Name: development of a noninvasive, laser-assisted experimental model of corneal endothelial cell loss
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我们感谢克里斯蒂娜·奥伦和扬·阿·苏楚雷克在实验方法方面的帮助。

所有涉及动物组织的程序都遵循当地动物护理和道德委员会的准则。
1. 器官培养和激光治疗的准备
<ol><li>从当地的屠宰场获得新鲜镶有的猪眼。在Dulbecco的改良鹰中（DMEM）中保持凉爽（4°C），高葡萄糖，辅以L-谷氨酰胺、丙酮钠、青霉素/链霉素（1%）和猪血清（10%），本文今后称为全中量。</li>
	<li>用剪刀去除细胞外组织，将眼睛浸泡在5%povidone-碘眼科溶液中5分钟，然后放?...

<ol>
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试验研究结果表明，在选择能量剂量和焦点位置的适当参数时，Nd：YAG激光器可用于选择性地退位角膜内皮细胞。
由于内皮功能对角膜透明度和角膜免受基质水肿影响十分重要，内皮功能障碍模型在抗水肿药物或外科手术的发展中起着重要作用。有几个既定的体外模型来模拟体内情况10，14，15，但众所周知，体外模型不能完全模仿酶和细胞因子的影响或细胞-细胞相互作用的?...

角膜的透明度对于将光传输到视网膜及其光感受器1至关重要。在这方面，相对脱水状态对于保持角膜基质内的胶原纤维正确对齐至关重要。这种平衡由位于Descemet膜（DM）2上的角膜内皮细胞（CEC）维持。内皮是最内层角膜层。它有一个重要的屏障和泵功能，这是角膜透明度3的关键。与上皮相比，内皮不能自我更新4。因此，疾病或创伤造成的任何细胞损伤刺激剩余的内皮细胞扩大和迁移，覆盖由此产生的缺陷，并保持角膜功能5。然而，如果CEC密度低于临界阈值，内皮的解值会导致水肿，导致视力模糊和不适，甚至剧烈疼痛4。尽管有缓解症状的药物，目前这些病例中唯一的明确治疗方法是角膜移植，这种移植可以以全厚移植或角膜内皮移植的形式进行。后一个程序是作为Descemet的膜内皮角膜（DMEK）以及Descemet的剥离自动内皮角膜（DSAEK）6。6然而，保护剩余的CEC并增强其生存能力可能是一个替代目标，这需要一个适当的疾病模型来测试潜在的治疗药物。
目前的CEC损失病模型侧重于通过注射有毒物质（如氯化苯甲酸酯）或使用侵入性脱氨血症技术77、88的细胞机械磨损来破坏内皮。虽然这些模型已经确立，但存在一般炎症反应和不精确的附带损害等缺点。因此，这些模型更有可能代表疾病的最后阶段，当上述手术选择是不可避免的。
随着干细胞和基因治疗等细胞治疗策略的进步，这些细胞疗法的应用在CEC损失9的早期阶段可能很有用。随后，我们需要一个模型，更充分地代表疾病的这些早期阶段。在这方面，细胞培养模型在过去十年中有所改善，但其有效性仍然有限，因为体外细胞无法接近于复制角膜10内不同细胞类型之间发生的复杂相互作用。因此，活体和体内疾病模型仍然需求量大，改进现有的模式是十分感兴趣的。
非侵入性，眼内手术通过光干扰使用镉：YAG （Nd：YAG） 激光已成为世界各地的眼科医生的例行程序，因为它在20世纪70年代末推出11.光中断依赖于非线性光吸收，导致等离子体的形成，产生声冲击波，并产生气穴气泡，每当应用地点位于液体环境中12。一般来说，这些过程有助于精确组织切割的预期效果。然而，它们也可能成为不必要的附带损害的来源，限制了当地对激光手术的监禁。
通过对冲击波传播和气穴过程的表征，对由此产生的机械效应的预测有了显著改善。我们的目标是以尽可能少地破坏周围组织而达到CEC为目标，为CEC损失的早期阶段提供非侵入性激光辅助实验疾病模型。为此，有必要确定激光焦点的最佳脉冲能量和位置。

<table border="0" cellpadding="0" cellspacing="0" style="width:933px;" width="932">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:313px;">BARRON VACUUM TREPHINE</td>
			<td style="width:213px;">Katena</td>
			<td style="width:344px;">K20-2058</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cryostat</td>
			<td>Leica</td>
			<td>CM 3050S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium - high glucose</td>
			<td>PAA</td>
			<td>E-15009</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eye holder</td>
			<td>Self</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted Microscope</td>
			<td>Leica</td>
			<td>DMI 6000 B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KH2PO4</td>
			<td>Merck</td>
			<td>529568</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Na2HPO4</td>
			<td>Merck</td>
			<td>1065860500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nd:YAG laser</td>
			<td>Zeiss Meditec</td>
			<td>visuLAS YAG II plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT Tissue Tek</td>
			<td>Sakura Finetechnical</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010056</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Porcine serum</td>
			<td>Sigma-Aldrich</td>
			<td>12736C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spectral-domain optical coherence tomograph</td>
			<td>Heidelberg Engineering</td>
			<td>Spectralis</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue culture plate 12-well</td>
			<td>Sarstedt</td>
			<td>833921</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Two-Photon Microscope</td>
			<td>JenLab</td>
			<td>DermaInspect</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Viscoelastic</td>
			<td>OmniVision</td>
			<td>Methocel</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of a noninvasive, laser-assisted experimental model of corneal endothelial cell loss

