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。

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	<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>&#160;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 &#38; 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>&#160;106 Pump&#160;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&#228;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/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/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/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/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/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/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/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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Accessing the Porcine Brain via High-Speed Pneumatic Drill Craniectomy

270 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

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

用于访问视网膜下腔的另类和验证注射法<i&gt;通过</i&gt;一经巩膜后路

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

科研

神经科学

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven therapies for TBI have been developed. This paper presents a novel method for pre-clinical neurotrauma research intended to complement existing pre-clinical models. It introduces human pathophysiology through the use of human induced pluripotent stem cell-derived neurons (hiPSCNs). It achieves loading pulse duration similar to the loading durations of clinical closed head impact injury. It employs a 96-well format that facilitates high throughput experiments and makes efficient use of expensive cells and culture reagents. Silicone membranes are first treated to remove neurotoxic uncured polymer and then bonded to commercial 96-well plate bodies to create stretchable 96-well plates. A custom-built device is used to indent some or all of the well bottoms from beneath, inducing equibiaxial mechanical strain that mechanically injures cells in culture in the wells. The relationship between indentation depth and mechanical strain is determined empirically using high speed videography of well bottoms during indentation. Cells, including hiPSCNs, can be cultured on these silicone membranes using modified versions of conventional cell culture protocols. Fluorescent microscopic images of cell cultures are acquired and analyzed after injury in a semi-automated fashion to quantify the level of injury in each well. The model presented is optimized for hiPSCNs but could in theory be applied to other cell types.

Here we present a method for a human in vitro model of stretch injury in a 96-well format on a timescale relevant to impact trauma. This includes methods for fabricating stretchable plates, quantifying the mechanical insult, culturing and injuring cells, imaging, and high content analysis to quantify injury.

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

富德瑞迪蒂菲洛波迪亚成分的纯化

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

通过对流增强的传递将抗体输送到毛里脑

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

A Mouse Model of Direct Anastomosis via the Prespinal Route for Crossing Nerve Transfer Surgery

Crossing nerve transfer surgery has been a powerful approach for repairing injured upper extremities in patients with brachial plexus avulsion injuries. Recently, this surgery was creatively applied in the clinical treatment of brain injury and achieved substantial rehabilitation of the paralyzed arm. This functional recovery after the surgery suggests that peripheral sensorimotor intervention induces profound neuroplasticity to compensate for the loss of function after brain damage; however, the underlying neural mechanism is poorly understood. Therefore, an emergent clinical animal model is required. Here, we simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice. Neuroanatomical, electrophysiological, and behavioral experiments helped identify that the transferred nerves of these mice successfully reinnervated the impaired forelimb and contributed to accelerating motor recovery after brain injury. Therefore, the mouse model revealed the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

We simulated clinical surgery to establish a protocol of direct anastomosis of bilateral brachial plexus nerves via the prespinal route in mice, contributing to the study of the neural mechanisms underlying rehabilitation upon crossing nerve transfer after central and peripheral nervous system injuries.

通过椎前途径直接吻合的小鼠模型用于跨神经转移手术

Crossing nerve transfer surgery has been a powerful approach for repairing injured upper extremities in patients with brachial plexus avulsion injuries. ...

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain cells and their interactions. Viral vector transduction enables the marking of diverse cell types in different fluorescence colors within the same tissue. Cells can be identified genetically by activity or projection. Using a modified CLARITY protocol, the potential sample size of astrocytes and neurons has grown by 2-3 orders of magnitude. The use of CLARITY allows the imaging of complete astrocytes, which are too large to fit in their entirety in slices, and the examination of the somata with all their processes. In addition, it provides the opportunity to investigate the spatial interaction between astrocytes and different neuronal cell types, namely, the number of pyramidal neurons in each astrocytic domain or the proximity between astrocytes and specific inhibitory neuron populations. This paper describes, in detail, how these methods are to be applied.

Combining viral vector transduction and brain clearing using the CLARITY method allows the investigation of a large number of neurons and astrocytes simultaneously.

研究清除大脑中星形胶质细胞和神经元之间的空间相互作用

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and large-scale genomic and drug screening. Due to the self-organizing nature of cells in brain organoids and the growing range of available protocols for their generation, issues with heterogeneity and variability between organoids have been identified. In this protocol paper, we describe a robust and replicable protocol that largely overcomes these issues and generates cortical organoids from neuroectodermal progenitors within 1 month, and that can be maintained for more than 1 year. This highly reproducible protocol can be easily carried out in a standard tissue culture room and results in organoids with a rich diversity of cell types typically found in the developing human cortex. Despite their early developmental make-up, neurons and other human brain cell types will start to exhibit the typical signs of senescence in neuronal cells after prolonged in vitro culture, making them a valuable and useful platform for studying aging-related neuronal processes. This protocol also outlines a method for detecting such senescent cells in cortical brain organoids using senescence-associated beta-galactosidase staining.

Robust and Highly Reproducible Generation of Cortical Brain Organoids for Modelling Brain Neuronal Senescence In Vitro

In this study, we provide a detailed technique for a simple yet robust cortical organoid culture system using standard feeder-free hPSC cultures. This is a rapid, efficient, and reproducible protocol for generating organoids that model aspects of brain senescence in vitro.

强大且高度可重复的皮质脑类器官生成，用于在体外模拟大脑神经元衰老

Brain organoids are three-dimensional models of the developing human brain and provide a compelling, cutting-edge platform for disease modeling and ...