作者对丹麦奥尔堡大学医院生物医学实验室工作人员分享的支持和技术经验表示感谢。

所描述的所有动物实验均在丹麦奥尔堡大学医院根据现有法律并经丹麦动物实验检查局批准（许可证号 2020-15-0201-00401）进行。本研究使用家猪，母猪，约 40 公斤，年龄 3 个月。有关所用试剂和设备的详细信息，请参见 材料表。
1. 主体住房
<ol><li>在手术前至少 7 天将受试者分组在批准的笔中进行 12 小时的光照/黑暗循环，以确保适应。</li>
</ol>2. 麻醉和监测
<ol><li>通过肌肉注射 2 mL/10 kg 含有噻来胺 6.25 mg/mL、氯胺酮 6.25 mg/mL、佐拉西泮 6.25 mg/mL、布托啡诺 1.25 mg/mL 和甲苯噻嗪 6.25 mg/mL 的混合物来镇静动物。</li>
	<li>将动物仰卧在加热毯上，以确保最佳体温调节。</li>
	<li>用 6.5 号管7 插管受试者，并根据呼气末 CO2 浓度 <6.0 kPa），用非湿化空气、潮气量为 8 mL/kg、呼吸频率为 16-20 呼吸/分钟进行机械通气。 注意：这还可以确保管子在气管内的正确放置。</li>
	<li>通过吸入 1% 至 2% 的七氟烷来维持麻醉。</li>
	<li>小心地将眼药膏涂抹在受试者的双眼上，以防止麻醉时干燥。</li>
	<li>通过检查睫状反射并相应地调整七氟烷来确保麻醉程度。</li>
	<li>将带有温度计的膀胱导管通过尿道插入受试者的膀胱，以监测温度并将尿液收集在导尿袋中。</li>
	<li>在整个手术过程中监测动物的生命体征。 注意：生命体征包括脉搏、连续动脉血压、体温和呼气末 CO2。</li>
	<li>通过经皮穿刺将中心静脉导管（6 Fr 护套）插入右颈静脉，并在研究结束时将其用于连续盐水（NaCl，0.9%）输注、药物输注和安乐死。</li>
</ol>3. 动物定位
<ol><li>将受试者置于俯卧位，抬起头部并用沙袋稳定，直到额骨几乎水平，以确保最佳定位。 注意： 确保头部在一定程度上固定以防止不必要的移动。</li>
	<li>使用修剪器或剃须刀剃掉手术部位的毛发。</li>
</ol>4. 手术器材的准备
<ol><li>准备 材料表中列出的手术设备。</li>
</ol>5. 露出额骨
<ol><li>确定每个眶上嵴的颈项突起和尾部（图 1）以定义预期的矢状中线。</li>
	<li>首先使用 24 号手术刀切开中线切口，切开皮肤和腱膜，切开额骨骨膜。</li>
	<li>用手术拭子擦去血液。</li>
	<li>使用 12 毫米扁平的咬骨逐渐将 galea 腱膜与切口周围的下额骨分离。</li>
	<li>使用手术牵开器分离 galea 腱膜并暴露下面的额骨（图 2）。</li>
</ol>6. 确定暴露额骨的解剖标志
<ol><li>确定矢状缝合线作为解剖中线和冠状缝合线的参考（图2）。</li>
	<li>通过手动触诊识别三种骨骼结构：颈项突起和两个眶上嵴的尾部（图 1 和 图 3）。 注意：这些标志形成一个三角形，在该三角形内进行开颅手术（图 1 和 图 4A）。</li>
</ol>7. 进入硬脑膜
<ol><li>使用带有圆形金刚石涂层毛刺的高速气动钻头（图 3A），在先前定义的三角形的边界内定义矩形的每个角。</li>
	<li>用 4 mm 圆形金刚石毛刺连接每个角，以确保开口的正确位置（图 4B）。</li>
	<li>用 4 毫米的圆形金刚石毛刺逐渐变薄额骨，直到硬脑膜暴露出来。 注意：避免与中线的硬脑膜进行第一个接触点，对应于矢状缝合（图 1），因为大的背侧矢状窦位于此处。此时穿孔可导致严重的静脉出血。</li>
