为处理眼植入视网膜假体的局部病理组织学分析技术：第2部分视网膜前植入视网膜大头钉
Summary
Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.
Abstract
Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.
Introduction
色素性视网膜炎（RP）是一种遗传性疾病，导致感光体，它们是细胞中负责转导光视网膜的最外层的普遍丧失，在光子的形式，成神经活性。重要的是，患者的RP通常具有剩余的神经元在其视网膜的其它层，它们仍然起作用。视网膜假体能够通过针对这些存活的神经元电刺激来激活他们的视觉通路1,2恢复一些有限的视野，这些患者中。从临床试验的感性效果已经显示出可喜的早期结果和最近一些设备已被批准用于商业用途。目前，存在其中临床视网膜假肢已被定位的三个主要解剖学位置：epiretinally 3,4，视网膜下5,6和suprachoroidally 7,8。不同的设备利用不同的材料和它们的形式被定制到它们被植入的位置。然而，它们都构成视觉知觉通过激活视网膜的剩余神经元的电脉冲。
存在用于任何医疗假体损伤周围组织因初始放置的机械作用或随后持续力的潜力。在可植入刺激器，如视网膜假肢的情况下，存在的电气参数应该是在安全范围内的额外的考虑因素。病人的安全是最重要的，所以设备必须在临床前研究推进到临床设置9-15前的严格测试。在我们的同伴文章中，我们描述了用于评估周围定位在脉络膜上腔16的植入物的眼睛的局部组织病理学的方法。在本手稿中，我们描述用于可视眼组织周围的电极阵列上涨到视网膜epiretinally，在临床前的技术（FE线）模型（ 图1）。
在视网膜前的位置是定位视觉假体是最常用的位置。这里位于电极阵列通常固定到与该穿透眼睛17-20的所有层的金属粘着性视网膜。之前在本手稿中描述的技术，这是难以准确评估视网膜和直接围绕一个粘性其他组织。用中性缓冲的福尔马林标准眼球固定造成人为的视网膜损伤，由于视网膜和巩膜对粘性的固定点的差速运动。因此所造成的粘性和视网膜前阵的任何实际损害不能精确观察。另外，切片的眼组织不能与原位视网膜粘性进行作为金属物体不能容易地切断与传统组织学装置;去除粘性前组织处理也并不可取，因为这也导致人为的视网膜损伤。
本研究的目的是双重的：1），以减少视网膜脱离伪影，使得所引起的粘性和视网膜植入阵列的任何损坏能够可靠地评估;和2）以可视化相邻的粘性视网膜结构而不删除。为了达到目的，图1中，新的固定技术被利用，从而降低了人为的视网膜剥离（如在同伴文章16描述）。为了达到目的，2，我们修改的嵌入，研磨，和抛光技术中，最初在原位观察耳蜗植入电极21-23的开发。在这个手稿中描述的方法允许周围视网膜的可视化和邻近粘着在原位 ，同时尽量减少人为的视网膜损伤，因此允许所造成的粘性和视网膜前阵的任何潜在损害精确的评估。
Protocol
Representative Results
Discussion
标准组织学技术目前无法处理原位硬金属植入物由于在切割这些对象与金属，玻璃或甚至金刚石刀片的限制。在我们的配套文件16，我们发现，使用修改的全眼球固定技术可以减少人为的视网膜分层。在目前的手稿，一个既定的研磨和抛光技术可视化人工耳蜗植入原位 21-23修改视网膜假体。的钛粘性，用来固定电极阵列到视网膜，epiretinally，包埋在环氧树脂沿与周围的眼组织。这种树脂块，然后导向适当且逐步地/抛光以揭示所述组织形态学紧邻金属粘性。该块在不同深度的抛光表面的图像用一个功能强大的解剖显微镜。这种技术是有用的：可视化和evaluat荷兰国际集团邻近视网膜植入物的组织反应;评估与植入物的植入相关的外科手术创伤;以确定生物反应到硬金属部件;并测量植入物和视网膜的表面之间的距离。
这种技术将在未来的安全性研究可用于在邻近于在眼睛视网膜的粘性或其他硬（ 如，金属）物体区域的原位可视化。这在评估假肢上涨到视网膜epiretinally的临床前安全性直接应用。它也可以是用于评估组织损伤的视网膜的区域与位于子视网膜位置的植入物接触是有用的。
有几种方法，以验证该技术已被正确地执行。在每个阶段，视网膜应保持附着在眼球的外层。如果没有人为的视网膜脱离总值（GDP），这可能会印度语吃与固定的问题。当样品被嵌入和重新定向在最终树脂阻断视网膜应接近正交与磨削面块;这将最大限度地减少斜向切割。检查增量研磨步骤来遍历一个对象（如视网膜粘性）所需（已知步长）的数目与对象的尺寸相应地关联是有用的。
该技术可以被优化以几种方式。与研磨过程相关联的环氧块的表面上的划痕可减小逐渐变细级抛光。对于本研究中，我们使用的800，1000，1200，2400和4000级碳化硅纸。钻石膏也可以用来改善表面光洁度。更精细的表面光洁度给出更高质量的图像，但在附加的抛光时间的成本。另一个重要的考虑因素对于提高该技术的结果是OPTI的选择和质量CS和照明用于图像捕获。其他基本的组织学染色 – 特别是尼氏染色，可代替甲苯胺蓝的可以使用，但可能需要进一步优化。一些污渍会弄脏树脂以及该组织（ 例如 ，曙红），因此一个浅抛光，可能需要在染色后除去背景变色。专门的污渍，荧光染料和免疫组化染色没有尝试，但除非一个非常特定的结果是期望的，在每个磨削级别执行这些污渍所需的时间可能是令人望而却步。然而，有可能的嵌入步骤（步骤3.4）24之前以染色组织作为一个整体。
