作者感谢 Tongalp Tezel, MD, 咨询有关 sT 注射液;特里萨 Swayne, 博士, 咨询有关倍显微镜;来自哥伦比亚大学医学中心欧文研究所的设计和生物统计学资源和生物核心设施。
在部分支持的研究, 以防止失明和国家卫生研究院赠款 NCRR UL1RR024156, 内 P30 EY019007, P30 CA013696, 和内 R01EY020495 (DCP)。哥伦比亚大学拥有相关知识产权: 美国颁发的专利号: 8466203 和 no: 9125856。国际专利待定: PCT/US2015/020276。
图像收集的共焦和专业显微镜共享资源的赫伯特欧文综合癌症中心在哥伦比亚大学, 支持的 NIH 赠款 #P30 CA013696 (国家癌症研究所)。共焦显微镜是购买与 NIH 赠款 #S10 RR025686。

所有的程序都是使用尸体兔眼在完整的 outbred 兔头。遵循了关于实验室动物的护理和使用的所有机构和国家准则。
1. 解决方案的编写
<ol><li>txl-的 SMG 准备:<ol>
<li>1毫升的0.2 米碳酸氢钠溶液 (NaHCO3) 溶液, 使用0.0165g 的 NaHCO3 粉末溶解在蒸馏水的1毫升。</li>
		<li>将0.1016 毫克的粉状钠 hydroxymethylglycinate (smg) 溶解在1毫升的蒸馏水中, 最终得到 800 mM 的浓度。调整碳酸氢钠溶液的最终浓度为0.1 米 NaHCO3和 400 mM SMG。根据所需的交联效果的 SMG 的浓度。在这里描述的协议中, 我们使用了40、100和 400 mM 的 SMG。</li>
	</ol>
</li>
</ol>2. SubTenon txl-使用 SMG 的注射
<ol><li>分别用400µL 控制和 SMG 溶液填充两个1毫升的胰岛素注射器 (25G 针)。</li>
	<li>在一个垫子的帮助下, 把兔子的头放在剖面平面上。聚苯乙烯泡沫塑料或纸栈可以用来修复头部在一个最佳位置。</li>
	<li>用儿童眼窥镜收回眼睑。</li>
	<li>使用平眼压装置测量初始眼压 (眼压)。</li>
	<li>在角膜缘的上半部分用组织标记标记拟注入的部位。</li>
	<li>用结膜钳 (或任何带有锯齿状圆尖的钳子) 在注射部位周围收回结膜, 并将针头插入结膜, 进入榫的蒴果稍超出标记的角膜缘部位 (即, 2-3从角膜缘的毫米)。一个小切口在结膜也可以用虹膜剪刀, 以方便通过针通过榫的胶囊。</li>
	<li>一旦在榫的胶囊, 确保针是自由移动, 通过移动它的侧边。在这段时间里, 地球不应该移动。这证实了适当的位置, 针以上的巩膜在小榫的 (sT) 空间。</li>
	<li>从注射器中注入溶液并丢弃针头。紧接着注射, 液体将积累在 sT 空间创造一个前凸出看到通过结膜 (即, 水肿)。</li>
	<li>重复的眼压测量, 以确认它没有改变, 由于一个疏忽穿孔的全球。</li>
	<li>取下盖子窥镜, 并通过闭合的眼睑进行数字按摩约2-3 分钟。</li>
	<li>在移动到下一步骤之前, 将头部保持在3.5 小时 (室温 = 18 ° c) 的潜伏期。</li>
</ol>3. 组织制备
<ol><li>使用眼睑窥镜收回眼睑, 以优化对全球的访问。根据眼睛的大小选择最佳尺寸的窥镜。</li>
	<li>将结膜周围的角膜缘分开。如果它已经被切割附近的注射点, 圆周扩大边界, 所以它将包含一个接种大小约1x1 厘米。</li>