	<li>使用与硬脑膜的第一个接触点目视评估剩余额骨的厚度。</li>
	<li>继续使用 4 毫米圆角金刚石毛刺小心地在定义的矩形中减薄额骨。 注意：保存骨粉以备以后从静脉或额骨暴露的海绵状出血中止血。</li>
	<li>将 3 毫米的解剖器滑到骨板周围足够薄的骨头下，然后用轻微的手动压力将其凿开，以扩大开口至硬脑膜。 注意：在钻孔和削掉骨头之间交替进行，直到露出骨板周围的硬脑膜的多个点。</li>
	<li>用注射器涂抹无菌盐水以清除视野。</li>
</ol>8. 去除骨板
<ol><li>将 3 毫米的解剖器插入骨板下方，并逐渐向下按压其手柄以折断骨板。 注意：现在应该暴露下面的硬脑膜，露出两个大脑半球的轮廓（图 5 和 图 6）。</li>
	<li>通过目视检查脑脊液 （CSF） 渗漏来评估硬脑膜的完整性。 注意： 如果硬脑膜没有明显的缺陷，两个半球都会在同步脉动中升高。</li>
	<li>使用节省的骨粉或小心地使用低电压烧灼进行单极或双极凝血，以阻止轻微的静脉出血。 注意：预计会有来自使者静脉或复叶静脉的轻微静脉出血。</li>
</ol>9. 保护裸露的硬脑膜
<ol><li>用浸泡在无菌盐水中的无菌手术拭子覆盖暴露的硬脑膜，以防止皮下组织变干。</li>
</ol>10. 插入微量透析导管 （MDC）
<ol><li>使用背侧矢状窦作为中线的参考。将微量透析导管 （MDC） 置于一次性 18 G 针头的管腔内。用针头沿颅嘴方向以 10°-15° 角从背矢状窦横向刺入硬脑膜 10 毫米，直到针头 20 毫米位于组织内。</li>
	<li>慢慢抽出针头，同时确保 MDC 保持在浅表大脑皮层内的相同位置。</li>
	<li>将导管牢固地固定在附近的皮肤上，以防止 MDC 脱位。 注意： 确保 MDC 的尖端不接触针的锋利尖端，以免损坏。</li>
	<li>使用背侧矢状窦作为中线参考重复该过程。如上所述将 MDC 放入针头内，并在针头垂直（90° 角）的情况下从中线 15 毫米处穿透对侧半球的硬脑膜。<ol><li>将针头插入 20 毫米，然后按照说明缓慢抽出，将 MDC 留在皮质下脑组织内。如上所述固定 MDC。</li>
	</ol></li>
	<li>准备一个带有 2 G 一次性针头的 18 mL 一次性塑料注射器。用 0.5 mL 无菌生理盐水填充注射器。</li>
	<li>从中线横向穿透硬脑膜 20 毫米，同时保持针头与中线成 45° 角。一次缓慢引入针头 1 毫米，同时在插入的每个步骤中轻轻吸出。</li>
	<li>每次抽吸期间观察注射器，直到获得脑脊液 （CSF）。</li>
	<li>将针头与注射器分开，同时保持针头位于相同的解剖位置。通过针头将 MDC 插入侧脑室，直到感觉到阻力。</li>
	<li>如上所述，用手术胶带牢固地固定 MDC。</li>
</ol>11. 微透析 （MD）
<ol><li>将每个微量透析导管 （MDC） 连接到单独的微量透析泵。</li>
	<li>通过将无菌盐水泵入 MDC 并将其收集在合适的样品中来启动微透析过程。通过观察每个样品中的盐水，确保流经每个 MDC。</li>
	<li>以 1 μL/min 的流速开始 30 分钟的连续微透析的组织校准期。</li>
</ol>12. 安乐死
<ol><li>通过中心静脉导管推注静脉注射戊巴比妥 （50 mg/kg）（遵循机构批准的方案）。</li>
	<li>观察呼吸器上的脉搏、血压和呼气末 CO2 曲线，寻找平坦线，以确认心脏骤停。</li>
</ol>