该技术的主要限制是，一旦所感兴趣的区域已被磨掉，它不能被检索，因此，为谨慎起见，在各种放大倍数在研磨和抛光的每个阶段，捕捉许多（可能冗余）的图像。这是还重要的是使用较小的增量为每个磨削深度调整。这种技术的另一个限制是该光学放大倍率和分辨率的组织固定在玻璃载片上，并与一个标准（传输）光学显微镜观察比较的那个。进行原型设计和评估一个新颖的植入装置的安全性的目的，病理评估总值为主要兴趣。该技术提供了用于观察与视网膜粘性相关的临床相关损害的有效方法。通过实践，收集研磨，抛光和拍摄一个给定的样本（一旦嵌入）所需的总时间是不相上下的时候，将采取部分中的蜡块或冰冻切片。
还有要延伸到视网膜植入物的范围之外的应用对本技术的潜力。这种技术适合于评估所述组织邻近硬植入物，其中植入物的提取不feasibLE或会损坏接口。例如，该技术可以扩展到评价从金属（ 例如 ，铂，镍钛诺等 ），不能用常规的组织学技术切割，制成的植入物，如某些脑深部或外周神经的电极，插管用于药物递送，血管支架或矫形假肢。
Disclosures
The authors have nothing to disclose.
Acknowledgements
Nicole Vella (Macquarie University) for providing reagents; Alexia Saunder (Bionics Institute; BI), Michelle McPhedran (BI), Chris Williams (BI) for experimental support; the Royal Victorian Eye and Ear Hospital (RVEEH) Biological Research Centre staff for animal care; Sue Pierce (RVEEH) for veterinary advice; Anthony Burkitt (Bionic Vision Australia; BVA), Tamara Brawn (BVA) and the BVA staff for administrative support.
This research was supported by the Australian Research Council (ARC) through its Special Research Initiative (SRI) in Bionic Vision Science and Technology grant to Bionic Vision Australia (BVA). The Bionics Institute receives Operational Infrastructure Support from the Victorian Government and also acknowledges support from the Bertalli Family Trust and the J T Reid Charitable Trust. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
The Bionic Vision Australia Consortia authors for this manuscript are (a-z):
Penelope J. Allen, Owen Burns, Kate E. Fox, Kumaravelu Ganesan, David J. Garret, Hamish Meffin, Joel Villalobos, and Jonathan Yeoh.
Materials
| Name of the reagent / equipment | Company | Catalogue number | Comments (optional) |
| Acetone | Chem-Supply | AA008 | Propanone BHD Medical grade |
| Epo-Tek 301 Epoxy | Epoxy Technology | Part A 1675-54-3 Part B 9046-10-0 | |
| Ethanol 70-75% v/v | Merck PTY LTD | 4.10261 | Alcohol |
| Ethanol | Merck PTY LTD | 90143 | Alcohol |
| Toluidine blue O | Sigma-Aldrich | T3260 | |
| Ethylenediamine Tetraacetic Acid | Sigma-Aldrich | ||
| TegraPol grinding/polishing machine | Struers | TegraPol-25 | |
| AccuStop specimen holder | Struers | Accustop | |
| Light microscope | Leica | MZ16 | |
| Objective lens | Leica | 2.0x Planapo Objective | |
| Digital Microscope Camera | Leica | DFC-420C | |
| Microscope Software | Leica | Application Suite v4.1.0 |
References
- Shepherd, R. K., Shivdasani, M. N., Nayagam, D. A., Williams, C. E., Blamey, P. J. Visual prostheses for the blind. Trends Biotechnol. 31, 562-571 (2013).