	<li>切割 extra-ocular 的肌肉在他们的位置巩膜插入。</li>
	<li>用钳子把眼球抬高, 从后侧推。这提供了进入后地球, 并有助于切割视神经的眼动脉和静脉位于地球后极附近。</li>
	<li>切出的巩膜复合体, 外边界包括标记的注射地点。该污渍仍应在巩膜的剩余部分可见。</li>
	<li>通过使用组织钳牵引, 将语料库 vitreus和所有层连接到巩膜内侧。 注意: 进一步的步骤取决于正在执行的以下过程: 4.-DSC 分析, 5.-倍显微镜。</li>
</ol>4. 区域 DSC 分析
<ol><li>
治疗眼睛:用剪刀从剩余的巩膜杯中切出四个巩膜扇区, 使注射部位位于上部区域, 并集中排列。将其余的3扇区从侧边 (即、鼻和颞部) 和底部剪切。 注意: 在图 1A中演示了进一步划分为正方形 (1-16) 的扇区 (1-4) 的编号。</li>
	<li>将巩膜扇区 (1-4) 切割成较小的正方形 (1-16), 每块大约 4 x 4 毫米。区段1应该被划分成9正方形 (使射入的确切的站点一个单独正方形 [正方形 2])。划分区段2和3成2正方形每个 (正方形10-11 和 12-13) 和区段4成3正方形 (正方形 14-16)。</li>
	<li>为每个正方形分配一个数字, 如图 1A所示, 以便将分析的组织的距离定位到注入区域的位置。</li>
	<li>
控制眼睛:将组织分成四个巩膜扇区 (类似于处理过的组织) 后, 从以下位置切割出正方形的组织: 3 平方从顶部扇区 (扇区 1), 1 从每边 (扇区2和 3), 1 从底部区段 (区段 4)。</li>
	<li>将剩余的视网膜和脉络膜层刮掉, 每次用新鲜的 PBS 冲洗两次, 将这些碎片浸入溶液中一次大约十年代。</li>
</ol>5. 倍成像
<ol><li>使用剪刀切开巩膜的上部, 以创建一个 1 x 1 厘米的区域, 并将注射部位居中对齐。</li>
	<li>刮掉剩余的视网膜和脉络膜层, 每次用新鲜的 PBS 洗两次, 然后在大约十年代的时候把这些碎片留在溶液中。</li>
	<li>将组织放置在1毫升的管子中, 用 PBS 溶液填充, 以输送到成像设备。所有的程序, 在孵化时间之后, 从眼球解剖开始, 应在一小时内完成。</li>
</ol>6. 显微镜协议
注意: 本协议为激光扫描显微镜量身定做了从巩膜组织胶原蛋白成像后散射的倍信号。
<ol><li>
显微镜设置
	<ol>
<li>在执行倍显微镜时最大化信号和分辨率使用物镜优化传输红外光和高数值孔径 (NA)。我们的目标是尼康钻 25 x/NA1.1 水浸泡。</li>
		<li>调整镜头的校正衣领, 以配合样品的深度, 在这种情况下, 是厚度的片, 0.17 毫米。</li>
		<li>安装25x 物镜, 并添加大量的润滑水性凝胶, 以覆盖成像表面前安装样品。水基凝胶不会在实验过程中蒸发, 因此会保持图像质量。</li>
		<li>将巩膜组织从1毫升管与 PBS 不干燥之间的两个25毫米圆形片 (巩膜侧向下) 提供最大的接触之间的烧灼和片表面。 注: 组织也可放置在片上。大量的 PBS 应保持组织水合过程中的成像。在这种情况下, 添加组织片和 PBS 后组装的 cellchamber。</li>
		<li>通过放置一个25毫米圆形片, 单或在夹层技术, 组装的细胞室, 在底部的部分和螺钉的顶部部分下来, 以创建一个密封的圆形室。当使用顶部片时, 不要拧紧, 以免人为地使组织扁平和损坏。</li>
