作者感谢Karine Loulier博士提供转基因小鼠系MAGIC-Markers。作者还感谢RAM-Neuro动物核心设施和成像设施MRI，这是法国国家研究机构支持的France-BioImaging国家基础设施的成员（ANR-10-INBS-04，“未来投资”）。这项研究得到了ATIP-Avenir计划，Inserm，Région Occitanie，蒙彼利埃大学，法国国家研究局（ANR-21-CE17-0061），医学研究基金会（FRM再生医学，REP202110014140）和Groupama基金会的支持。

所有实验均获得国家动物实验委员会的批准。
1. 准备工作
<ol><li>制备氯胺酮-甲苯噻嗪的麻醉溶液进行麻醉。通过将 200 μL 氯胺酮 （100 mg/mL） 和 125 μL 甲苯噻嗪 （20 mg/mL） 稀释在 2,175 mL 无菌 0.9% NaCl 中，注射 80 mg/kg 的氯胺酮和 10 mg/kg 的甲苯噻嗪。</li>
	<li>通过将 100 μL 0.3 mg/mL 丁丙诺啡加入 1,400 mL 无菌 0.9% NaCl 中，制备 0.02 mg/mL 丁丙诺啡溶液作为镇痛溶液。</li>
	<li>准备荧光染色溶液。<ol><li>用细秤称取10mg荧光素盐，用10mL磷酸盐缓冲盐水（PBS）稀释，得到0.1%荧光素溶液。</li>
		<li>保护溶液免受光线照射并摇晃5分钟。使用 10 mL 注射器和 0.2 μm 的注射器过滤器过滤溶液。 注意：荧光溶液必须避光，并且可以在 +4 °C 下储存 5 天。</li>
	</ol></li>
	<li>鼠标的准备<ol><li>通过腹膜内注射10μL / g小鼠重量（MW）的麻醉溶液来称量小鼠并诱导麻醉。当鼠标停止移动时，将其放在加热板（37°C）上，并通过捏住脚趾来验证其完全麻醉。</li>
		<li>在接受手术的眼睛上滴一滴人工泪液，在对侧眼睛上滴一滴眼部凝胶。</li>
		<li>一旦小鼠对捏无反应，以5μL / gMW注射镇痛剂（0.02mg / mL丁丙诺啡），以在颈部皮下提供0.1mg / kg丁丙诺啡。</li>
	</ol></li>
</ol>2.角膜擦伤
<ol><li>按照步骤1.3制备荧光染色溶液。</li>
	<li>按照步骤 1.4 准备鼠标。</li>
	<li>在进行磨损之前，将眼部毛刺浸入 70% EtOH 中，然后浸入 PBS 中进行清洁。</li>
	<li>将鼠标放在侧面的加热板（37°C）上，以便轻松进入正在接受手术的眼睛。<ol><li>用棉签吸收人工泪液，在不接触眼睛的情况下将睫毛刷掉。</li>
		<li>打开眼毛刺，用两根手指打开鼠标的眼睑，同时挡住振动器，以免它们卡在毛刺中。</li>
	</ol></li>
	<li>定位瞳孔或眼睛中心，将眼部毛刺涂在眼睛表面，并做圆周运动。通过仔细观察，可以观察到去除的上皮的表面。否则，在角膜上做大约 20 次圆周运动。</li>
	<li>将鼠标放回腹部，检查对侧眼睛中是否仍然存在眼部凝胶。</li>
	<li>在擦伤的眼睛上滴一滴荧光染色溶液，等待20秒。用纸巾吸收滴剂，不要接触眼睛，然后用一滴人工泪液冲洗。用纸巾吸收人工泪液滴，并用蓝色钴灯照亮眼睛。 注意： 磨损必须呈圆形。它需要一些培训才能获得它。</li>
	<li>在擦伤的眼睛上滴一滴眼部凝胶，让鼠标在加热垫上醒来。当鼠标自行移动时，将其放回笼子中。</li>