一种有效的高速气动钻头开颅术以进入猪脑

<ol>
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作者没有需要声明的利益冲突。

演示的程序涉及几个关键步骤。首先，由于猪颅骨的组成，准确规划开颅手术的位置至关重要。由于猪额骨的厚度在外侧边缘增加，因此将开口过于外侧放置11 会使钻孔过程中难以到达硬脑膜。此外，在中线内正确定位开口对于降低对下面的背侧矢状窦意外损伤的风险很重要。然而，重要的是要注意，猪的颅骨组成表现出显着的个体差异，不幸的是，并非所有猪都表现出明确的矢状缝或冠状缝合，11。因此，该程序中介绍的方法依赖于更一般的解剖标志，例如颈项嵴和眼眶突起，以提高定位开颅手术的准确性。
虽然高速的圆形钻头很有效，但它也会导致意外硬脑膜损伤的高风险，尤其是在初始接触点。循序渐进的实践经验有助于降低这种风险。然而，与普通手动钻头等替代品相比，气动高速钻头通常很昂贵，这是一个很大的局限性。有人可能会争论通过使用手动颅钻进行初始硬脑膜接触，然后是有脚颅骨来改进该程序。这种方法可能会降低硬脑膜病变的风险，因为硬脑膜不会暴露在高速移动的物体上。然而，根据我们的经验，猪额骨内鼻窦的厚度和分布不同，这使得使用常规颅骨具有挑战性。
所提出的方法的总体目的是通过实用、简单且省时的程序实现对猪大脑大部分的视觉访问。我们相信，与钻孔开颅手术等侵入性较小的手术相比，所演示的手术具有多项优势。首先，与单个钻孔开颅手术 （0.7-1.5 cm2，取决于钻头的直径） 相比，这种方法暴露的猪脑比例明显更大 （约 20 cm2）。这不仅允许对大脑进行大量工具化（例如，插入心室引流管、神经元传感器、多个导管、探针或刺激电极），而且还使用可见结构（如外侧半球边界、脑回轮廓或背侧矢状窦）增强对特定大脑区域的导航。其次，如果操作得当，大脑本身在手术过程中不会受损，这可以通过经验丰富的从业者相对较快地实现。
尽管在非存活猪研究中，演示的程序用于描述莫西沙星的脑渗透12，但我们认为这种方法也适用于生存研究。重新插入骨板并通过缝合线闭合覆盖的皮肤组织将使猪在手术后醒来时不会受伤。然而，这只有在底层大脑皮层相对不受可能的工具化伤害的情况下，这才在实际和伦理上可行，从而确保完整的神经状态。这种性质的生存研究需要严格的消毒技术，并在术后密切监测猪是否因手术创伤或出血而出现神经功能缺损的迹象，例如硬膜外血肿。
总的来说，我们相信所提出的方法简单、可重复，并且在与中枢神经系统 （CNS） 相关的各种未来猪模型中具有重要应用，特别是在需要对其他不间断的猪脑进行大量工具化和/或监测的情况下。

一般来说，当实际和/或伦理限制禁止使用人类患者来检查疾病或测试手术方法时，会使用动物模型。建立新颖的动物模型通常是为了提供对人类状况具有转化价值的新知识。出于实际和经济考虑，经常使用啮齿动物，但它们对人类的转化价值有限，尤其是由于巨大的解剖学差异1。然而，与啮齿动物相比，猪具有几个优势。猪不仅与人类具有几个关键的解剖学、生理学、代谢和遗传学特征，而且猪器官系统的大小可以与体重相匹配，类似于人类器官 2,3。这使得猪在外科动物模型和程序训练中具有独特的作用4。尽管与使用啮齿动物相比，使用猪模型需要一定的实践和财务能力，但与使用非人类灵长类动物相比，猪在经济和道德上都提供了更容易接受的选择。
猪脑在转化神经科学研究中特别感兴趣。首先，猪脑的结构与人脑相似，因为两者都以白质为主，脑回 3,5,6。其次，与啮齿动物相比，猪的大脑尺寸更大，因此可以使用手术设备和相当于临床环境中使用的各种成像方式 7,8。因此，近几十年来，各种猪模型已广泛用于神经科学研究9。然而，这些猪 CNS 模型中的大多数需要直接分析脑组织，这可以通过多种方式获得（例如，植入导管或电极、组织活检等）。10. 由于这些方式中的大多数都需要一定程度的仪器化和直接进入大脑，因此必须考虑不同的手术进入方法。
该方法包括对一头镇静的 3 个月大的雌性丹麦长白猪进行额骨前颅切除术。这份手稿的总体目的是描述一种使用气动高速钻头通过开颅手术暴露大部分腹侧猪脑的方法。第一步是将拍摄对象放置在合适的位置，头部升高。由于猪的颅骨与人类的颅骨完全不同，因此第二步涉及使用各种解剖标志规划开颅手术的放置。第三步是进入覆盖两个半球的底层硬脑膜而不损坏它。