		<li>在显微镜台上用组织标本装入细胞室。</li>
		<li>用透射光将显微镜设置为眼观。</li>
		<li>定位舞台和调整目标的高度, 使样品的下表面处于焦点, 由明亮的现场检查通过眼睛片断确定。</li>
		<li>关闭所有的灯, 除了电脑显示器和阻挡尽可能多的光从显示器上, 铝箔板披在显微镜阶段。最小化任何到达探测器的杂散光将确保低噪音的获取, 因为 GaAsP NDD 探测器具有高灵敏度。</li>
		<li>在软件的 Ti Pad 面板中, 检查镜头定义是否正确。</li>
		<li>在 A1 紧凑的 GUI 面板, 选择红外激光成像, 选择 NDD 探测器, 并选择 DAPI 通道, 配备了 400-450 nm 带通滤波器。</li>
		<li>在 A1 MP GUI 面板中, 将红外激光器的波长设置为 860 nm, 并打开快门。</li>
		<li>在 A1 紧凑的 GUI 面板中设置激光扫描条件, 如下所示。选择: (a) 电扫描仪, (b) 单向扫描, (c) 像素驻留时间6.2 µs, (d) 帧大小 1024 x 1024 像素, (e) 线平均2x 注: 电扫描仪和单向扫描确保精确点对齐。整个视场的大小为 1024 x 1024, 转换为0.5 微米/像素的像素大小。线平均将减少图像中的拍摄噪声。</li>
		<li>通过调整激光功率和探测器增益来设置 A1 紧凑型 GUI 面板中的成像条件。打开 "查找表格" 面板 (尿路), 在当前图像中显示像素亮度值的直方图。打开 "查找" 模式中的实时图像, 通过调整激光功率和探测器增益来最大化检测到的像素值范围。避免饱和。典型值是2.5% 激光功率, 从 2.35 W 在 860 nm, 和100高压 (探测器增益)。</li>
		<li>注: 对于此设置, 用内部功率计测量的激光功率为5.2 兆瓦。每次进行实验时, 重新调整激光百分比, 使内部功率测量在5.2 兆瓦的成像会话之间恒定。设置激光电源时应注意。变色龙 II 激光器是一个 3 W 激光器在 800 nm 和10% 或更高的功率可能导致组织损伤。</li>
	</ol>
</li>
	<li>
图像采集
	<ol>
<li>在预览模式下, 使用 XYZ 概述工具扫描组织区域。</li>
		<li>将图像设置为较低的分辨率 (256 x 256 像素, 无线平均), 以加快此模式下的映像的获取速度。</li>
		<li>捕捉 5 x 5, 3 x 3, 或单一的视野, 以覆盖整个表面的组织。在每个位置, 在概要捕获之前, 打开现场 "扫描" 模式, 并使组织成为焦点。注意, 不同区域的组织将有轻微不同的位置在轴向方向。</li>
		<li>找到一个平坦的区域, 其中胶原纤维在整个视图字段中可见, 并在 "概览" 工具中双击该位置以将舞台移动到该特定位置。</li>
		<li>打开实时 "扫描" 模式, 调整目标的 z 位置, 使底部平面处于焦点, 在 Ti Pad 中, 使用 z 驱动器将光学平面10-15 μ m 移到此底部层之上。</li>
		<li>使用 "捕获" 按钮, 获取高分辨率的图像, 具有 1024 x 1024 像素和2x 线平均。</li>
		<li>使用 "+" 按钮保存 XYZ 概述中的位置。这样可以确保不重新捕获同一区域的组织。</li>
		<li>为每块组织捕捉10图像的不重叠的视野。</li>
	</ol>
</li>
</ol>7. DSC 协议
