这项工作得到了北京自然科学基金 (7152038), 中央南大学中央大学 (2016zzts162) 的基础研究基金和尔眼科医院组的科学研究基金会 (赠款 No。AF156D11)。最后, 黄益平感谢徐先生多年来的宝贵支持。

涉及动物的程序由首都医科大学机构动物保育和使用委员会 (IACUC) 批准。所有的手术器械和解决方案在手术前都被消毒以限制微生物感染. 
 注意: 手术方案包括麻醉、手术方法、视神经评估、闭合和恢复等五部分。为辅助视神经部分定量横断, 设计了一种新型的低成本手术器械。所有的程序都是按照道德规则和外科技术进行的. 
 1. 外科技术 <ol> <li> 使用无菌技术进行实验; 动物使用的协议应该是特定于机构的. </li>
	<li> 消毒仪器和材料 (解决方案、测试物质、示踪剂、针头、 等 ) 与活体组织接触, 以防止感染和对动物福利的不利影响, 以及对研究的潜在负面影响.</li>
</ol> 2。麻醉 <ol> <li> 麻醉 SD 大鼠使用一个兽医异氟醚蒸发器系统。以1升/分钟的速度使用医用级氧气蒸发异氟醚气体。将动物放在附着的麻醉箱中, 以4.5% 的浓度运行异氟醚, 直到缓慢呼吸和动物镇静. </li>
	<li> 将气体流量切换到防毒面具附件, 并将动物放在操作台上。将异氟醚浓度降低到 2%, 监测麻醉。较大的动物 (#62; 300 克) 可能需要更高浓度的异氟醚。在手术中监测麻醉, 异氟醚剂量相应调整。不断评估呼吸的深度和速度, 每5分钟进行一次脚趾夹压评估, 以确保没有深痛. </li>
	<li> 手术完成后, 关闭异氟醚, 让动物呼吸氧气几分钟, 然后再从手术台上取出。在手术中用外科毯子覆盖动物和/或使用调节过的电热毯来保持体温. </li>
</ol> 3。自行设计的外科辅助仪器 (SSAI) <ol> <li> 使用由不锈钢制成的 SSAI, 主要包括 hand-held 杆 (长度、100 mm、直径、9 mm) 和凹槽头 ( 图 1 )。凹槽头的沟槽表面呈半圆形, 垂直深度、宽度和长度分别为200和 #181; m、500和 #181; m 和1000和 #181; m。在两个部分之间, 有一个长度为50毫米的联合剖面。槽头和 #39 的边缘为300和 #181; m 宽. 
	注: 沟槽表面可使腹侧视神经的稳定为横断。当视神经的腹侧在其内部放置时, 背视神经就暴露在凹槽的外部;同时, 在进行垂直切削时, 暴露在沟槽边缘的背视神经可以断。在槽面内铺设的腹侧视神经受凹槽头和 #39 的边缘保护. </li>
	<li> 在视网膜断视神经轴突 (背侧) 的基础上达到初级损伤, 而继发性损伤则在视网膜对应 untransected 视神经轴突 (腹侧) 而不直接损伤的情况下进行. </li>
</ol> 4。手术方法 <ol> <li> 将鼠的右侧放在手术台上, 头朝外科医生。在手术视野的中心调整正确的轨道。然后, 在右侧眼眶皮肤外侧眼角多次清扫切口区域, 应用0.5% 氯己定75% 乙醇。用虹膜剪刀将外眼角的皮毛移到外部的声学孔上. </li>
	<li> 用虹膜剪刀沿侧眼角向外部声学孔进行皮肤切口, 长度为0.5 到1厘米。然后, 捏筋膜和向上拉, 创建一个三角形楔形与0.12mm 齿钳。将 Vannas 弹簧剪刀的下部刀片插入切口区域, 并在相同的剪切方向上切开筋膜. </li>
	<li> 用 Vannas 弹簧剪刀切开筋膜, 露出眶静脉。用锋利的锯齿钳夹住眶静脉两侧, 并钝开切口两侧。将眶静脉放置在眶肌表面的外科方向上, 以利于后续手术, 避免眶静脉的手术相关出血. </li>
	<li> 使用虹膜剪刀切断沿切口线的右外眼角, 以充分暴露在后续的轨道肌肉钝解剖的视野. </li>
	<li> 继续钳夹结扎眶肌的文件夹, 并在皮肤和筋膜切口的方向上垂直分开。沿两侧逐渐分离至眶深, 直至眼眶脂肪组织出现. </li>
	<li> 在眼眶脂肪组织暴露后, 将大鼠头部方向从外科医生的右侧垂直转向外科医生。同时, 使用含有无菌 PBS 的外科或棉签, 使该区域持续湿润。这一过程确保了外科领域的明确愿景, 同时保持组织的湿润和柔软. </li>
</ol> 5。访问视神经 <ol> <li> 切断眼眶脂肪组织, 覆盖眶内视神经周围的眼眶肌锥。这可以确保更好地暴露适当的手术方法。保持脂肪组织的切除以避免持续出血. </li>
	<li> 切开脂肪组织, 露出侧腹直肌。将外直肌钳夹向外, 然后用 Vannas 弹簧剪刀将其切开。如果脂肪组织保持在侧腹直肌下, 用0.12 毫米齿钳拔下视神经上的脂肪, 用 Vannas 弹簧剪刀将其切开. 
	注意: 此时, 视神经周围的组织鞘应该是可见的. </li>
	<li> 继续将组织鞘沿着视神经的方向分离, 直至视神经完全暴露为止。通过使用外科拭子清除组织清除产生的少量血液, 保持区域清洁. 
	注意: 现在, 视觉神经应该是可见的. </li>
	<li> 为了访问它, 取出周围神经的脑膜鞘, 而不损害眼动脉。在手术显微镜下, 轻轻旋转鞘, 在高倍镜下检查硬脑膜的血管形态. 
	<ol> <li> 确定没有血管的区域, 并在硬脑膜上进行纵向切口. 13 用26G 针针尖或刀尖的蓝宝石手术探针将鞘与视神经方向平行撕开, 避免侧切伤到血管. 
		注: 唯一覆盖神经的残余物应该是蛛网膜肢体ne, 这是非常薄和透明的. </li>
	</ol> </li> <li> 类似于步骤 5.2, 用26G 针尖或刀尖的蓝宝石手术探针轻轻地撕裂蛛网膜, 平行于视神经的方向. 13 </li> <li> 将视觉神经放置在仪器凹槽中, 轻轻地小心, 导致背部视神经略高于凹槽头边缘。此时, 用26G 针尖或刀尖的蓝宝石手术探针, 在凹槽顶部边缘上方的背视神经横断面, 完成部分视神经横断. </li>
</ol> 6。关闭和恢复 <ol> <li> 将仪器稍微深一点, 朝向视神经的垂直方向, 以释放后者。然后, 轻轻地取出仪器的凹槽头。尽量不要刮伤眼部肌肉或其他组织, 以避免额外的伤害。可观察部分视神经横断残端. </li>
	<li> 将眼侧直肌、筋膜及其他周围组织替换为原来的位置。然后, 依次缝合轨道上的肌肉和皮肤层。如果出血持续, 在关闭伤口前轻轻地用棉球填充, 并保持一段时间。将抗生素软膏涂抹在伤口上以防止感染. </li>
	<li> 关闭异氟醚源, 让动物呼吸氧气几分钟。在大鼠复苏过程中, 用加热垫准备隔热, 或用干填料覆盖笼面。用毯子盖住动物, 确保在恢复过程中大鼠气道通畅. </li>
	<li> 在手术后单独饲养动物。根据机构动物保育机构提供的指导方针, 管理后镇痛药。术后仔细观察动物. </li>
</ol>