	<li>在接下来的 2 天内检查动物的健康状况。</li>
	<li>使用眼部毛刺后，使用软组织去除粘在毛刺头部的上皮细胞，并将其浸入 70% EtOH 中，然后浸入 PBS 中。 注意：一旦上皮细胞与软组织一起去除，请确保没有组织碎片卡在毛刺的头部。</li>
</ol>3. 角膜神经轴突切开术
<ol><li>按照步骤 1.4 准备鼠标。</li>
	<li>将鼠标放在侧面的双目放大镜下，以进入正在接受手术的眼睛。将培养皿的底部放在鼠标头部下方，使眼睛水平。 注意： 以下步骤将通过双目放大镜进行。</li>
	<li>用棉签去除人工泪液，并在不接触眼睛的情况下去除睫毛。</li>
	<li>将光滑的弯曲钳子放在动物的眼睛下方，以便将其从眼眶中弹出。确保将钳子合得足够大，以便在施加压力后眼睛无法回到眼眶中，但不要过度以防止视神经损伤。</li>
	<li>将 2.5 毫米的活检冲头垂直施加在眼睛上，不要施加压力，以确保冲头与角膜表面完全接触。 注意：一旦将打孔施加在角膜上，实验者的手可能会干扰视野。</li>
	<li>然后，开始施加压力并旋转冲头几次。 注意： 根据压力的不同，扭曲的次数可能会有所不同，但通常在 5 左右。它需要训练才能感受到正确的压力和运动。如果施加过大的压力，眼睛就会破裂。在这种情况下，可以观察到液体从病灶中流出，眼睛变得柔软。动物必须被牺牲。</li>
	<li>取下活检打孔器。在应用活检打孔的角膜上必须看到一个圆圈。 注意： 接下来的步骤不需要双目放大镜。</li>
	<li>检查对侧眼是否仍有眼部凝胶，并在轴切除的眼睛上涂抹一些。</li>
	<li>让鼠标在加热垫（37°C）上醒来，并在自行移动时将其放回笼子中。</li>
	<li>在接下来的 2 天内检查动物的健康状况。</li>
</ol>4.角膜整体安装加工
<ol><li>样品采集和固定。<ol><li>称量小鼠并通过腹膜内注射10μL / g小鼠重量（MW）的麻醉溶液来诱导麻醉。</li>
		<li>当鼠标停止移动并且没有通过脚趾捏合产生反射时，进行颈椎脱位。</li>
		<li>用两根手指将眼睛从眼眶中弹出，然后用弯曲的剪刀剪断视神经，将眼睛去核。</li>
		<li>将眼睛放入含有 PBS 的 2 mL 塑料管中。</li>
		<li>用4%多聚甲醛代替PBS并将其放在室温（RT）的振荡器（30rpm）上20分钟，以固定眼睛。</li>
		<li>在振荡器（30rpm）上用PBS在室温下冲洗3次，持续10分钟。</li>
	</ol></li>
	<li>角膜清扫术<ol><li>将一滴PBS滴在培养皿内的一块封口膜上。</li>
		<li>将眼睛放在这滴PBS中。</li>
		<li>将培养皿放在双目放大镜下。</li>
		<li>使用显微解剖剪刀和镊子解剖角膜。切开睫状体上方以保持角膜缘。</li>
		<li>用细镊子取出晶状体、睫状体和虹膜。</li>
		<li>将角膜放回 2 mL 塑料管中。</li>
	</ol></li>
	<li>免疫荧光实验方案<ol><li>将角膜在2mL的2.5%鱼皮明胶，5%山羊血清和0.5%洗涤剂的PBS中孵育1小时，在振荡器（30rpm）上室温下。</li>
		<li>将角膜在150μL的2.5%鱼皮明胶，5%山羊血清和0.1%去污剂的PBS溶液中孵育，其中含有以1/1000稀释的一抗（抗βIII微管蛋白抗体）在振荡器（30rpm）上过夜。</li>