注意: 当组织准备完成时, 进行区域 DSC 分析, 或在 SHGM 进行组织成像后立即进行此步骤。
<ol><li>准备好 DSC, 称量和标注。 注意: 此步骤应在组织剥离前进行, 以减少组织的脱水。</li>
	<li>用吸水组织干燥每个巩膜正方形, 并用齿钳将其平放在 DSC 盘的底部。</li>
	<li>称锅内有组织, 盖子卷曲, 覆盖以获得组织湿重 (样品质量应在5至11毫克范围内)。 注: 在进行下一个组织样本之前, 使用机密封每个平底锅。锅是密封的, 防止任何水分损失之前, 热分析。</li>
	<li>一旦样品卷曲, 将其放置在其指定的位置上的 DSC 托盘。应该有6样本的控制和16的治疗眼睛。</li>
	<li>使用仪器管理软件创建方法, 指定组织的重量, 并使用以下参数运行热分析: 温度范围40至80° c, 加热速率: 1 ° c/分钟, 热流: 17.37 兆瓦, 气体 (N2) 流量: 19.8 毫升/分钟,气压: 2.2 巴。</li>
	<li>完成后, 通过使用仪器管理软件来提取热变性发生的过渡温度峰值来分析每个样品的数据。</li>
</ol>8. 图像分析
<ol><li>
倍信号
	<ol>
<li>选择至少5-10 个最具代表性的图像从每个治疗和它的控制, 这样, 图像的面积是由大多数胶原纤维占据。</li>
		<li>在 ImageJ 软件中上传每个图像, 并通过选择活动图像的分析 > 测量来测量平均像素强度。</li>
		<li>提取的值被报告为平均像素强度, 也可以通过从菜单 "分析 > 直方图" 中选择绘制亮度直方图来显示。</li>
		<li>使用 Excel 工作表, 创建一个表, 以相应地将所有测量的数据记录到示例 ID。</li>
		<li>计算每个处理和控制条件下像素强度的平均值和标准偏差。</li>
		<li>使用学生的 t 检验, 比较所有对浓度的两两比较的差异 (即, 40 mm smg vs. 0 和 400 mm, 0)。[P≤0.05]。</li>
	</ol>
</li>
	<li>
波纹
	<ol>
<li>选择显示胶原纤维的图像。每个样品至少有10图像应该分析 (包括每个浓度的控制样品-至少 40)。</li>
		<li>打开ImageJ > 插件 > NeuronJ。NeuronJ需要预先安装。</li>
		<li>将所有 imagesby 拖放到打开的NeuronJ窗口中。</li>
		<li>沿纤维创建跟踪线, 跟随纤维与鼠标的轮廓 (可以使用钢笔绘图片), 单击 M 以测量总光纤长度的距离。</li>
		<li>选择 "选项" 以绘制切线直线, 并连接先前绘制的纤维轮廓的开始和结束。现在, 单击 M 以测量端对长度。</li>
		<li>对每个图像至少10纤维重复相同的过程。</li>
		<li>收集这两个测量从每10纤维, 并输入数据到一个 excel 电子表格, 表示总纤维长度 (轮廓) 和端面长度 (直线连接线) 长度 [曲线] 和长度 [线性], 分别。</li>
		<li>使用公式计算波纹度指数 (w): w = 长度 [曲线]/长度 [线性]。</li>
		<li>使用该公式计算来自处理样品 (smg) 图像的波纹度与对照样品的图像的百分比: (w [smg]-1)/(w [控制]-1)</li>
		<li>对波纹度指数 (W) 进行配对 t 检验, 以确定不同处理条件和对照的胶原纤维形态的统计差异 (p 值)。</li>
	</ol>
</li>
</ol>