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所有的作者都宣称他们没有任何利益冲突。

操作程序
在模型构建过程中, 存在一些值得注意的问题。在步骤4.2 中, 手术应小心进行, 以避免损害结扎肌上方的血管。特别是在外侧眼角切开皮下筋膜时, 应采用尖锐锯齿钳将皮下筋膜垂直向上拉;应用 Vannas 弹簧剪刀切开筋膜, 避免外眼角的眶静脉损伤, 从而导致模型失效。4.3 步具有潜在的预防出血的优势, 当直接从血管中移除。在4.5 步分离轨道肌肉时, 选择锋利锯齿钳而不是 Vannas 弹簧剪刀的原因是为了避免连续出血和出血。两侧的肌肉在垂直方向上直接与皮肤筋膜切口分开;同时, 轨道的深部肌肉向外伸展和外围延伸。这一过程将揭示更深层次的轨道腔, 提供一个更大的手术窗口, 并允许不受阻碍地进入覆盖视神经的组织。在上述程序中, 如果发生出血, 应使用无菌手术或棉签来施加压力。小出血将停止几秒钟后, 这个程序。步骤4.6 的目的是促进后续行动, 以方便地去除一些脂肪和分离的肌肉在轨道肌锥, 以揭露视神经沿视神经方向的轨道深度。
当前协议的最关键部分是步骤 5.1-5.6。重要的是不要破坏视神经管周围的血管。视神经应部分断至少 1.5-2.0 毫米的眼睛后部, 以避免对眼动脉的任何损害, 穿透神经在1毫米的眼睛, 并提供血液到内视网膜。切割外直肌的目的是为了更好地暴露视神经, 因为侧腹直肌是宽的, 明显阻挡了视神经的视野。同时, 为了避免切除与脑膜鞘相关的眼动脉(图 5), 有必要将硬脑膜周围的视神经分离和游离, 并检查脑膜鞘的血管形态,用镊子轻轻旋转鞘。此外, 应确定一个缺乏血管的区域, 允许在脑膜鞘内进行纵向切割。还有必要保持一个小的工作距离, 从后面的眼睛, 以避免部分硬脑膜, 这是密切联系的眼动脉。视网膜通常是透明的, 血管可以被清楚地标定。在视网膜血液供应受损的情况下, 视网膜退化, 导致乳白色絮凝剂的出现。眼睛和晶状体的玻璃腔通常也会变混浊, 随着时间的推移眼睛的尺寸也会减少。在本研究中, 术前和术后眼底图像在应用上述步骤后, 证实模型对眼底血供没有损伤。
此外, 这一模式的几个步骤需要特别照顾。当使用尖锐的锯齿钳或其他手术器械暴露视神经时, 外科医生应避免过度用力, 因为它可能会损伤视神经、眼球或眼动脉, 导致原发性损伤和视网膜缺血。此外, 眼睛周围的血管不应受损, 以避免持续出血, 这可能导致建模失败。在这个实验中使用的 SSAI 需要细致的使用。当视神经被放置在仪器凹槽内时, 视神经和沟槽表面需要紧装, 以确保每个动物模型的一致性和重复性。通过实践, 完整的手术程序可以完成后15-20 分钟内的每眼, 在最初的入境削减已经作出。
王et al.19发布了使用视神经定量切断建立的部分视神经横断的类似动物模型。手术方法包括: 1) 切除外眼角, 悬吊和固定睑上级;2) 探索视神经, 用切断 transecting 视神经的上部;3) 缝合结膜和皮肤。虽然手术过程比较简单, 但在手术中遇到了以下问题。虽然外眼角切口可暴露一定的手术空间, 但不可避免地需要不断伸展眼球, 以暴露后视神经鞘, 尤其是当外科医生想要暴露一个更长的后视神经鞘, 以促进鞘的进一步隔离;眼球伸展力较大, 可能引起眼球和视神经的直接牵引损伤。没有特别注意可能与视神经鞘一起切割的血管, 而血管损伤可能导致模型建立失败。