		<li>在振荡器（30rpm）上用0.1%的PBS溶液在室温下冲洗3次1小时。</li>
		<li>将角膜在与步骤4.3.2相同的溶液中孵育，该溶液含有在振荡器（30rpm）上以1/500在4°C下稀释过夜的二抗。</li>
		<li>在振荡器（30rpm）上用PBS在室温下冲洗3次1小时。</li>
		<li>用在振荡器（30rpm）上以1/5000的室温稀释的150μLDNA插层器孵育角膜10分钟。</li>
		<li>用 PBS 冲洗 2 次，持续 5 分钟。</li>
	</ol></li>
	<li>角膜的安装<ol><li>将角膜放在载玻片上，上皮朝向载玻片，然后用手术刀在不到达中心的情况下创建四个部分。 注意： 将彼此相对的部分做成花朵形状。</li>
		<li>将角膜内皮朝向载玻片，并使用纸巾去除角膜周围的 PBS。 注意： 不要去除角膜下方的 PBS;否则，可能会出现气泡。如果是这样，将 PBS 添加到角膜中并去除气泡。然后从步骤 4.3.2 重新启动。</li>
		<li>在矩形盖玻片的四个角上添加模型粘贴。 注意：模型糊状物抬高了盖玻片，因为角膜是厚组织。</li>
		<li>将 50 μL 的封固剂滴在角膜上，并小心地将盖玻片放在上面以避免气泡形成。</li>
		<li>用指甲油密封盖玻片。</li>
	</ol></li>
	<li>成像<ol><li>将载玻片置于落射荧光显微镜下。</li>
		<li>定义镶嵌的大小和图像的深度，然后开始采集。</li>
		<li>对采集的图像应用去模糊和去卷积程序。 注：步骤4.6.3是采集软件上选择的选项（大体积计算清算[LVCC]）</li>
	</ol></li>
</ol>5.角膜神经的局部激光消融术
<ol><li>打开正置多光子显微镜和激光与光学参量振荡器相结合。激活显微镜的加热台（37°C），并将激光器设置为850nm和1100nm，对应于小鼠模型所需的波长。</li>
	<li>使用功率计同时将激光器的功率设置为 20 mW 左右。</li>
	<li>按照步骤 1.4 准备鼠标并在双眼上涂抹眼部凝胶。 注意：用于该方案的动物必须来自转基因小鼠系（MAGIC-Marker转基因小鼠21），该系在角膜神经纤维中表达内源性荧光团。</li>
	<li>将动物安装在头架中。将动物旋转 90°，使感兴趣的眼睛面向天花板。</li>
	<li>绑住动物的振动器以清洁眼部区域。绑住动物的身体，以减少呼吸引起的头部运动。</li>
	<li>在眼部凝胶上方的眼睛上放置一个圆形盖玻片。盖玻片必须是水平的。不要犹豫，移动动物的头部，以便盖玻片正确定位。</li>
	<li>在盖玻片上方加入一滴 PBS。小心地将动物放在显微镜载物台上，并将物镜转台设置在适当的距离。</li>
	<li>使用 20 倍水性物镜和落射荧光通过目镜定位眼睛和神经支配。</li>
	<li>激活激光并定义需要照明的深度。</li>
	<li>以最大扫描速度的 70%-85% 的采集速度采集 1024 x 1024（像素）图像。</li>
	<li>获取图像后，将动物从显微镜上取下，取下盖玻片和带子。将动物从头架上取下，如果需要，添加更多的眼部凝胶，然后让它在加热板上的笼子里醒来。 注意：成像后 1 周可以观察到神经支配的破坏。该协议可以每周重复一次，持续数周，以增加激光造成的损害。</li>
	<li>当动物自行移动时，将笼子放回马厩，并在接下来的 2 天内检查其健康状况。</li>
</ol>

