is used between the coverslip of the transducer and the coverslip covering the cranial window to prevent air bubbles, which impede ultrasound propagation. When sterile surgery procedures and anti-inflammatory measures are taken, ultrasound-microbubble treatments and imaging sessions can be performed repeatedly over several weeks. Fluorescent dextran conjugates are injected intravenously to visualize the vasculature and quantify ultrasound-microbubble induced effects (e.g., leakage kinetics, vascular changes). This paper describes the cranial window placement, ring transducer placement, imaging procedure, common troubleshooting steps, as well as advantages and limitations of the method.

This protocol describes the surgical and technical procedures that enable real-time in vivo multiphoton fluorescence imaging of the rodent brain during focused ultrasound and microbubble treatments to increase blood-brain barrier permeability.

实时活体内多光子显微镜可可视化聚焦超声和微气泡处理，以提高血脑屏障通透性

The blood-brain barrier (BBB) is a key challenge for the successful delivery of drugs to the brain. Ultrasound exposure in the presence of microbubbles ...

生物工程

A Benchtop Approach to the Location Specific Blood Brain Barrier Opening using Focused Ultrasound in a Rat Model

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic delivery including precise localization to a target brain region and reduction of off target side effects. However, stereotaxic surgery is highly invasive which limits its translational efficacy, requires long recovery times, and provides challenges when targeting multiple brain regions. Focused ultrasound (FUS) can be used in combination with circulating microbubbles to transiently open the blood brain barrier (BBB) in millimeter sized regions. This allows intracranial localization of systemically delivered agents that cannot normally cross the BBB. This technique provides a noninvasive alternative to stereotaxic surgery. However, to date this technique has yet to be widely adopted in neuroscience laboratories due to the limited access to equipment and standardized methods. The overall goal of this protocol is to provide a benchtop approach to FUS BBB opening (BBBO) that is affordable and reproducible and can therefore be easily adopted by any laboratory.

Focused ultrasound with microbubble agents can open the blood brain barrier focally and transiently. This technique has been used to deliver a wide range of agents across the blood brain barrier. This article provides a detailed protocol for the localized delivery to the rodent brain with or without MRI guidance.

在鼠模型中使用聚焦超声波打开位置特定血脑屏障开口的台面方法

Stereotaxic surgery is the gold standard for localized drug and gene delivery to the rodent brain. This technique has many advantages over systemic ...

Focused Ultrasound Induced Blood-Brain Barrier Opening for Targeting Brain Structures and Evaluating Chemogenetic Neuromodulation

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a combination of focused ultrasound (FUS) induced blood-brain barrier opening (FUS-BBBO), gene delivery with adeno-associated viral (AAV) vectors, and activation of cellular signaling with engineered, chemogenetic, protein receptors and their cognate ligands. With ATAC, it is possible to transduce both large and small brain regions with millimeter precision using a single noninvasive ultrasound application. This transduction can later allow for a long-term, noninvasive, device-free neuromodulation in freely moving animals using a drug. Since FUS-BBBO, AAVs, and chemogenetics have been used in multiple animals, ATAC should also be scalable for the use in other animal species. This paper expands upon a previously published protocol and outlines how to optimize the gene delivery with FUS-BBBO to small brain regions with MRI-guidance but without a need for a complicated MRI-compatible FUS device. The protocol, also, describes the design of mouse targeting and restraint components that can be 3D-printed by any lab and can be easily modified for different species or custom equipment. To aid reproducibility, the protocol describes in detail how the microbubbles, AAVs, and venipuncture were used in ATAC development. Finally, an example data is shown to guide the preliminary investigations of studies utilizing ATAC.

This protocol delineates steps necessary for the gene delivery through focused ultrasound blood brain barrier (BBB) opening, evaluation of the resulting gene expression, and measurement of neuromodulation activity of chemogenetic receptors through histological tests.

聚焦超声诱导血脑屏障打开，用于靶向大脑结构和评估化学遗传神经调控

Acoustically Targeted Chemogenetics (ATAC) allows for the noninvasive control of specific neural circuits. ATAC achieves such control through a ...

Enhancing Drug Delivery to the Brain: MRI-Guided Ultrasound Techniques in Rats

Monitoring Blood-Brain Barrier Opening in Rats with a Preclinical Focused Ultrasound System

The brain has a highly selective semipermeable blood barrier, termed the blood-brain barrier (BBB), which prevents the delivery of therapeutic macromolecular agents to the brain. The integration of MR-guided low-intensity pulsed focused ultrasound (FUS) with microbubble pre-injection is a promising technique for non-invasive and non-toxic BBB modulation. MRI can offer superior soft-tissue contrast and various quantitative assessments, such as vascular permeability, perfusion, and the spatial-temporal distribution of MRI contrast agents. Notably, contrast-enhanced MRI techniques with gadolinium-based MR contrast agents have been shown to be the gold standard for detecting BBB openings. This study outlines a comprehensive methodology involving MRI protocols and animal procedures for monitoring BBB opening in a rat model. The rat model provides the added benefit of jugular vein catheter utilization, which facilitates rapid medication administration. A stereotactic-guided preclinical FUS transducer facilitates the refinement and streamlining of animal procedures and MRI protocols. The resulting methods are characterized by reproducibility and simplicity, eliminating the need for specialized surgical expertise. This research endeavors to contribute to the optimization of preclinical procedures with rat models and encourage further investigation into the modulation of the BBB to enhance therapeutic interventions in neurological disorders.

This study describes a combined magnetic resonance imaging (MRI) and low-intensity pulsed focused ultrasound (FUS) protocol, utilizing living rats with jugular vein catheterization to monitor blood-brain barrier (BBB) opening.

The brain has a highly selective semipermeable blood barrier, termed the blood-brain barrier (BBB), which prevents the delivery of therapeutic ...

Focus Ultrasound Based Microbubble Mediated Blood-Brain Barrier Opening: A Technique to Create Localized Transient Openings in Blood-Brain Barrier of Mouse by Sonoporation

Source: Haumann, R. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/61269" target="_blank">A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in Rodents.</a> J. Vis. Exp. (2020)
This video describes a method of temporarily disrupting the blood-brain barrier (BBB) by creating transient openings using microbubble-mediated focused ultrasound (FUS). These transient local BBB openings can eventually be used to deliver larger-sized drug molecules directly into the brain parenchyma.

基于聚焦超声的微泡介导的血脑屏障开口:一种通过超声在小鼠血脑屏障中创建局部瞬时开口的技术

Source: Haumann, R. et al. A High-Throughput Image-Guided Stereotactic Neuronavigation and Focused Ultrasound System for Blood-Brain Barrier Opening in ...