- Santos, A., et al. Preservation of the inner retina in retinitis pigmentosa. Arch Ophthalmol. 115, 511-515 (1997).
- Humayun, M. S., et al. Visual perception in a blind subject with a chronic microelectronic retinal prosthesis. Vision Res. 43, 2573-2581 (2003).
- Roessler, G., et al. Angiographic findings folowing tack fixation of a wireless epiretinal retina implant device in blind RP patients. Graefes Arch Clin Exp Ophthalmol. 249, 1281-1286 (2011).
- Chow, A. Y., et al. The artificial silicon retina microchip for the treatment of vision loss from retinitis pigmentosa. Arch Ophthalmol. 122, 460-469 (2004).
- Zrenner, E., et al. Subretinal electronic chips allow blind patients to read letters and combine them to words. Proc Biol Sci. 278, 1489-1497 (2011).
- Fujikado, T., et al. Testing of semichronically implanted retinal prosthesis by suprachoroidal-transretinal stimulation in patients with retinitis pigmentosa. Invest Ophthalmol Vis Sci. 52, 4726-4733 (2011).
- Saunders, A. L., et al. Development of a surgical procedure for implantation of a prototype suprachoroidal retinal prosthesis. Clin Experiment Ophthalmol. , (2013).
- Lee, S. W., et al. Development of microelectrode arrays for artificial retinal implants using liquid crystal polymers. Invest Ophthalmol Vis Sci. 50, 5859-5866 (2009).
- Sakaguchi, H., et al. Transretinal electrical stimulation with a suprachoroidal multichannel electrode in rabbit eyes. Jpn J Ophthalmol. 48, 256-261 (2004).
- Majji, A. B., et al. Long-term histological and electrophysiological results of an inactive epiretinal electrode array implantation in dogs. Invest Ophthalmol Vis Sci. 40, 2073-2081 (1999).
- Walter, P., et al. Successful long-term implantation of electrically inactive epiretinal microelectrode arrays in rabbits. Retina. 19, 546-552 (1999).
- Ray, A., Chan, L., Thomas, B., Weiland, J. D. Effects of prolonged stimulation at the electrode-retina interface. Conf Proc IEEE Eng Med Biol Soc. 1, 1285-1287 (2006).
- Colodetti, L., et al. Pathology of damaging electrical stimulation in the retina. Experimental eye research. 85, 23-33 (2007).
- Nayagam, D. A. X., et al. Chronic Electrical Stimulation with a Suprachoroidal Retinal Prosthesis: A Preclinical Safety and Efficacy Study. PLoS One. 9, e97182 (2014).
- Nayagam, D. A. X., et al. Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis. Journal of visualized experiments : JoVE. , e50411 (2013).
- Laube, T., et al. Development of surgical techniques for implantation of a wireless intraocular epiretinal retina implant in Gottingen minipigs. Graefe’s Archive For Clinical And Experimental Ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie. 250, 51-59 (2012).
- Gerding, H., et al. Successful long-term evaluation of intraocular titanium tacks for the mechanical stabilization of posterior segment ocular implants. Mat.-wiss. u. Werkstofftech. 32, 903-912 (2001).
- Seo, J. -. M., et al. Silicon retinal tack for the epiretinal fixation of the polyimide electrode array. Current Applied Physics. 6, 649-653 (2006).
- Hadjinicolaou, A. E., et al. Electrical stimulation of retinal ganglion cells with diamond and the development of an all diamond retinal prosthesis. Biomaterials. 33, 5812-5820 (2012).
- Briggs, R. J., et al. Comparison of round window and cochleostomy approaches with a prototype hearing preservation electrode. Audiol Neurootol. 11, 42-48 (2006).
- Shepherd, R., et al. An improved cochlear implant electrode array for use in experimental studies. Hear Res. 277, 20-27 (2011).
- Tykocinski, M., et al. Comparison of electrode position in the human cochlea using various perimodiolar electrode arrays. Am J Otol. 21, 205-211 (2000).
- Hardie, N. A., MacDonald, G., Rubel, E. W. A new method for imaging and 3D reconstruction of mammalian cochlea by fluorescent confocal microscopy. Brain Res. 1000, 200-210 (2004).