用于轴突再生研究的角膜感觉纤维损伤方案

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C., Norden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phototoxicity+in+live+fluorescence+microscopy,+and+how+to+avoid+it.">Phototoxicity in live fluorescence microscopy, and how to avoid it.</a> BioEssays. 39 (8), 1700003 (2017).</li><li>Volatier, T., Schumacher, B., Cursiefen, C., Notara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV+protection+in+the+cornea:+Failure+and+rescue.">UV protection in the cornea: Failure and rescue.</a> Biology. 11 (2), 278 (2022).</li><li>Loulier, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+cell+and+lineage+tracking+with+combinatorial+labels.">Multiplex cell and lineage tracking with combinatorial labels.</a> Neuron. 81 (3), 505-520 (2014).</li><li>Dana, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expert+consensus+on+the+identification,+diagnosis,+and+treatment+of+neurotrophic+keratopathy.">Expert consensus on the identification, diagnosis, and treatment of neurotrophic keratopathy.</a> BMC Ophthalmol. 21 (1), 327 (2021).</li><li>Matsumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autologous+serum+application+in+the+treatment+of+neurotrophic+keratopathy.">Autologous serum application in the treatment of neurotrophic keratopathy.</a> Ophthalmology. 111 (6), 1115-1120 (2004).</li><li>Bonini, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+II+randomized,+double-masked,+vehicle-controlled+trial+of+recombinant+human+nerve+growth+factor+for+neurotrophic+keratitis.">Phase II randomized, double-masked, vehicle-controlled trial of recombinant human nerve growth factor for neurotrophic keratitis.</a> Ophthalmology. 125 (9), 1332-1343 (2018).</li><li>Aggarwal, S., Colon, C., Kheirkhah, A., Hamrah, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+autologous+serum+tears+for+treatment+of+neuropathic+corneal+pain.">Efficacy of autologous serum tears for treatment of neuropathic corneal pain.</a> Ocul Surf. 17 (3), 532-539 (2019).</li><li>Singh, N. P., Said, D. G., Dua, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lamellar+keratoplasty+techniques.">Lamellar keratoplasty techniques.</a> Indian J Ophthalmol. 66 (9), 1239-1250 (2018).</li><li>Gautier, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV2/9-mediated+gene+transfer+into+murine+lacrimal+gland+leads+to+a+long-term+targeted+tear+film+modification.">AAV2/9-mediated gene transfer into murine lacrimal gland leads to a long-term targeted tear film modification.</a> Mol Ther Methods Clin Dev. 27, 1-16 (2022).</li></ol>

作者没有利益冲突需要披露。

神经营养性角膜炎被认为是一种罕见疾病，每 10,000 人中就有 5 人受到影响。然而，由于化学烧伤等身体伤害或糖尿病或多发性硬化症等综合征而患有NK的人不包括在这些统计数据中3。此外，这种情况仍然严重不足22，并且该疾病的患病率被低估了。迫切需要新的治疗方法和疗法来促进轴突再生，作为保护患者视力的一种手段。目前，为NK早期阶段患者提供的唯?...