本文所描述的二次损伤模型的主要方法是: 从眼球外侧眶壁直接进入眶肌锥周围的后视神经, 避免原发性损伤眼球和视神经, 当向下拉或朝向眼球的侧鼻侧时。这种新的手术方法增加了在建模过程中手术的空间, 并使与眼动脉紧密相连的脑膜鞘在视神经部分横断之前易于分离。部分视神经切断术采用自行设计的手术器械, 成本效益高, 可重复使用, 降低了建模的总成本。大鼠的眼眶结构不同于其他哺乳动物, 其眼眶离眼角最近, 无骨结构, 但有肌肉覆盖。手术方法可达到眼球后部, 不需要破坏眶骨和骨膜。经过严格的术前消毒和术后抗生素预防, 感染、炎症和水肿大为减少。
自行设计的外科辅助器械
采用自行设计的手术辅助器械, 建立大鼠部分视神经横断模型, 其主要特点如下。它可以帮助部分定量的视神经横断暴露在沟槽边缘, 也确保不同动物之间的横断一致性。通过试验验证了模型建立与 SSAI 的重复性。最大变异系数为 1.85%, 平均值为0.67%±0.44%。20这些结果表明, SSAI 可用于建立部分视神经横断模型, 具有令人满意的重现性和联合国iformity
沟槽表面的宽度和凹槽内表面半圆的设计对视神经有更固定的影响, 使沟槽表面和视神经附着更加紧密, 也降低了实验误差和不良反应。凹槽边缘允许更好地保护视神经在凹槽在操作期间, 不会损坏视觉神经在凹槽, 不管切削刀的锋利。沟槽边缘的另一个优点是视神经横断时的挤压伤预防。
适用于深、窄空间的操作。虽然新的手术方法已经扩大, 路径仍然深, hand-held 杆和关节节可用于放置在视神经鞘下的槽头容易执行后续行动。当该仪器用于操作时, 可使用多种刀具进行横截,例如26 克针尖。甚至可以选择蓝宝石外科探针刀, 以避免剪刀造成的挫伤和挤压伤。凹槽表面可以在不同的垂直深度进行, 以完成不同程度的视神经切割。
与王et al.的切断相比, SSAI 具有更简单的结构。此外, 使用 SSAI 的切割步骤更方便, 提高了动物模型的一致性和重复性。最后, 适用于切割与 SSAI 的工具范围也更广。结论: SSAI, 使神经的定量和均匀切口, 可作为建立大鼠视神经横断模型的有效工具。
大鼠部分视神经横断模型的特点
部分视神经横断模型可用于评价视网膜的二次变性。该模型的潜在优点是能够在视神经和视网膜中准确地将初级和二次变性分别从原位分离出来。中央和腹侧视神经更容易继发性损伤后部分横断 (约1/3 至 1/2) 的背视神经(图 6)。在视网膜中, 视网膜的区域位置应根据视网膜视网膜后部分横断后的视神经地形图进行定位。如果整个视网膜的大鼠被分为背 (上级) 和腹 (下等) 部分, 次要和主要的伤害是存在的两个部分。然而, 根据视网膜视网膜与视神经轴突之间的关系, 在腹视网膜上的死亡应主要归因于继发性损伤(图 3)。12,22,23该模型的优点包括: 简单、操作的仪器和标准程序;对眼部血管无影响;良好的重现性和稳定性。这一技术可用于染视网膜从这个节省空间的操作方法, 应用短干扰 rna (siRNAs), 质粒, 和病毒载体的部分视神经残端;此外, 可将试剂放在部分视神经残端上, 用于选择性治疗或标记视网膜。
总体上, 视网膜在部分视神经横断后共存, 在视网膜上有明显的边界, 两种损伤类型之间有明确的界限。虽然视神经轴突和王国在视网膜上的位置的联系需要进一步的研究, 以更精确的区分, 这种节省空间的操作方法扩展了模型的应用范围, 并允许研究人员探索视网膜二次损伤机制的新方法。