作者没有什么可透露的。

进行的实验表明, 证据支持使用倍信号显微镜作为一种方法评价胶原交联效果的巩膜, 提高未来的可能性, 使用该技术作为一种监测工具的交联治疗靶向胶原蛋白。值得注意的是, 一个仪器已经在临床上使用, 它可能会捕获这个倍信号。虽然该仪器主要是为成像皮肤人的真皮设计的, 它已成功地用于图像角膜和巩膜23。
在比较控制和处理样品时, 必须保持相同的?...

渐进性近视是假设通过酶巩膜交联 (光化学和/或化学) 可以治疗, 这是有道理的, 因为阻断胶原酶交联可以增加实验形式剥夺 (FD) 诱导近视1。Elsheikh 和菲利普斯2最近讨论了使用标准紫外线-A 辐照 (UVA)-核黄素介导的光化学交联 (也称为德累斯顿协议) 的可行性和潜力, 这里缩写为 (核黄素第一百四十)后巩膜稳定, 以制止近视的轴向伸长。这种光化学方法已成功地用于治疗圆锥和后 LASIK keratectasia 的前地球仪表面的失稳 (即, 膨隆的角膜)。然而, 这一第一百四十协议的应用受阻的问题与困难的后巩膜与紫外线 (UV) 光源, 以及需要修改一个更大的组织表面积。尽管如此, 第一百四十的方法已被用来制止视觉形式的剥夺兔的轴向伸长 (通过 tarsorrhaphy), 虽然后巩膜的多个区域需要多个单独的照射区在该研究3。相比之下, 通过 sT 空间注入化学稳定剂 (即, 交联剂) 可以更简单地改变后巩膜, 避免了引入紫外线光源的需要。这种注射技术是众所周知的一种有用的方法, 诱导眼麻醉期间眼科手术, 如白内障外科4,5,6。Wollensak7描述了先前使用的甘油 (一种化学交联剂, 在概念上类似于本研究中描述的甲醛释放剂 (法)) 来硬化兔巩膜和京尼平有已显示在 FD 豚鼠中限制轴向长度8,9。这些研究人员已经证明了在光化学第一百四十技术上使用可溶性化剂的明显优势。因此, 巩膜交联使用某种类型的注射化学剂, 包括法 (即, txl-)10, 可以提供一种可行的治疗方法, 以阻止近视伸长的进展。
在这里提出的协议, 我们使用化学交联溶液的钠 hydroxymethylglycinate (SMG), 通过 sT 注射到兔尸体的眼睛巩膜。我们已经实施了类似的协议之前的局部化学交联在角膜。特别是在先前报告的研究中 , 可利用 SMG 获得浓度依赖联效应 , 其作用范围可达到与光化学第一百四十一样的程度 , 由热变性分析确定为11.
在这里, 我们描述的协议, 以评估通过 sT 注射的 SMG 传递到巩膜组织, 热变性使用差示扫描量热法 (DSC) 和第二次谐波生成显微镜 (SHGM) 的交联效应。
使用差示扫描量热法 (DSC), 也称为热量分析, 测量热变性转变, 这对于巩膜组织主要是由纤维胶原的性质, 因为它们构成了大多数的蛋白质。该方法评价胶原分子结构的稳定性和稳定胶原纤维的交联键, 即主要的第三系蛋白质结构。在 DSC 加热过程中, 实现了一个关键的转变温度, 导致胶原分子的变性, 导致三重螺旋的开卷, 这一过程形成了通常所说的明胶。这种热变性扰乱了胶原分子的氢键, 通过诱导交联方法12,13可以转移到更高的温度。这种方法已经使用了几十年, 特别是在生物材料行业和工艺, 包括皮革制造。然而, 这种方法需要提取的巩膜组织, 因此只能作为一个ex 体内技术。
二次谐波生成显微镜 (SHGM) 是基于中心分子环境的特殊材料的非线性光学特性。在这种材料中, 强光, 例如激光产生的光, 产生倍信号, 其中入射光的频率加倍。已知产生倍信号的生物材料是胶原蛋白、微管和肌球蛋白。例如, 以 860 nm 波长的红外光激发的胶原蛋白将在可见光范围内发射一个倍信号, 其波长为 430 nm。二次谐波 (倍) 信号成像是评价治疗性胶原交联的一种很有前途的方法。已知30年多来, 组织中的胶原纤维发出倍信号14。但是, 只有最近才能在各种组织中获得高分辨率图像15 , 包括肌腱16、皮肤、软骨17、血管18和胶原凝胶19。
基于这一知识, 本研究通过 SMG 化学诱导的胶原交联来评价巩膜倍信号的变化。结果表明, 对巩膜的 SMG 修饰增加了组织胶原纤维束产生的倍信号 (由胶原蛋白组成的高阶第四纪结构), 同时也产生了胶原蛋白的结构形态变化。光纤网络, 反映在纤维束 "矫直" 中。