角膜是眼睛的透明表面，由三个不同的层组成：上皮、基质和内皮。该器官在体内具有最高的神经支配密度，主要由源自三叉神经节眼科分支的感觉纤维（Aδ 和 C 型）组成。感觉纤维以大束的形式穿透中基质中角膜的外围，这些束向外分支以覆盖表面。然后它们分叉以刺穿鲍曼膜并形成基底下丛，这很容易通过角膜中心形成的涡旋来识别。这些纤维终止于上皮外表面的游离神经末梢。它们能够转导热刺激、机械刺激和化学刺激，并释放对上皮稳态至关重要的营养因子 1,2。神经营养性角膜炎（NK）是一种影响角膜感觉神经支配的退行性疾病。这种罕见的疾病源于角膜敏感性的降低或丧失，导致角膜泪液分泌减少和愈合性能差3.NK 进展经历了三个明确描述的阶段，从患者出现上皮缺陷的第 1 阶段到发生基质融化和/或角膜穿孔的第 3 阶段4。
在临床上，这种疾病的起源可能是多种多样的。患者在眼睛受到物理损伤、手术或慢性疾病（如糖尿病）后可能会失去角膜神经支配 5,6。迄今为止，NK的发病机制过程仍然知之甚少，这种威胁视力的疾病的治疗选择非常有限。因此，需要更好地了解上皮缺损的特征，以更好地了解这些纤维再生背后的机制并可能促进它们。在这里，我们提出了几种在小鼠中诱导NK的角膜损伤模型。
第一种模型是带有眼毛刺的角膜上皮层的磨损。该模型主要在不同动物（如啮齿动物和鱼类）的上皮再生的背景下进行研究 7,8,9，并测试促进角膜愈合的分子10,11。从生理上讲，上皮细胞需要 2-3 天才能闭合伤口。然而，神经支配的生理模式需要四个多星期才能从擦伤中恢复12,13。在手术过程中，眼毛刺会去除角膜的上皮层，该角膜上皮层包含基底下丛和纤维的游离神经末梢。该手术在临床上可以与光屈光性角膜切除术 （PRK） 患者进行比较，以矫正眼部屈光缺损。该过程包括去除角膜上皮，然后用激光重塑基质14.患者在此类手术后可能会出现多种副作用，例如角膜神经密度降低 2 年，敏感性降低持续 3 个月至 1 年15。鉴于手术会引起角膜微环境的脆弱性，该模型可以帮助研究这些副作用并开发治疗方法，以促进更快的神经再支配，从而减少所讨论的副作用。
第二种模型包括用活检打孔切开角膜外围的轴突，诱导中枢神经支配的沃勒变性 16。在临床上，这种方法可以与前板层角膜移植术进行比较，在前板层角膜移植术中，外科医生实现了角膜的部分环钻术，以去除角膜前部厚度的一部分，并用供体移植代替 17。板层角膜移植术后，患者可能会出现许多症状，包括干眼症、角膜神经支配丧失和移植物排斥反应18。这种对角膜神经进行的轴突切开术模型可以深入了解纤维变性的机制，纤维变性发生在移植物后，然后是轴突再生。
第三种方法是用激光损伤角膜神经。通过在麻醉动物的角膜上使用多光子显微镜，由于活性氧 （ROS） 的形成，诱导位于光场中的神经变性，从而导致 DNA 损伤和细胞空化19。这种方法概括了过度暴露于自然紫外线（晒伤）引起的角膜光损伤，这也触发了 ROS 的形成，导致 DNA 损伤20。患有角膜晒伤的患者会经历巨大的痛苦，因为上皮细胞的恶化剥夺了角膜纤维的四肢。
这里描述的三种方法旨在研究NK的发病机制过程和轴突再生。它们易于重复且精确。此外，它们可以快速恢复和轻松监控动物。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#181;m seringe filter</td>
			<td>CLEARLINE</td>
			<td>51733</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mm rust ring remover</td>
			<td>Alger Equipment Company</td>
			<td>BU-5S</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL plastic tubes</td>
			<td>Eppendrof&#160;</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>Algerbrush burr, Complete instrument</td>
			<td>Alger Equipment Company</td>
			<td>BR2-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-beta III Tubulin antibody</td>
			<td>Abcam</td>
			<td>ab18207</td>
			<td></td>
		</tr>
		<tr>
			<td>Antigenfix</td>
			<td>Diapath</td>
			<td>P0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial tear</td>
			<td>Larmes artificielles Martinet</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprecare</td>
			<td>Animalcare</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Any provider</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting tools</td>
			<td>Fine Science Tools</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein</td>
			<td>Merck</td>
			<td>103887</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin from cold water fish skin</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Merck</td>
			<td>S26</td>
			<td></td>
		</tr>
		<tr>
			<td>Head Holder</td>
			<td>Narishige</td>
			<td>SGM 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated plate</td>
			<td>BIOSEB LAB instruments</td>
			<td>BIO-HE002</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Imalgene 1000</td>
			<td>BOEHRINGER INGELHEIM ANIMAL HEALTH France</td>
			<td>N/A</td>
			<td>French marketing authorization numbre: FR/V/0167433 4/1992</td>
		</tr>
		<tr>
			<td>LAS X software</td>
			<td>Leica</td>
			<td>N/A</td>
			<td>Large volume computational clearing (LVCC) process</td>
		</tr>
		<tr>
			<td>Laser Chameleon Ultra II</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser power meter</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Thunder Imager Tissue microscope</td>
			<td>Leica</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-photon Zeiss LSM 7MP upright microscope</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocry-gel</td>
			<td>TVM lab</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Parametric oscillator</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Penlights with blue cobalt filtercap</td>
			<td>Bernell</td>
			<td>ALPEN</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Thermo Scientific</td>
			<td>150318</td>
			<td>Axotomy protocol</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Cornea whole-mount processing</td>
		</tr>
		<tr>
			<td>Rompun 2%</td>
			<td>Elanco</td>
			<td>N/A</td>
			<td>French marketing authorization numbre: FR/V/8146715 2/1980</td>
		</tr>
		<tr>
			<td>Sterile biopsy punch 2.5 mm</td>
			<td>LCH medical</td>
			<td>LCH-PUK-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>VWR</td>
			<td>0694</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>EuroBioSciences</td>
			<td>H-1000</td>
			<td>Mounting medium</td>
		</tr>
	</tbody>
</table>

three strategies to induce neurotrophic keratitis and nerve regeneration in murine cornea