继发性变性通常发生在中枢神经系统 (CNS) 创伤后, 并继急性和慢性神经变性疾病。1,2,3,4,5神经元和神经胶质细胞的死亡作为原发性病理事件的早期结果被称为原发性变性, 而继发性变性则指神经元和胶质细胞的死亡, 它们不是或只部分受初级损害.二次变性的视网膜也认为是发生在青光眼。6 Yoles et al.7证实视神经损伤模型中视网膜发生继发性损伤。他们表明, 神经元的轴突没有受伤后, 急性损伤将最终退化, 由于退化环境产生的损伤周围的这些轴突。这种继发性变性影响了神经元的渐进方式与损害的严重性有关。到目前为止, 研资局对青光眼的损害的机制仍然不清楚, 特别是那些与继发性损伤有关, 导致临床治疗不足。8,9,10因此, 在青光眼发展过程中, 有必要探讨视网膜二次变性的基本机制。11建立可定量评估视网膜二次变性的大小、分布和机制的继发性损伤动物模型, 引起了研究视网膜二次损伤的科学家越来越多的关注。
为了澄清这个问题, Levkovitch-Verbin et al.建立了一个鼠邦模型。12评价轴索损伤引起的视网膜的变性和死亡。该模型被认为是一个很好的工具, 探索的机制, 次生变性和识别潜在的神经保护剂。用于产生这种二次损伤模型的仪器, 是通过拨盘旋钮, 通过表盘的深度来定量地进行定量的金刚石刀, 以完成视神经的数量横断。手术路径的方法从眼球向上或颞结膜。在手术过程中, 视网膜和视神经可能受钳力的影响, 这反过来又可能导致原发伤。更重要的是, 由于暴露的视神经空间有限, 在切开前很难分离出脑膜鞘。因此, 在部分视神经横断时, 有可能损伤眼部血管, 导致视网膜缺血和模型失效。此外, 金刚石刀是昂贵的, 每使用减少尖端清晰度。这可能反过来影响建模的深度和效果.
通过对眼球外侧眶壁的一种新的手术方法, 获得了本研究所描述的视网膜的二次变性模型。这种新的手术方法直接访问的后视神经周围的轨道肌锥, 避免对眼球和视神经的主要伤害时, 拉向下或对鼻侧侧的眼球。这也增加了在模型建立的外科手术的空间, 并使脑膜鞘隔离前部分 transecting 视神经。重要的是要注意, 疏忽参与和伤害眼血管可能导致模型的失败。此外, 该模型能够对部分断视神经的残端的转染细胞、药物和试剂进行后续评估。自行设计的手术器械价格低廉, 可多次使用, 从而降低了建模成本。该方法所建立的视网膜二次损伤模型具有良好的重现性和稳定性。