Nd：YAG激光器已经用于进行非侵入性的眼内手术，如眼内切除术几十年了。精辟效果依赖于激光对焦处的光学故障。产生声冲击波和气穴气泡，导致组织破裂。气泡尺寸和压力振幅随脉冲能量和焦点位置而变化。在这项研究中，被包裹的猪眼被放置在商业上可用的Nd：YAG激光器前。测试了角膜后焦点的不同位置的可变脉冲能量以及不同位置。由此产生的病变通过双光子显微镜和组织学进行评估，以确定角膜内皮细胞（CEC）唯一分离的最佳参数，并尽量减少附带损害。该方法的优点是CEC的精确消融，减少附带损害，最重要的是非接触式处理。

使用此处介绍的程序，我们使用 Nd：YAG 激光治疗眼睛，评估不同的脉冲能量 （1.0±4.6 mJ） 和焦点的位置（与角膜后表面的距离：0.0±0.2 mm），以找到最佳参数。对激光参数的每个星座（12 x 21）计算了多个复制 （n = 3）。
除上述协议外，在固定和H&E染色之前，还使用双光子显微镜对试样进行了分析。双光子显微镜采用固态模式锁定的 80 MHz Ti：蓝宝石激光器，调谐范围为 690×1b...

Watch this Scientific Journal Video about Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss at JoVE.com

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

在这里，我们提出了一个协议，从Descemet的膜（DM）分离角膜内皮细胞（CEC），使用一个镉：YAG（Nd：YAG）激光作为牛角病（BK）的外活体疾病模型。

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

目前角膜内皮细胞流失的疾病模型侧重于内皮的破坏，更可能代表角膜移植不可避免的疾病的最后阶段。然而，使用干细胞或基因疗法的新疗法现在可用于疾病的早期阶段，而缺乏这些模型。使用 Nd：YAG 激光进行光破坏的非侵入性眼内手术已成为眼科医生的常规程序。从当地屠宰场获得新鲜被驯化的猪眼后，将样本以四摄氏度的全中等温度放置。使用剪刀去除细胞外组织，将眼睛浸泡在5%的碘眼液中5分钟。其次，将消毒样品放在无菌PBS中，在室温下。使用光谱域光学相干断层扫描设备，筛查眼睛中的主要前段病理，如角膜疤痕、水肿和其他不透明度。经过筛选，将眼睛放在装有波长为 1，064 纳米、空气中焦点直径为 10 微米的 Nd：YAG 激光器的滑灯单元前面。选择 12X 放大率并偏转照明以可视化各个角膜层。将脉冲能量和焦点设置为角膜内皮细胞选择性消融的适当参数后，对组织进行多次激光照射。然后在解剖显微镜下，将一个清晰的角膜副体靠近边缘，并注射粘弹性，以稳定前室。然后使用8毫米的颤数切除激光处理的中央角膜，将分离的角膜放入12井板内皮侧的一个井中。收集所有角膜后，在每个样品井中加入三毫米的全介质，并在37摄氏度下孵育样品长达三天。在孵化结束时，用索伦森缓冲液中不含甲醇的4%甲醛代替每口井中的介质，在室温下孵育20分钟。在孵育结束时，将固定样品放在PBS中20%蔗糖中约一小时，直到角膜下沉。将样品在 PBS 中隔夜转移到 30%蔗糖中，然后将组织嵌入最佳切割温度，在零下 80 摄氏度下储存。使用温度设置为零下 27 摄氏度的低温统计器，在获得后一分钟内获取 10 微米厚的冷冻组织，收集显微镜幻灯片上的每个部分。然后将幻灯片存放在零下80摄氏度，直到染色。对于赤氧树脂和 eosin 染色，空气干燥冷冻部分几分钟，以去除水分，然后用过滤的 0.1% 迈尔的赤氧素染色，在 50 毫升管中 10 分钟。在孵化结束时，用双蒸馏水冲洗滑梯五分钟。接下来，将幻灯片浸入 0.5%eosin 10 次，然后浸入双蒸馏水中冲洗，直到 eosin 停止条纹。将冲洗过的幻灯片浸入 50% 乙醇中 10 次，在 70% 乙醇中浸10次。最后70%乙醇浸渍后，在95%乙醇中平衡30秒，随后在100%乙醇中平衡60秒，然后多次浸渍二甲苯。然后在光学显微镜上获取图像之前，使用盖玻片安装试样。应独立评估两个光子和光显微镜图像，认为无损伤、太大的损伤或正确的损伤量。根据这些评估，可以计算热图，以选择正确的激光参数星座，选择性消融角膜内皮细胞，对周围组织的损害最小。先前的分析已经确定，激光的聚焦点必须至少比角膜内皮后面0.15毫米，以测试测试的最低脉冲能量。对于高于2.9毫焦耳的脉冲能量，测试的最长焦距仍然离内皮太近。各种细胞保护剂可以测试，而切除标本在培养。如果证明成功，它们可以添加到灌溉溶液中进行处理。

Research

JoVE Journal

Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

供体角膜内皮角膜移植术的组织准备

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

角膜新生血管在小鼠碱烧伤模型

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

角膜供体组织准备后弹力层内皮角膜移植术

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model

评价一种新型激光辅助冠状动脉吻合口连接器 - 三位一体夹 - 在猪非体外循环旁路模式

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

在脊髓损伤的实验挫伤型号神经干细胞移植

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

A Neonatal Mouse Spinal Cord Compression Injury Model

新生儿小鼠脊髓压迫损伤模型

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

使用磁共振成像实验性脑疟疾模型中的水肿发育​​和微血管病理学的体内追踪

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

家兔膳食诱导代谢综合征的实验模型: 方法论考虑、发展和评价

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...