角膜是覆盖眼睛的透明组织，对于清晰的视力至关重要。它是体内神经支配最多的组织。这种神经支配为眼睛提供感觉和营养功能，并有助于保持角膜的完整性。这种神经支配的病理破坏称为神经营养性角膜炎。这可能是由眼睛受伤、手术或疾病引发的。在这项研究中，我们提出了三种不同的方案，以概括临床中常见的三种类型的病例的方式对神经支配造成损害。
第一种方法是用眼科毛刺磨损上皮。这涉及以类似于临床上进行的光折射性角膜切除术的方式切除上皮层、游离神经末梢和基底下神经丛。第二种方法仅针对神经支配，方法是用活检穿刺在外周切开神经支配，以保持上皮的完整性。这种方法类似于板层角膜移植术的第一步，导致神经支配退化，然后中央角膜中的轴突再生。最后一种方法使用多光子显微镜破坏转基因小鼠模型的神经支配，多光子显微镜特异性定位荧光神经纤维的烧灼位点。这种方法会造成与光性角膜炎相同的损害，即过度暴露在紫外线下。
本研究描述了研究角膜神经支配生理病理学的不同选择，特别是轴突的退化和再生。促进再生对于避免角膜上皮缺损甚至穿孔等并发症至关重要。所提出的模型可以帮助测试新的药理学分子或基因疗法，以增强神经再生并限制疾病进展。

The cornea, which is the transparent surface of the eye, is composed of three distinct layers: the epithelium, the stroma, and the endothelium. This organ has the highest density of innervation in the body and is composed mainly of sensory fibers (types A&#948; and C) originating from the ophthalmic branch of the trigeminal ganglion. Sensory fibers penetrate the periphery of the cornea in the mid-stroma in the form of big bundles that branch out to cover the surface. They then bifurcate to pierce the Bowmann&#39;s membrane and form the subbasal plexus, which is easily recognizable by the formation of a vortex in the center of the cornea. Those fibers terminate as free nerve endings at the external surface of the epithelium. They are able to transduce thermal, mechanical, and chemical stimuli and to release trophic factors that are essential for epithelium homeostasis1,2. Neurotrophic keratitis (NK) is a degenerative disease affecting the corneal sensory innervation. This rare disease stems from a decrease or a loss of corneal sensitivity that results in lower tear production and poor healing properties of the cornea3. NK progresses through three well-described stages, from stage 1 where patients suffer epithelial defects, to stage 3 where stromal melting and/or corneal perforation occur4.
Clinically, the origins of this disease can be diverse. Patients can lose corneal innervation after physical injury to the eye, surgery, or through chronic diseases, such as diabetes5,6. To date, the NK pathogenesis process remains poorly understood, and therapeutical options for this sight-threatening condition are very limited. Therefore, a better understanding of the characteristics of epithelial defects is needed to better understand the mechanisms behind the regeneration of those fibers and potentially promote them. Here, we propose several models of corneal injury that induce NK in mice.
The first model is the abrasion of the epithelial layer of the cornea with an ocular burr. This model has mainly been studied in the context of the regeneration of the epithelium in different animals, such as rodents and fish7,8,9, and to test molecules promoting corneal healing10,11. Physiologically, it takes 2-3 days for the epithelial cells to close the wound. The physiological pattern of the innervation, however, takes more than four weeks to recover from the abrasion12,13. During the surgery, the ocular burr removes the epithelial layer of the cornea that contains the subbasal plexus and the fibers&#39; free nerve endings. This procedure can be clinically compared to patients with photorefractive keratectomy (PRK) to correct eye refractive defects. The procedure consists of removing the epithelium of the cornea and then reshaping the stroma with a laser14. Patients can experience several side effects following such surgery, such as a decrease of corneal nerve density for 2 years and a reduction in sensitivity for a duration of 3 months to one year post-surgery15. Given that the surgery induces a fragility of the corneal microenvironment, this model could help investigate these side effects and develop therapeutical approaches that would promote faster reinnervation, thus reducing the side effects in question.
The second model consists of sectioning the axons at the periphery of the cornea with a biopsy punch, inducing a Wallerian degeneration of the central innervation 16. Clinically, this method could be compared to anterior lamellar keratoplasty, in which the surgeon realizes a partial trephination of the cornea to remove a part of the anterior thickness of the cornea and replace it with a donor transplant 17. Following lamellar keratoplasty, patients may suffer from a number of symptoms including dry eye, loss of corneal innervation and graft rejection18. This axotomy model performed on corneal nerves could provide insight into the mechanisms of fiber degeneration, which occurs after a graft, followed by the axons&#39; regeneration.
The third method damages the corneal nerves with a laser. By using a multiphoton microscope on the cornea of anesthetized animals, degeneration of the nerves localized in the optical field is induced as a result of reactive oxygen species (ROS) formation, which leads to DNA damage and cellular cavitation19. This method recapitulates the corneal photodamage induced by overexposure to natural UV (sunburn), which also triggers ROS formation, leading to DNA damage20. Patients who suffer from corneal sunburn experience great pain, as the deterioration of epithelial cells deprives the corneal fibers&#39; extremities of all.
The three methods described here are designed to enable the investigation of the NK pathogenesis process and axon regeneration. They are easily reproducible and precise. Moreover, they allow quick recovery and easy monitoring of the animals.