<table border="0" cellpadding="0" cellspacing="0" width="770">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Animal Aneathesia Ventilator System</td>
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			<td style="width:268px;">Leica AG, Heerbrugg, Switzerland</td>
			<td style="width:119px;">M620 F20</td>
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			<td height="21" style="height:21px;width:216px;">Fluorogold</td>
			<td style="width:268px;">Biotium</td>
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			<td height="21" style="height:21px;width:216px;">Iris scissors</td>
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			<td height="21" style="height:21px;width:216px;">Vannas spring scissor</td>
			<td style="width:268px;">66vision Co.</td>
			<td style="width:119px;">54137B</td>
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			<td height="61" style="height:61px;width:216px;">Sharp-serrated forceps/0.12mm toothed forceps&#160;</td>
			<td style="width:268px;">66vision Co.</td>
			<td style="width:119px;">53329A</td>
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			<td height="21" style="height:21px;width:216px;">Sharp-curved forceps</td>
			<td style="width:268px;">66vision Co.</td>
			<td style="width:119px;">53324A</td>
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			<td height="21" style="height:21px;width:216px;">Sapphire surgical probe</td>
			<td style="width:268px;">66vision Co.</td>
			<td style="width:119px;">50205TA</td>
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			<td height="41" style="height:41px;width:216px;">26G needle tip</td>
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			<td height="21" style="height:21px;width:216px;">10 &#956;l Hamilton Syringe</td>
			<td style="width:268px;">Hamilton Co.</td>
			<td style="width:119px;">80030</td>
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			<td height="21" style="height:21px;width:216px;">5-0 non-absorbable suture</td>
			<td style="width:268px;">Johnson &#38; Johnson International Co.</td>
			<td style="width:119px;">W580</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Chlorhexidine</td>
			<td style="width:268px;">Sigma-Aldrich</td>
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			<td height="21" style="height:21px;width:216px;">Stereotaxie apparatus</td>
			<td style="width:268px;">RWD Life Science Co.</td>
			<td style="width:119px;">68026</td>
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			<td style="width:268px;">OptoProbe Ltd.</td>
			<td style="width:119px;">OPTO-RIS</td>
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			<td height="41" style="height:41px;width:216px;">RetCamII wide field imaging system</td>
			<td style="width:268px;">Clarity Medical Systems,Inc.</td>
			<td style="width:119px;">RetCamII</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Fluorescence microscope</td>
			<td style="width:268px;">Leica Microsystems Inc.</td>
			<td style="width:119px;">DM6000</td>
			<td></td>
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	</tbody>
</table>

partial optic nerve transection in rats: a model established with a new operative approach to assess secondary degeneration of retinal ganglion cells

