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 border="0" cellpadding="0" cellspacing="0" width="1441">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:245px;">MILLI-Q SYNTHESIS A10 120V</td>
			<td style="width:289px;">EMD Millipore, Massachusetts, USA</td>
			<td style="width:111px;"></td>
			<td style="width:796px;">Double distilled, deionized water. - protocol step 1.1.1</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sodium hydroxymethylglycinate&#160;</td>
			<td>Tyger Chemicals Scientific, Inc. Ewing, NJ, USA</td>
			<td></td>
			<td>Crosslinking reagent - protocol step 1.1.2</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">Tono-pen&#160;</td>
			<td>Reichter Technologies Depew, NY</td>
			<td></td>
			<td>IOP measurements - protocol step 2.4</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DSC 6000 Autosampler</td>
			<td>Perkin-Elmer Waltham, MA, USA</td>
			<td></td>
			<td>Thermal denaturation analyzer - protocol step 7.4</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pyris software&#160;</td>
			<td>Perkin-Elmer, Waltham, MA, USA</td>
			<td>Ver 11.0</td>
			<td>&#160;protocol step 7.5</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">GenTeal&#160;</td>
			<td>Alcon, Fort Worth, TX&#160;</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 height="17">
			<td height="17" style="height:17px;">Chameleon Vision II&#160;</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 height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">Eclipse Ti-E</td>
			<td>Nikon Instruments, Melville, NY, USA</td>
			<td></td>
			<td>protocol step 8.1.4.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">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 height="17">
			<td height="17" style="height:17px;">NIS Elements software</td>
			<td>Nikon Instruments, Melville, NY, USA</td>
			<td>Ver 4.3</td>
			<td>refered to as &#34;software&#34; in the text - protocol step 8.1.9</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fiji/ImageJ</td>
			<td>National Institute of Health</td>
			<td></td>
			<td>&#160;protocol step 9.1.2</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">NeuronJ</td>
			<td style="width:289px;">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 height="17">
			<td height="17" style="height:17px;">Microsoft Excel&#160;</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

Universidad de Cartagena UDEC

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

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

综合胸部肿瘤生成数据库 - 翻译研究的工具

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

科研

医学

Osteopathic Manipulative Treatment as a Useful Adjunctive Tool for Pneumonia

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in the hospitalization of 1.1 million patients2. With an average length of in-patient hospital stay of five days2, pneumonia and influenza comprise significant financial burden costing the United States $40.2 billion in 20053. Under the current Infectious Disease Society of America/American Thoracic Society guidelines, standard-of-care recommendations include the rapid administration of an appropriate antibiotic regiment, fluid replacement, and ventilation (if necessary). Non-standard therapies include the use of corticosteroids and statins; however, these therapies lack conclusive supporting evidence4. (Figure 1)

Osteopathic Manipulative Treatment (OMT) is a cost-effective adjunctive treatment of pneumonia that has been shown to reduce patients&#8217; length of hospital stay, duration of intravenous antibiotics, and incidence of respiratory failure or death when compared to subjects who received conventional care alone5. The use of manual manipulation techniques for pneumonia was first recorded as early as the Spanish influenza pandemic of 1918, when patients treated with standard medical care had an estimated mortality rate of 33%, compared to a 10% mortality rate in patients treated by osteopathic physicians6. When applied to the management of pneumonia, manual manipulation techniques bolster lymphatic flow, respiratory function, and immunological defense by targeting anatomical structures involved in the these systems7,8, 9, 10.

The objective of this review video-article is three-fold: a) summarize the findings of randomized controlled studies on the efficacy of OMT in adult patients with diagnosed pneumonia, b) demonstrate established protocols utilized by osteopathic physicians treating pneumonia, c) elucidate the physiological mechanisms behind manual manipulation of the respiratory and lymphatic systems. Specifically, we will discuss and demonstrate four routine techniques that address autonomics, lymph drainage, and rib cage mobility: 1) Rib Raising, 2) Thoracic Pump, 3) Doming of the Thoracic Diaphragm, and 4) Muscle Energy for Rib 1.5,11

Pneumonia is one of the top ten leading causes of death in the U.S. Osteopathic manipulative techniques (OMT) may be utilized as an adjunctive therapy in the treatment of pneumonia to enhance biomechanical and immune function; and ultimately, to reduce hospital stay, duration of antibiotics, incidence of respiratory failure, and mortality. In this video-article, we will review randomized controlled studies on the use of OMT in pneumonia patients, as well as demonstrate specific hands-on techniques that are routinely practiced by osteopathic physicians when treating pulmonary infections.

整骨手法治疗为肺炎是非常好的辅助工具

Pneumonia, the inflammatory state of lung tissue primarily due to microbial infection, claimed 52,306 lives in the United States in 20071 and resulted in ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

生物标志物的研究下一代组织微阵列（ngTMA）协议

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

眼底摄影作为一种方便的工具，研究微血管回应流行病学研究心血管疾病的风险因素

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Therapeutic Effectiveness of a Dietary Supplement for Management of Halitosis in Dogs

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized placebo-controlled crossover clinical evaluation, we assessed&#160;the effectiveness of a dedicated dietary supplement to improve chronic halitosis in 32 dogs of different breeds and ages. This protocol describes how to evalute the presence of oral volatile suphur compunds, e.g. methyl mercaptan, hydrogen sulfide and dimethyl sulfide, by means of a portable gas chromatograph device coupled with a syringe, which was used to collect the breath, and a dedicated software, which allows the operator to monitor each compound concentration during each measurement, in a relatively short time (8 min).
A significant modification of halitosis parameters was observed after 30 days since the beginning of treatment (p &#60;0.05), while a long-lasting effect was still observed even 20 days after the suspension of the treatment. Portable gas chromatograph, which is also widely used in clinical practice, can be therefore used to confirm&#160;and control halitosis in humans and animals. Even though human and animal species present some differences,&#160;this innovative and alternative therapy for halitosis management might be extended to human clinical practice as an adjuvant dietary approach.

We describe a simple approach for diagnosis of halitosis in dogs as well as a dietary approach for its management. This protocol may be extended to the management of halitosis in humans in the near future.

膳食补充剂的口臭犬管理疗效

Halitosis is a common complaint involving social and communicational problems in humans and also affects the pet-owner relationship. In this randomized ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

评价疗效<em&gt;小时。幽门螺杆菌</em&gt;蛋白HP-NAP为膀胱癌的治疗在原位小鼠模型的治疗工具

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

MRI引导dmPFC-rTMS治疗作为治疗难治性抑郁症

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

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 ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

输卵管的管状细胞学作为卵巢癌早期检测的有前景的工具

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Subclavian Vein Puncture As an Alternative Method of Blood Sample Collection in Rats

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful compared to other more aggressive methods such as retro-orbital plexus sample collection. However, this blood collection method is sometimes limited by an unsatisfactory success rate. Here, we introduce a method for blood collection through the subclavian vein puncture. The subclavian vein is located just under the clavicle and this vein is large enough to fulfill the volume requirements of blood collection. Our results show that this method is safe and applicable for blood collection sampling with the required blood volume. Blood collection through the subclavian vein puncture could serve as an alternative blood collection method in case of failed tail vein blood sampling in rats.

Here, we present a protocol to collect blood samples from the rat subclavian vein.

锁骨下静脉穿刺作为大鼠采血的一种替代方法

Blood collection with enough blood volume is essential in animal experiments. Blood collection from the tail vein of rats is popular and less stressful ...