作者聚焦：通过创新模型推进角膜神经支配研究 

诱导小鼠角膜神经营养性角膜炎和神经再生的三种策略

The cornea, which is the transparent tissue covering the eye, requires a balanced microenvironment to maintain its integrity. The dense innervation of the cornea participates to this microenvironment. If there is a loss, which could be partial or total, of this innervation, then a specific condition arises, which is called neurotrophic keratitis.To find new cures and efficient cures for this disease, we need to have specific models. The expansion of the use of transgenic animal bearing fluorescent reporters and microscopic advances have opened new possibilities in visualizing axonary generation. In vivo imagine has become more accessible to any research lab willing to follow the neurogeneration process.The various causes inducing neurotrophic keratitis underlying the difficulties of having only one model to test nerve regeneration therapies. These three models are reproducing various situations seen in clinics, and sharing those methods will allow other labs to uncover new aspects of corneal innervation from which innovative treatments will be designed.

本研究提出了几种对小鼠角膜神经支配造成损害的方案。虽然类似的方案已被用于研究上皮愈合的生理病理学，但我们选择适应和开发研究角膜神经支配再生的新方法。为了观察神经支配，我们使用了两种技术。首先，我们采用免疫荧光技术使用泛神经元抗体（BIII微管蛋白）对神经纤维进行染色，并用插层器对细胞核进行染色。其次，我们利用了在神经纤维中表达荧光蛋白的转基因小鼠品系。使...

Watch this Scientific Journal Video about Advancing Corneal Innervation Research Through Innovative Models at JoVE.com

在这里，我们提出了三种不同的方法来破坏支配角膜的感觉纤维。这些方法有助于研究小鼠的轴突再生。这三种方法适用于其他动物模型，是研究角膜神经支配生理学和再生的理想方法。

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

角膜是覆盖眼睛的透明组织，需要一个平衡的微环境来维持其完整性。角膜的致密神经支配参与这种微环境。如果这种神经支配丧失，可能是部分或全部，则会出现一种特定情况，称为神经营养性角膜炎。为了找到这种疾病的新疗法和有效治疗方法，我们需要有特定的模型。转基因动物携带荧光报告基因的扩大和微观进步为轴突生成的可视化开辟了新的可能性。对于任何愿意遵循神经生成过程的研究实验室来说，体内想象已经变得更容易获得。诱发神经营养性角膜炎的各种原因，是只有一种模型来测试神经再生疗法的困难所在。这三种模型正在重现临床上看到的各种情况，共享这些方法将使其他实验室能够发现角膜神经支配的新方面，从而设计出创新的治疗方法。

three-strategies-to-induce-neurotrophic-keratitis-nerve-regeneration

Research

JoVE Journal

Neuroscience

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

996 次观看.  University of  Montpellier, INSERM. 角膜是覆盖眼睛的透明组织，需要一个平衡的微环境来维持其完整性。角膜的致密神经支配参与这种微环境。如果这种神经支配丧失，可能是部分或全部，则会出现一种特定情况，称为神经营养性角膜炎。为了找到这种疾病的新疗法和有效治疗方法，我们需要有特定的模型。转基因动物携带荧光报告基因的扩大和微观进步为轴突生成的可视化开辟了新的可能性。对于任?...