以往的研究表明, 视网膜神经节细胞 (视网膜) 的继发性变性通常发生于青光眼。部分视神经横断被认为是一种有用且可重复的模型。与一般用于评估二次变性的视神经损伤模型相比,如完全视神经横断和视神经粉碎模型, 部分视神经横断模型的优越性在于它区别初级从原位中的次变性。因此, 它是评价二次变性的一个很好的工具。本研究通过眼球眶外侧壁直接进入后视神经区, 描述了一种新的部分视神经横断手术方法。此外, 我们提出了一个新设计的, 低成本的手术器械, 以协助横断。正如代表所证明的那样, 在区分主次伤害区域的边界时, 新的方法和仪器通过为外科手术提供足够的空间来确保模型的高效率和稳定性。这反过来使它很容易分离的脑膜鞘和眼血管从视神经前横断。另外一个好处是, 这种节省空间的操作方法提高了调查人员对部分断视神经残端的药物、载体或有选择性的王国追踪者的管理能力, 从而可以探索背后的机制视网膜的继发伤, 以一种新的方式。

为了验证在建立第二损伤模型方面的成功, 使用 SSAI (图 2A)的新操作方法, 视网膜在模型建立后立即逆行标记。本程序的目的是通过在无菌磷酸盐缓冲盐水中注入神经示踪染料 (3% 荧光 (如, Fluorogold), 将视网膜逆行标记为高级丘(图 2B)。这种方法产生可再生的标签, 可存活视网膜的变化不大。14,15,16,17,18染料将被逆行视网膜在视网膜中占去, 构成活视网膜的标记, 而轴突不断在右眼。同时, 视网膜对应于右眼部分断的视神经轴突不能用示踪染料标记(图 2C)。作为对照眼, 左眼无手术, 视网膜沿视神经的视网膜全部贴上荧光金染料, 以逆行的方式从上级丘(图 2D)。
七天后部分视神经横断和逆行标记的视网膜, 视网膜收获, 固定, 扁平, 并安装。在视网膜的定义区域内, 在荧光显微镜下对标记的视网膜进行了成像。在图 3中显示了带有或不带部分视神经横断的荧光标记视网膜的结果。只有视网膜在右视网膜对应的 untransected 部分的视神经被标记为荧光金, 并明确边界无标记和标记视网膜可以可视化(图 3A,图 3B),展示视神经的部分交易作为对照眼, 所有左眼视网膜的视网膜显示荧光(图 3C,图 3D)。
为了评估在手术过程中, 视神经头周围的血管和向 entoretina 提供血液的眼动脉是否受到损伤和影响, 术前、后对右眼眼底进行了成像。术后1小时, 图像显示右眼 (手术眼) 血供。动脉里的血足够了没有观察到静脉阻塞。这些发现表明, 在操作(图 4A,图 4B)中没有对血液供应系统造成损害。从而成功地建立了视网膜的二次退化模型。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56272/56272fig1.jpg"> 
图 1: 自行设计的外科辅助仪器的照片, SSAI.(a)手术器械的全景图, 有两个主要部分, 包括 hand-held 杆和凹槽头。在他们之间, 有一个联合部分与长度50毫米。hand-held 杆的长度为100毫米, hand-held 杆的直径为9毫米. (B) SSAI 凹槽的特征。凹槽头的沟槽表面是半圆形的, 它使视神经在它内放置, 被稳定为横断。沟槽表面是与垂直深度200µm 和宽度500µm 和长度1000µm。槽头的边缘宽度为300µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56272/56272fig1large.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/56272/56272fig2.jpg"> 
图 2: 通过自行设计的外科辅助仪器 (SSAI) 和逆行标记高级丘与荧光的关系, 在视网膜部分横断后, 由研资局贴上标签的示意图.(A)自行设计的手术辅助器械 (SSAI) 对大鼠部分右视神经横断的手术治疗看法。(B)在建模后, 视网膜被标记为逆行, 方法是将神经示踪染料 (黄色、3% 荧光在无菌磷酸盐缓冲盐水中) 注入大脑中的高级丘。由于视网膜的轴突位于高级丘, 示踪染料由视网膜逆行, 构成活细胞的标志物。图中的横断面代表视神经的横断面。外径, 操作眼睛;操作系统, 控制眼睛不操作。在(C)中, 只有与视神经 untransected 部分对应的视网膜标记为荧光。蓝色代表未腹神经和相应的视网膜在视网膜上;红色反射部分断背视神经和相应的视网膜在视网膜上。(D)在没有手术的情况下, 左眼 (控制眼) 的视网膜视网膜被染料标记。背部和腹侧视神经也都被染料标记。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56272/56272fig2large.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/56272/56272fig3.jpg"> 