<table><tbody><tr><td>10 mL plastic syrringes</td><td>Becton, Dickinson and Company</td><td>303219</td><td></td></tr><tr><td>107 Microdialysis pump</td><td>M Dialysis</td><td>P000127</td><td>&amp;nbsp;107 Microdialysis Pump</td></tr><tr><td>2 mL plastic syrringes</td><td>Becton, Dickinson and Company</td><td>300928</td><td></td></tr><tr><td>25 mm, 18 G needles</td><td>Becton, Dickinson and Company</td><td>304100</td><td></td></tr><tr><td>Bair Hugger heater</td><td>3M</td><td>B5005241003</td><td></td></tr><tr><td>Bair Hugger heating blanket</td><td>3M</td><td>B5005241003</td><td></td></tr><tr><td>Batery for microdialysis pump</td><td>M Dialysis</td><td>8001788</td><td>Battery 6V, 106 &amp;amp; MD Pump</td></tr><tr><td>Dissector</td><td>Karl Storz</td><td>223535</td><td>Flattended 3 mm dissector</td></tr><tr><td>Endotracheal tube size 6.5</td><td>DVMed</td><td>DVM-107860</td><td>Cuffed endotracheal tube</td></tr><tr><td>Euthasol Vet</td><td>Dechra Veterinary Products A/S</td><td>380019</td><td>phentobarbital for euthanazia, 400 mg/mL</td></tr><tr><td>Farabeuf Rougine</td><td>Mahr Surgical</td><td></td><td>Flat headed rougine (12 mm)</td></tr><tr><td>Foley Catheter 12 F</td><td>Becton, Dickinson and Company</td><td>D175812E</td><td>Catherter with in-built thermosensor</td></tr><tr><td>Intravenous sheath</td><td>Coris Avanti</td><td></td><td>Avanti Cordis Femoral Sheath 6 F</td></tr><tr><td>Microdialysis brain catheters</td><td>M Dialysis</td><td>P000050</td><td>membrane length 10 mm -shaft 100 mm 4/pkg</td></tr><tr><td>Microdialysis syringe</td><td>M Dialysis</td><td>8010191</td><td>&amp;nbsp;106 Pump&amp;nbsp;Syringe 20/pkg</td></tr><tr><td>Microvials for microdialysis sampling</td><td>M Dialysis</td><td>P000001</td><td>Microvials 250/pkg</td></tr><tr><td>Operating table</td><td></td><td></td><td></td></tr><tr><td>Pneumatic high-speed drill</td><td>Medtronic</td><td></td><td>Medtronic Midas Rex 7 drill</td></tr><tr><td>Primus respirator</td><td>Dr&amp;auml;ger</td><td></td><td>Respirator with in-built vaporiser for supplementary Sevofluran anesthesia</td></tr><tr><td>Rounded diamond drill</td><td>Medtronic</td><td>7BA40D-MN</td><td></td></tr><tr><td>Self-retaining retractor</td><td>World Precission Instruments</td><td>501722</td><td>Weitlander retractor, self-retaining, 14 cm blunt</td></tr><tr><td>Sterile Saline</td><td>Fresnius Kabi</td><td>805541</td><td>1000 mL</td></tr><tr><td>Sterile surgical swaps</td><td></td><td></td><td></td></tr><tr><td>Surgical scalpel no 24</td><td>Swann Morton</td><td>5.03396E+12</td><td>Swann Morton Sterile Disposable Scalpel No. 24</td></tr><tr><td>Zoletil Vet</td><td>Virbac</td><td></td><td>Medical mixture for induction of anesthesia</td></tr></tbody></table>

accessing the porcine brain via high-speed pneumatic drill craniectomy

使用猪作为实验动物模型在神经科学研究中尤为重要，因为猪和人类中枢神经系统 （CNS） 具有许多重要的功能和结构特性。因此，预计猪在未来各种神经系统疾病的研究中将发挥越来越重要的作用。在这里，描述了一种通过猪额骨进行前颅切除术的方法。在中线切口和随后暴露猪额骨后，使用解剖标志来确保开颅手术的最佳位置。通过用圆钻头小心地逐渐变薄额骨，可以实现硬脑膜和下脑半球的矩形开口。所提出的方法需要一定的手术材料，包括气动高速钻，以及一定程度的手术经验。潜在的并发症包括硬脑膜或矢状窦背侧的意外病变。然而，该方法简单、省时，并为研究人员提供了高度的可重复性。如果执行得当，该技术会暴露出大部分未受影响的猪脑，用于各种神经监测或分析。

