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의학

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

Published: January 06, 2018
doi:
10.3791/56385
, , ,
1Department of Ophthalmology,Columbia University College of Physicians and Surgeons, 2Confocal and Specialized Microscopy Shared Resource, Herbert Irving Comprehensive Cancer Center,Columbia University

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 txl-的 SMG 准备: 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。 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?…

Representative Results

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?…

Discussion

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?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Second Harmonic GenerationSHGMicroscopyScleraTXLMyopiaCollagenSub-tenon’s InjectionFormaldehydeDifferential Scanning CalorimetryNon-invasiveRabbit EyeEyelid SpeculumConjunctivaExtra Ocular MusclesScleral InsertionPBSObjective LensNumerical ApertureImmersion Gel

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Zyablitskaya, M., Munteanu, E. L., Nagasaki, T., Paik, D. C. Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia. J. Vis. Exp. (131), e56385, doi:10.3791/56385 (2018).
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