<table><tbody><tr><td>MILLI-Q SYNTHESIS A10 120V</td><td>EMD Millipore, Massachusetts, USA</td><td></td><td>Double distilled, deionized water. - protocol step 1.1.1</td></tr><tr><td>Sodium hydroxymethylglycinate&amp;nbsp;</td><td>Tyger Chemicals Scientific, Inc. Ewing, NJ, USA</td><td></td><td>Crosslinking reagent - protocol step 1.1.2</td></tr><tr><td>Injection needle with luer-lock syringe</td><td>BD Eclipse, NJ, USA</td><td></td><td>Syringe for sub tenon injection. - protocol step 2.1</td></tr><tr><td>Rabbit head</td><td>La Granja poultry</td><td>Outbred</td><td>Rabbit head separated and delivered within 1 hour postmortem. - protocol step 2.2</td></tr><tr><td>Tono-pen&amp;nbsp;</td><td>Reichter Technologies Depew, NY</td><td></td><td>IOP measurements - protocol step 2.4</td></tr><tr><td>DSC 6000 Autosampler</td><td>Perkin-Elmer Waltham, MA, USA</td><td></td><td>Thermal denaturation analyzer - protocol step 7.4</td></tr><tr><td>Pyris software&amp;nbsp;</td><td>Perkin-Elmer, Waltham, MA, USA</td><td>Ver 11.0</td><td>&amp;nbsp;protocol step 7.5</td></tr><tr><td>CFI75 Apochromat LWD 25X/1.10 W MP</td><td>Nikon Instruments, Melville, NY, USA</td><td></td><td>A water immersionn objective with high IR transmittance with a working distance of 2.0 mm - protocol step 8.1.1.</td></tr><tr><td>GenTeal&amp;nbsp;</td><td>Alcon, Fort Worth, TX&amp;nbsp;</td><td>B000URVDQ8</td><td>Water-based gel used as objective immersion medium instead of water to prevent evaporation - 8.1.1</td></tr><tr><td>Chameleon Vision II&amp;nbsp;</td><td>Coherent, Santa Clara,CA, USA</td><td></td><td>Ti:Sapphire pulsed laser with a 140 fs pulse width at 80 MHz and a tunable range from 680 nm to 1080 nm. - protocol step 8.1.11</td></tr><tr><td>AttoFluor cell chamber</td><td>Thermo Fisher Scientific Inc</td><td>A7816</td><td>Fixation of the cover slip - protocol step 8.1.3</td></tr><tr><td>25-mm round coverslips, #1.5</td><td>Neuvitro Corporation, Vancouver, WA, USA</td><td>GG-25-1.5</td><td>protocol step 8.1.3</td></tr><tr><td>Eclipse Ti-E</td><td>Nikon Instruments, Melville, NY, USA</td><td></td><td>protocol step 8.1.4.</td></tr><tr><td>Non-descanned (NDD) GaAsP detector</td><td>Nikon Instruments, Melville, NY, USA</td><td></td><td>Equipped with a 400-450 nm band pass filter - protocol step 8.1.7</td></tr><tr><td>A1R-MP laser scanning system</td><td>Nikon Instruments, Melville, NY, USA</td><td></td><td>Compatible with infrared (IR) multi-photon excitation. - protocol step 8.1.8</td></tr><tr><td>NIS Elements software</td><td>Nikon Instruments, Melville, NY, USA</td><td>Ver 4.3</td><td>refered to as &quot;software&quot; in the text - protocol step 8.1.9</td></tr><tr><td>Fiji/ImageJ</td><td>National Institute of Health</td><td></td><td>&amp;nbsp;protocol step 9.1.2</td></tr><tr><td>NeuronJ</td><td>Eric Meijering, Erasmus University Medical Center, Rotterdam, The Netherlands</td><td></td><td>https://imagescience.org/meijering/software/neuronj/, for protocol step 9.2.2</td></tr><tr><td>Microsoft Excel&amp;nbsp;</td><td>Microsoft Corporation, Redmond, WA, USA</td><td>Ver 14</td><td>protocol step 9.2.8</td></tr></tbody></table>

second harmonic generation signals in rabbit sclera as a tool for evaluation of therapeutic tissue cross-linking (txl) for myopia