猪头的俯卧位置为外科医生在手术过程中提供了最佳通道，而使用稳定沙袋可降低钻孔时猪头位置意外移动的风险。
在此演示期间，使用猪上颅骨（眶上嵴和颈项嵴）的浅表解剖标志（图 1 和 图 3）在切开之前精确识别居中的矢状线。切开并移除 galea 腱膜后，确定矢状缝线以确定真正的解剖中线（图2）。暴露额骨后，使用三个可触及的标志（图 1 和 图 3）和矢状缝合线（图 2）来定义一个三角形，在该三角形内确定开颅手术的边界在所需位置（图 4A）。这些标志非常适合该程序，因为它们与猪脑密切相关（图 7）。
在沿开颅手术的边界逐渐钻孔和减薄额骨后（图 4B），实现了与底层硬脑膜的接触点，从而可以评估额骨的剩余厚度。
最后，在小心地去除额骨后，揭示了覆盖两个大脑半球三分之二的颅骨的底层硬脑膜。该程序被认为是成功的，因为 （1） 揭示的底层硬脑膜是完整的（图 6）;（2） 额骨的开口居中，由背侧矢状窦的位置证实（图5）;（3） 仅沿暴露的海绵骨发生轻微静脉出血。
通过目视检查较小的缺损是否有明显的脑脊液 （CSF） 渗漏来评估硬脑膜的完整性。这些泄漏会与两个半球的脉动同步脉动。确认硬脑膜完整，因为没有观察到肉眼的 CSF 渗漏，并且两个半球都与搏动同步升高和变圆（图 6）。半球的这种升高外观表明 CSF 内的净正压，相当于完整的脑膜。
开颅手术的正确中心位置至关重要，原因有几个。首先，它确保了两个半球的最大暴露，以便以后的仪器化。其次，它降低了钻头意外损坏背侧矢状窦的风险。取出骨板后确认了正确的位置，两个大脑半球的轮廓被矢状窦背侧上方的裂缝对称地隔开（图4）。
预计硬脑膜和暴露的小梁都会有轻微的静脉出血。这可以通过涂抹开颅手术产生的骨尘几分钟来实现止血。如果这还不够，可以通过使用单极或双极烧灼对出血源进行低压烧灼来确保止血。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66788/66788fig01.jpg"> 图 1：暴露的额骨图像。 三个解剖标志通过手动触诊确定：颈项突起和每个眶上嵴的尾部。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66788/66788fig01large.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/66788/66788fig02.jpg"> 图 2：解剖后暴露的额骨图像，揭示了矢状缝和冠状缝。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66788/66788fig02large.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/66788/66788fig03.jpg"> 图 3：3 维计算机断层扫描。 从前外侧视角 （A） 和后上视角 （B） 可视化的猪颅骨后部扫描。额骨的解剖标志以红色（眶上嵴）和蓝色（颈项嵴）突出显示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66788/66788fig03large.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/66788/66788fig04.jpg"> 图 4：暴露的额骨图像。 （A） 使用颈项突起和眶上嵴作为标志来定义矩形开颅手术的每个角落。（B） 通过连接每个角来钻开颅手术的初始线。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66788/66788fig04large.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/66788/66788fig05.jpg"> 图 5：暴露的硬脑膜与下面的大脑半球和矢状缝合线的描述。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66788/66788fig05large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66788/66788fig06.jpg"> 图 6：从额叶角度看到的硬脑膜和底层大脑半球的图像。 如果硬脑膜完好无损，则两个半球都会升高并与脉动同步变圆（用白色规定线标记）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66788/66788fig06large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66788/66788fig07.jpg"> 图 7：水平计算机断层扫描。 猪颅骨扫描揭示了眶后嵴（红色）、颈嵴（蓝色）和大脑（黄色）之间的解剖关系。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66788/66788fig07large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

作者聚焦：一种通过颅骨切除术进入猪脑的可重复且有效的方法

该协议描述了使用高速气动钻对 3 个月大的丹麦长白猪进行开颅手术。通过额骨进入，露出腹侧硬脑膜和下面的大脑半球。该程序允许进入猪脑的大部分。

通过高速气动钻头开颅术进入猪脑

The use of pigs as an experimental animal model is especially relevant in neuroscience research, as the porcine and human central nervous systems (CNS) ...

accessing-the-porcine-brain-via-high-speed-pneumatic-drill-craniectomy

Nanyang Technological University

Research

JoVE Journal

Neuroscience

Accessing the Porcine Brain via High-Speed Pneumatic Drill Craniectomy

314 次观看.  Aalborg University Hospital. 我们的研究目标是开发一种可重复且有效的方法，通过小管切除术进入猪脑，允许进行广泛的神经监测和其他分析。通过利用猪脑和人脑之间的解剖学相似性，我们的目标是加强转化神经科学研究。我们成功开发了一种猪模型，用于持续监测健康脑组织和脑脊髓液中抗生素药物的脑内药代动力学。我们目前正在寻求通过免疫反应诱导可控的脑炎...