图 3: 整个视网膜荧光显微荧光标记视网膜7天后建立部分视神经横断模型和逆行标记的上级丘。还介绍了 Fluorogold 染色的研资区在视网膜上的相应示意图。(A)和(C)分别在标记荧光后, 在右侧 (手术) 和左 (控制) 眼的视网膜上代表视网膜的示意图。黄色表示标有荧光金色染料的区域。视网膜分为背部、腹侧和中央部分。(B)和(D)表示在荧光显微镜下获得的全视网膜荧光显微;黄色代表视网膜标有荧光的区域。在手术眼 (右眼) 显示在B中, 未标记区域表示与视神经视网膜对应的面积, 该区域是部分断, 主要位于视网膜的背侧。荧光金染料标记的区域是与视神经视网膜对应的区域, 它不断, 主要集中在视网膜的中央和腹侧。奔将军在未标记和标记的视网膜之间的区域是清楚的。研资局的初步变性将限于背部视网膜, 而中央和腹侧视网膜的全部损失都可归因于继发性变性。(D)视网膜与荧光标记后左眼的整个视网膜显微。无手术的左眼视网膜完全被荧光染色。缩放栏 = 500 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56272/56272fig3large.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/56272/56272fig4.jpg"> 
图 4: 术前及术后右眼眼底影像的眼底成像.(A)大鼠右眼术前眼底的影像, 显示良好的眼底血供, 动脉充盈, 无静脉回流或梗阻, 显示良好的视网膜供血系统。(B)手术后1小时右眼眼底的图像。与眼底的术前影像相比, 视网膜血供未见明显变化, 说明在建模过程中眼球的供血系统不受影响。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56272/56272fig4large.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/56272/56272fig5.jpg"> 
图 5: 后视神经和位于脑膜鞘上的眼动脉的照片, 通过手术方法捕获.在完全切除视神经的靶长后, 与视神经脑膜鞘相伴随的眼动脉 (箭头) 暴露, 并与视神经平行。缩放栏 = 500 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56272/56272fig5large.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/56272/56272fig6.jpg"> 
图 6: 视神经中原发性和继发变性位置示意图.采用自行设计的手术辅助器械 (箭头) 实现视神经部分切口。直接受损部位的轴突 (在灰色横断面上的视神经背侧切割部位) 经历原发变性, 而在间接损伤的部位 (在黄色的横断面上, 视神经的中央和腹侧区域)经历二次变性。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56272/56272fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells at JoVE.

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

大鼠部分视神经切断术: 建立一种新的手术方法评价视网膜神经节细胞二次变性的模型

视网膜神经节细胞 (视网膜) 继发性变性通常发生于青光眼。本研究介绍了一种创新的手术方式部分视神经横断。利用这种节省空间的操作方法扩展了模型的应用范围, 并允许在视网膜中探索二次损伤机制的新方法。

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

partial-optic-nerve-transection-rats-model-established-with-new

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.1K 次观看.  Central South University, Changsha, China. 视网膜神经节细胞 (视网膜) 继发性变性通常发生于青光眼。本研究介绍了一种创新的手术方式部分视神经横断。利用这种节省空间的操作方法扩展了模型的应用范围, 并允许在视网膜中探索二次损伤机制的新方法。

视频: Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

视神经横断:一个成人在中枢神经系统的神经元凋亡模型

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

科研

神经科学

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

在哺乳动物的中枢神经系统视神经横断后的实验操作方法

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

一个孤立的视网膜制备，记录从基因标记的视网膜神经节细胞的光反应

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

一个视神经挤压伤的小鼠模型，研究视网膜神经节细胞生存

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

在小鼠视网膜神经细胞，实现动态夹具与突触和人工电导

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...