视频: 作者聚焦：通过创新模型推进角膜神经支配研究 

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

科研

神经科学

Murine Corneal Nerve Abrasion for the Induction of Neurotrophic Keratitis

To begin corneal abrasion, place an anesthetized mouse on a table. Apply a drop of artificial tear on the eye undergoing the surgery. Apply a drop of ocular gel on the contralateral eye.Next, position the mouse on its side on a heated plate. Gently use a cotton swab to absorb the artificial tear, and brush aside the mouse's eyelashes without making contact with the eye. Turn on the ocular burr, and carefully open the mouse's eyelid with two fingers.Simultaneously shield the vibrancy to prevent them from entangling in the burr. Locate the pupil or the eye center and gently apply the ocular burr to the eye's surface in circular motions until the epithelium is removed. Alternatively, perform about 20 circular movements on the cornea.Now apply a drop of fluorescent staining solution on the abraided eye. After 20 seconds, dab the eye with a tissue to absorb the solution. Then rinse it with a drop of artificial tear.Finally, absorb the artificial tear with a tissue, and illuminate the eye using a blue cobalt lamp. Prior to the surgery, the basal epithelium was intact and the nerve fibers in the sub basal plexus formed a vortex. The burr tip removed the outer corneal layer, leaving the stroma and endothelium intact with a visible gap at the basal epithelium and sub basal plexus.Regeneration of the epithelium occurred over a week post-surgery. However, the vortex remained absent from the corneal center.

小鼠角膜神经磨损诱导神经营养性角膜炎

Murine Corneal Nerve Axotomy for the Induction of Neurotrophic Keratitis

To begin corneal nerve axotomy, place an anesthetized mouse on a table. Apply a drop of artificial tear on the eye undergoing the surgery. Apply a drop of ocular gel on the contralateral eye.Position the mouse under a binocular loupe to assess the eye that is being operated on. Place the bottom of a Petri dish under the mouse's head to keep the eye horizontal. With a cotton swab, carefully dad the artificial tear from the eye and remove the eyelashes without touching the eye.Place a pair of smooth curved pillars under the animal's eye and gently pop it out of the orbit. Gently place a biopsy punch vertically against the cornea surface, apply pressure, and twist the punch several times. Then, remove the biopsy punch and a circular mark should be visible on the cornea.A thin peripheral ring was seen at the point where the biopsy punch was applied, which damaged the basal epithelium. The peripheral nerves appeared to be sectioned, triggering the Wallerian axon degeneration. Heavy degeneration was observed, leading to total loss of innervation in the cornea center.This resulted in the disintegration of the epithelial layer, creating an ulcer.

小鼠角膜神经轴切术诱导神经营养性角膜炎

Localized Laser Ablation of Murine Corneal Nerves for the Induction of Neurotrophic Keratitis

To begin, place an anesthetized mouse on a table. Apply a drop of ocular gel on both eyes. Place the animal in a head holder to secure its position.Rotate the animal by 90 degrees so that the eye being operated on faces the ceiling. Secure the animal's vibrancy to clear the area around the eye. Secure the animal's body to minimize head movements caused by breathing.Place a circular cover slip horizontally over the eye on top of the ocular gel. Adjust the animal's head as necessary to correctly position the cover slip. Add a drop of PBS on top of the cover slip, then carefully place the animal onto a microscope stage.Adjust the objective turret of the microscope to the appropriate distance for observation. Activate the lasers to induce corneal nerve ablation. Once nerve ablation is complete, remove the animal from the head holder and place it in a cage on a heated plate.The vortex was visibly absent one week after laser ablation. A group of immune cells were observed to have migrated to the illumination site. The stromal fiber bundles appeared to have thinned out.