视频: 作者聚焦：一种通过颅骨切除术进入猪脑的可重复且有效的方法

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

一种慢性脑功能成像开颅手术过程

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

科研

生物学

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

侧向液压冲击：创伤性脑损伤小鼠模型

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

神经科学

Targeted Labeling of Neurons in a Specific Functional Micro-domain of the Neocortex by Combining Intrinsic Signal and Two-photon Imaging

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, direction5-7, ocular dominance8,9 and binocular disparity9. Orientation selectivity is the most widely studied feature and a continuous map with a quasi-periodic layout for preferred orientation is present across the entire primary visual cortex10,11. Integrating the synaptic, cellular and network contributions that lead to stimulus selective responses in these functional maps requires the hybridization of imaging techniques that span sub-micron to millimeter spatial scales. With conventional intrinsic signal optical imaging, the overall layout of functional maps across the entire surface of the visual cortex can be determined12. The development of in vivo two-photon microscopy using calcium sensitive dyes enables one to determine the synaptic input arriving at individual dendritic spines13 or record activity simultaneously from hundreds of individual neuronal cell bodies6,14. Consequently, combining intrinsic signal imaging with the sub-micron spatial resolution of two-photon microscopy offers the possibility of determining exactly which dendritic segments and cells contribute to the micro-domain of any functional map in the neocortex. Here we demonstrate a high-yield method for rapidly obtaining a cortical orientation map and targeting a specific micro-domain in this functional map for labeling neurons with fluorescent dyes in a non-rodent mammal. With the same microscope used for two-photon imaging, we first generate an orientation map using intrinsic signal optical imaging. Then we show how to target a micro-domain of interest using a micropipette loaded with dye to either label a population of neuronal cell bodies or label a single neuron such that dendrites, spines and axons are visible in vivo. Our refinements over previous methods facilitate an examination of neuronal structure-function relationships with sub-cellular resolution in the framework of neocortical functional architectures.

A method is described for labeling neurons with fluorescent dyes in predetermined functional micro-domains of the neocortex. First, intrinsic signal optical imaging is used to obtain a functional map. Then two-photon microscopy is used to label and image neurons within a micro-domain of the map.

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

In the primary visual cortex of non-rodent mammals, neurons are clustered according to their preference for stimulus features such as orientation1-4, ...

A Method to Make a Craniotomy on the Ventral Skull of Neonate Rodents

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain in adulthood and during development. Although most in vivo approaches use a craniotomy to study brain regions located on the dorsal side, brainstem regions such as the pons, located on the ventral side remain relatively understudied. The main goal of this protocol is to facilitate access to ventral brainstem structures so that they can be studied in vivo using electrophysiological and imaging methods. This approach allows study of structural changes in long-range axons, patterns of electrical activity in single and ensembles of cells, and changes in blood brain barrier permeability in neonate animals. Although this protocol has been used mostly to study the auditory brainstem in neonate rats, it can easily be adapted for studies in other rodent species such as neonate mice, adult rodents and other brainstem regions.

A surgical method is described to expose the ventral skull in neonate rats. Using this approach it is possible to open a craniotomy to perform acute electrophysiology and two-photon microscopy experiments in the brainstem of anesthetized pups.

方法以使对新生儿鼠害的腹头骨开颅手术

The use of a craniotomy for in vivo experiments provides an opportunity to investigate the dynamics of diverse cellular processes in the mammalian brain ...

Exposure of the Pig CNS for Histological Analysis: A Manual for Decapitation, Skull Opening, and Brain Removal

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to non-human primates. The large brain size of the pig allows the use of conventional clinical brain imagers and the direct use and testing of neurosurgical procedures and equipment from the human clinic. Further macroscopic and histological analysis, however, requires postmortem exposure of the pig central nervous system (CNS) and subsequent brain removal. This is not an easy task, as the pig CNS is encapsulated by a thick, bony skull and spinal column. The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and the pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

The goal of this paper and instructional video is to describe how to expose and remove the postmortem pig brain and pituitary gland in an intact state, suitable for subsequent macroscopic and histological analysis.

猪中枢神经系统的组织学分析的曝光:一个手册斩首，颅骨打开，并去除脑

Pigs have become increasingly popular in large-animal translational neuroscience research as an economically and ethically feasible substitute to ...