方法加强组织通过引入化学键 (酶交联) 到结构蛋白 (纤维胶原) 的治疗包括光化学交联和组织交联 (txl-) 方法。在角膜变薄 (机械减弱) 性疾病中, 如圆锥的角膜, 以及渐进性近视的巩膜, 在后部巩膜发生, 可能有助于轴向伸长。这种组织增强的主要靶蛋白是纤维胶原, 它构成了角膜和巩膜中绝大部分的干重蛋白。偶然、纤维胶原蛋白是组织细胞外空间第二次谐波生成信号的主要来源。因此, 通过使用第二次谐波生成显微镜 (SHGM), 对胶原蛋白 (如通过交联疗法诱导的蛋白质) 的修饰可能会被发现并定量。通过使用激光扫描显微镜系统和红外激发光源来监测 SHGM 信号是一种令人兴奋的现代成像方法, 在生物医学科学中得到广泛的应用。因此, 本研究是为了评价使用 SHGM 显微镜作为一种手段, 以测量诱导交联效果的前体内兔巩膜, 继注射了化学交联剂到分榫的空间 (sT),注射法是眼科临床过程中引起眼部麻醉的标准做法。化学交联剂, hydroxymethylglycinate 钠 (SMG), 是从一类的化妆品防腐剂称为甲醛释放剂 ()。与 SMG 反应后的巩膜改变导致倍信号增加, 并与热变性温度的变化相关, 这是评价诱导组织交联效应的标准方法。

热变性温度 (Tm) 作为一种测定 txl-交联效应的方法:在这些实验中, 共使用了16对兔眼的 txl-程序。作为本研究的第一部分, 对单次注射 SMG 交联剂在兔尸体头上的 sT 间隙诱导交联效应的定位进行了评价。这种类型的实验与患者的临床治疗相关, 因为在一个以上的位置注射可能是稳定一个理想的巩膜区域是必要的。
根?...

Watch this Scientific Journal Video about Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia at JoVE.com

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

该协议描述了使用第二次谐波生成成像和差示扫描量热法评估兔巩膜化学交联的技术。

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

second-harmonic-generation-signals-rabbit-sclera-as-tool-for

Research

JoVE Journal

Medicine

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

7.7K 次观看.  Columbia University College of Physicians and Surgeons. 该协议描述了使用第二次谐波生成成像和差示扫描量热法评估兔巩膜化学交联的技术。

视频: Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

新型光声显微镜与光学相干层析成像在活兔眼中的双模脉络成像

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

科研

工程学

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been introduced as an efficient treatment option. In biological and chemical sciences, crosslinking refers to new chemical bonds formed between reactive molecules. Hence, the aim of corneal collagen CXL is to synthetically increase the formation of crosslinks between collagen fibrils in the corneal stroma. Despite the fact that the efficiency of the conventional CXL (C-CXL) protocol has already been shown in several clinical studies, it might benefit from improvements in duration of the procedure and removal of corneal epithelium. Hence, in order to provide a coherent evaluation of two new and optimized CXL protocols, we studied keratoconus patients who had undergone one of the three CXL treatments: iontophoresis (I-CXL), accelerated CXL (A-CXL), and conventional CXL (C-CXL). A-CXL is a 6 time faster CXL procedure using a ten time higher UVA irradiance but still including an epithelium removal. Iontophoresis is a transepithelial non-invasive technique in which a small electric current is applied to improve riboflavin penetration throughout the cornea. Using anterior segment optical coherence tomography (AS OCT) and in vivo confocal microscopy (IVCM), we conclude that regarding the depth of treatment penetration, conventional CXL protocol remains the standard for treating progressive keratoconus. Accelerated CXL seems to be a quick, effective and safe alternative to treat thin corneas. The use of iontophoresis is still being investigated and should be considered with greater caution.

Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

角膜胶原交联的圆锥角膜的三种不同的协议:常规，加速和离子导入

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been ...

医学

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

一种用于兔视网膜下手术分步协议

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

使用光学相干断层扫描对啮齿动物模型中眼部疾病的体内结构评估

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

生物工程

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the subretinal space, and to evaluate toxicity and efficacy in vivo. HR-SD-OCT uses near infrared light (800-880 nm) and has optics specifically designed for the unique optics of the mouse eye with sub-2-micron axial resolution. Transgenic mouse models of outer retinal (photoreceptor) degeneration and controls were imaged to assess the disease progression. Pulled glass microneedles were used to deliver sub retinal injections of adeno-associated virus (AAV) or nanoparticles (NP) via a trans-scleral and trans-choroidal approach. Careful positioning of the needle into the subretinal space was required prior to a calibrated pressure injection, which delivers fluid into the sub retinal space. Real time subretinal surgery was conducted on our retinal imaging system (RIS). HR-SD-OCT demonstrated progressive uniform retinal degeneration due to expression of a toxic mutant human mutant rhodopsin (P347S) (RHOP347S) transgene in mice. HR-SD-OCT allows rigorous quantification of all the retinal layers. Outer nuclear layer (ONL) thickness and photoreceptor outer segment length (OSL) measurements correlate with photoreceptor vitality, degeneration, or rescue. The RIS delivery system allows real-time visualization of subretinal injections in neonatal (~P10-14) or adult mice, and HR-SD-OCT immediately determines success of delivery and maps areal extent. HR-SD-OCT is a powerful tool that can evaluate the success of subretinal surgery in mice, in addition to measuring vitality of photoreceptors in vivo. HR-SD-OCT can also be used to identify uniform animal cohorts to evaluate the extent of retinal degeneration, toxicity, and therapeutic rescue in preclinical gene therapy research studies.

Here we demonstrate a novel approach to using high resolution spectral-domain optical coherence tomography (HR-SD-OCT) to assist delivery of gene therapy agents into the subretinal space, assess its areal coverage, and characterize photoreceptor vitality.

超高分辨率小鼠光学相干层析成像辅助眼内注射在视网膜基因治疗中的应用

HR-SD-OCT is utilized to monitor the progression of photoreceptor degeneration in live mouse models, assess the delivery of therapeutic agents into the ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

脉络细胞自体移植治疗老年性黄斑变性的再生疗法: 初步体内报告

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

Inducement and Evaluation of a Murine Model of Experimental Myopia

Murine model of myopia can be a powerful tool for myopia research because of the comparatively easy genetic manipulation. One way to induce myopia in animals is to put clear minus lenses in front of eyes for weeks (lens-induced myopia, LIM). However, extant protocols for inducement and evaluation vary from laboratory to laboratory. Here, we described a highly practical and reproducible method to induce LIM in mice using newly designed eyeglasses. The method fixes the lens stably in front of the mouse eye while allows the lens to be taken off for cleaning or topical drug administration. The phenotype is robust and efficient, and the variance is small. The method described here can be applied to mice right after weaning which extends the possible duration for experiments. We also gave technical advises for achieving reproducible results in refraction and axial length measurements. We hope the step-by-step protocol described here and the detailed article can help researchers perform myopia experiments with myopia more smoothly and make the data comparable across laboratories.

In this protocol, we describe the full process of experimental myopia inducement in mice using newly designed eyeglasses and the technic needed for achieving stable and reproducible results in ocular parameter measurements.

实验性近视小鼠模型的诱导与评价

Murine model of myopia can be a powerful tool for myopia research because of the comparatively easy genetic manipulation. One way to induce myopia in ...

神经科学

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...