Subdural Soft Electrocorticography (ECoG) Array Implantation and Long-Term Cortical Recording in Minipigs

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help delineate the epileptic region to resect. In long-term applications such as brain-computer interfaces, these epicortical electrodes are used to record the movement intention of the brain, to control the robotic limbs of paralyzed patients. However, current stiff electrode grids do not answer the need for high-resolution brain recordings and long-term biointegration. Recently, conformable electrode arrays have been proposed to achieve long-term implant stability with high performance. However, preclinical studies for these new implant technologies are needed to validate their long-term functionality and safety profile for their translation to human patients. In this context, porcine models are routinely employed in developing medical devices due to their large organ sizes and easy animal handling. However, only a few brain applications are described in the literature, mostly due to surgery limitations and integration of the implant system on a living animal.
Here, we report the method for long-term implantation (6 months) and evaluation of soft ECoG arrays in the minipig model. The study first presents the implant system, consisting of a soft microfabricated electrode array integrated with a magnetic resonance imaging (MRI)-compatible polymeric transdermal port that houses instrumentation connectors for electrophysiology recordings. Then, the study describes the surgical procedure, from subdural implantation to animal recovery. We focus on the auditory cortex as an example target area where evoked potentials are induced by acoustic stimulation. We finally describe a data acquisition sequence that includes MRI of the whole brain, implant electrochemical characterization, intraoperative and freely moving electrophysiology, and immunohistochemistry staining of the extracted brains.
This model can be used to investigate the safety and function of novel design of cortical prostheses; mandatory preclinical study to envision translation to human patients.

Subdural Soft Electrocorticography ECoG Array Implantation and Long-Term Cortical Recording in Minipigs

Here, we present a method for the long-term performance and safety assessment of soft subdural electrode arrays in a minipig model, describing surgical method and tools, postoperative magnetic resonance imaging, electrophysiology of the auditory cortex, electrochemical properties of the implant, and postmortem immunochemistry.

硬膜下软皮质电图 （ECoG） 阵列植入和小型猪的长期皮质记录

Neurological impairments and diseases can be diagnosed or treated using electrocorticography (ECoG) arrays. In drug-resistant epilepsy, these help ...

生物工程

Murine Model of Controlled Cortical Impact for the Induction of Traumatic Brain Injury

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the United States. In fact, TBI is a contributing factor to over a third of all injury-related mortality. Nonetheless, the cellular and molecular mechanisms underlying the pathophysiology of TBI are poorly understood. Thus, preclinical models of TBI capable of replicating the injury mechanisms pertinent to TBI in human patients are a critical research need. The controlled cortical impact (CCI) model of TBI utilizes a mechanical device to directly impact the exposed cortex. While no model can full recapitulate the disparate injury patterns and heterogeneous nature of TBI in human patients, CCI is capable of inducing a wide range of clinically applicable TBI. Furthermore, CCI is easily standardized allowing investigators to compare results across experiments as well as across investigative groups. The following protocol is a detailed description of applying a severe CCI with a commercially available impacting device in a murine model of TBI.

Here we describe a protocol for the induction of murine traumatic brain injury via an open-head controlled cortical impact.

创伤性脑损伤诱导受控皮质冲击的母模型

The Centers for Disease Control and Injury Prevention estimate that almost 2 million people sustain a traumatic brain injury (TBI) every year in the ...

Extraction and Dissection of the Domesticated Pig Brain

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular systems, gastrointestinal systems, and nutrition, and the pig is increasingly being used as a large animal model in neuroscience. Furthermore, the pig is an accepted model to study neurodevelopment as it displays brain growth and development patterns similar to what occurs in humans. As a less common animal model in neuroscience, surgical and dissection procedures on pigs may not be as familiar or well-practiced among researchers. Therefore, a standardized visual protocol detailing consistent extraction and dissection methods may prove valuable for researchers working with the pig. The following video showcases a technique to remove the pig brain while keeping the cortex and brainstem intact and reviews methods to dissect several commonly investigated brain regions including the brainstem, cerebellum, midbrain, hippocampus, striatum, thalamus, and medial prefrontal cortex. The purpose of this video is to provide researchers with the tools and knowledge necessary to consistently perform a brain extraction and dissection on the four-week-old pig.

This protocol details the technique for removal of the pig brain in its entirety and dissection of several brain regions commonly studied in neuroscience.

驯养猪脑的提取和解剖

Use of the pig as a preclinical and translatable animal model has been well-documented and accepted by research fields investigating cardiovascular ...

Direct Cannula Implantation in the Cisterna Magna of Pigs

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces and has been implicated in the clearance of neurotoxic peptides such as amyloid-beta. Impaired glymphatic function exacerbates disease pathology in animal models of neurodegenerative diseases, such as Alzheimer's, which highlights the importance of understanding this clearance system. The glymphatic system is often studied by cisterna magna cannulations (CMc), where tracers are delivered directly into the cerebrospinal fluid (CSF). Most studies, however, have been carried out in rodents. Here, we demonstrate an adaptation of the CMc technique in pigs. Using CMc in pigs, the glymphatic system can be studied at a high optical resolution in gyrencephalic brains and in doing so bridges the knowledge gap between rodent and human glymphatics.

This article presents a step-by-step protocol for the direct cannula implantation in the cisterna magna of pigs.

直接插管植入猪的蓄水池

The glymphatic system is a waste clearance system in the brain that relies on the flow of cerebrospinal fluid (CSF) in astrocyte-bound perivascular spaces ...

Assessment of Thermal Damage from Robot-Drilled Craniotomy for Cranial Window Surgery in Mice

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, when performing any craniotomy by hand, there is often thermal damage to brain tissue, which is inherently variable surgery-to-surgery and may be dependent on individual surgeon technique. Implementing a surgical robot can standardize surgery and lead to a decrease in thermal damage associated with surgery. In this study, three methods of robotic drilling were tested to evaluate thermal damage: horizontal, point-by-point, and pulsed point-by-point. Horizontal drilling utilizes a continuous drilling schematic, while point-by-point drills several holes encompassing the cranial window. Pulsed point-by-point adds a &#34;2 s on, 2 s off&#34; drilling scheme to allow for cooling in between drilling. Fluorescent imaging of Evans Blue (EB) dye injected intravenously measures damage to brain tissue, while a thermocouple placed under the drilling site measures thermal damage. Thermocouple results indicate a significant decrease in temperature change in the pulsed point-by-point (6.90 &#176;C &#177; 1.35 &#176;C) group compared to the horizontal (16.66 &#176;C &#177; 2.08 &#176;C) and point-by-point (18.69 &#176;C &#177; 1.75 &#176;C) groups. Similarly, the pulsed point-by-point group also showed significantly less EB presence after cranial window drilling compared to the horizontal method, indicating less damage to blood vessels in the brain. Thus, a pulsed point-by-point drilling method appears to be the optimal scheme for reducing thermal damage. A robotic drill is a useful tool to help minimize training, variability, and reduce thermal damage. With the expanding use of multiphoton imaging across research labs, it is important to improve the rigor and reproducibility of results. The methods addressed here will help inform others of how to better use these surgical robots to further advance the field.

Cranial windows have become a ubiquitously implemented surgical technique to allow for intravital imaging in transgenic mice. This protocol describes the use of a surgical robot that performs semi-automated bone drilling of cranial windows and can help reduce surgeon-to-surgeon variability and partially mitigate thermal blood-brain barrier damage.

机器人钻孔开颅术对小鼠颅窗手术热损伤的评估

Cranial window surgery allows for the imaging of brain tissue in live mice with the use of multiphoton or other intravital imaging techniques. However, ...

通过高速气动钻头开颅术进入猪脑

副本

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此视频中的章节

一种慢性脑功能成像开颅手术过程

侧向液压冲击：创伤性脑损伤小鼠模型

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

方法以使对新生儿鼠害的腹头骨开颅手术

猪中枢神经系统的组织学分析的曝光:一个手册斩首，颅骨打开，并去除脑

硬膜下软皮质电图（ECoG）阵列植入和小型猪的长期皮质记录

创伤性脑损伤诱导受控皮质冲击的母模型

驯养猪脑的提取和解剖

直接插管植入猪的蓄水池

机器人钻孔开颅术对小鼠颅窗手术热损伤的评估

通过高速气动钻头开颅术进入猪脑

副本

摘要

探索更多视频

此视频中的章节

一种慢性脑功能成像开颅手术过程

侧向液压冲击：创伤性脑损伤小鼠模型

有针对性的特定功能的微域的新皮质相结合的内源信号和双光子成像标记的神经元

方法以使对新生儿鼠害的腹头骨开颅手术

猪中枢神经系统的组织学分析的曝光:一个手册斩首，颅骨打开，并去除脑

硬膜下软皮质电图 （ECoG） 阵列植入和小型猪的长期皮质记录

创伤性脑损伤诱导受控皮质冲击的母模型

驯养猪脑的提取和解剖

直接插管植入猪的蓄水池

机器人钻孔开颅术对小鼠颅窗手术热损伤的评估

硬膜下软皮质电图（ECoG）阵列植入和小型猪的长期皮质记录