诱导眼表炎症和受累组织收集
Summary
眼表炎症会损害眼表组织并损害眼睛的重要功能。本协议描述了一种在睑板腺功能障碍（MGD）的小鼠模型中诱导眼部炎症和收集受损组织的方法。
Abstract
眼表疾病包括一系列干扰角膜、结膜和相关眼表腺网络的功能和结构的疾病。睑板腺（MG）分泌脂质，形成覆盖层，防止泪膜水部分蒸发。中性粒细胞和细胞外DNA陷阱在过敏性眼病小鼠模型中填充MG和眼表。聚集的中性粒细胞细胞外陷阱（aggNET）形成由细胞外染色质组成的网状基质，可阻塞MG出口并调节MG功能障碍。本文提出了一种诱导眼表炎症和MG功能障碍的方法。详细描述了收集与眼表相关的器官的程序，例如角膜，结膜和眼睑。使用处理每个器官的既定技术，还显示了MG功能障碍的主要形态学和组织病理学特征。眼渗出物提供了评估眼表炎症状态的机会。这些程序能够在临床前水平上研究局部和全身抗炎干预。
Introduction
每眨眼一下，就会补充分散在角膜上的光滑泪膜。眼表上皮有助于泪膜在眼表的分布和正确定向。粘蛋白由角膜和结膜上皮细胞提供，以帮助定位泪膜的水部分，这些泪膜来自眼睛表面的泪腺。最后，MG分泌脂质，形成覆盖层，防止泪膜1，2，3的水性部分蒸发。以这种方式，所有眼器官的协调功能保护眼表免受病原体入侵或伤害，并支持水晶般清晰的视力，而没有任何疼痛或不适。
在健康的眼表，眼流分泌物或眼部风湿可清除灰尘、死去的上皮细胞、细菌、粘液和免疫细胞。聚集的中性粒细胞细胞外陷阱（aggNET）配制由细胞外染色质组成的网状基质，并将这些成分掺入眼风湿中。AggNETs通过促炎细胞因子和趋化因子的蛋白水解降解来解决炎症4。然而，当它们变得功能失调时，这些异常的 aggNET 会驱动疾病的发病机制，例如 COVID-195、胆结石6 和唾液石症7 中的血管闭塞。同样，眼表上的aggNETs起着保护作用，有助于解决高度暴露表面的炎症8。眼表过度形成或缺乏 aggNET 都会损害泪膜稳定性和/或导致角膜伤口、瘙痒性结膜炎和干眼症。例如，MG的梗阻是干眼症的主要原因9。众所周知，AggNETs会堵塞MG导管的脂质分泌流并导致睑板腺功能障碍（MGD）。aggNETs对MG孔的充血导致缺乏脂肪液包裹眼表和逆行封堵液，导致腺体功能障碍和腺泡损伤。这种功能障碍会导致泪膜蒸发、眼睑边缘纤维化、眼睛发炎以及对 MG10，11 的有害损害。
多年来，已经开发了几种动物模型来模仿人类MGD的病理过程。例如，1岁的C57BL / 6小鼠帮助研究了与年龄相关的对干眼病（DED）和MGD的影响，反映了50岁及以上患者的眼部疾病病理学12，13，14。此外，兔子是研究药物干预效果的适当模型。因此，通过局部给予肾上腺素或全身引入13-顺式维甲酸（异维A酸）15，16，17，18，19在兔中诱导MGD已有报道。
尽管这些动物模型足以确定导致MGD病理生理学的不同因素，但它们的使用受到限制。例如，与年龄相关的MGD的小鼠模型仅适用于破译老年人的元素，因此，兔子似乎是最适合研究眼表疾病的动物模型，因为它们能够研究多种病理生理机制。然而，由于缺乏全面的分析工具来检测眼表的蛋白质，并且由于兔基因组的许多部分没有注释，它们仅限于研究20，21。
此外，这些用于研究干眼病发病机制的动物模型没有提供足够的细节来分析引发眼表炎症的疾病的免疫学臂。因此，由Reyes等人开发的MGD小鼠模型显示小鼠过敏性眼病与人类MGD之间存在关联，并强调了导致阻塞性MGD21的免疫病因。该模型将过敏性眼病与TH17反应相关联，TH17反应将中性粒细胞募集到结膜和眼睑，导致MGD和慢性眼部炎症21。在该小鼠模型中诱导MGD和眼部炎症是研究由持续免疫反应驱动的局部炎症发展过程中上游事件的宝贵工具21。目前的方案描述了伴有阻塞性MGD的眼表炎症。在该方法中，对小鼠进行免疫接种，并在2周后用免疫原在眼表上激发7天。此外，还描述了在急性炎症期间分离眼渗出物和相关眼器官的步骤以及角膜、结膜和眼睑的解剖。
Protocol
所有涉及动物的程序均根据动物福利机构指南进行，并经埃尔朗根-纽伦堡弗里德里希-亚历山大大学（FAU）动物福利委员会批准（许可证号：55.2.2-2532-2-1217）。本研究使用年龄为7-9周的雌性C57Bl / 6小鼠。从商业来源获得小鼠（见 材料表），并以12小时昼夜循环保存在特定的无病原体条件下。 1.诱导小鼠眼表炎症 进行免疫接种的免疫原准备。…
Representative Results
本协议描述了建立眼表炎症小鼠模型的顺序步骤。这些协议旨在展示如何在局部应用治疗，获取眼部渗出物，并切除相关的辅助器官，如健康和发炎的眼睑（图2），角膜和结膜。解剖上眼睑时必须注意结膜的隔离，并且在角膜解剖期间必须将其储存在1x PBS中。这将防止结膜干燥，结膜可用于组织学、药代动力学和基因表达研究。 按照上述方案连续7天局…
Discussion
睑板腺的油性分泌物对健康的眼睛非常重要22。然而，聚集的中性粒细胞细胞外陷阱（aggNET）阻塞这些皮脂腺，这些中性粒细胞细胞外陷阱（aggNET）排列成位于双眼睑睑板上的平行链，可以破坏泪膜23。这种破坏导致睑板腺功能障碍（MGD）1 和加速泪液蒸发并调节眼表损伤2。该协议描述了建立导致MG梗阻的免疫介导的眼表炎症?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了德国研究基金会（DFG）2886 PANDORA项目-No.B3的部分支持;SCHA 2040/1-1;MU 4240/2-1;CRC1181（C03）;TRR241（B04），H2020-FETOPEN-2018-2020项目861878，并由大众基金会（授予97744）授予MH。
Materials
| 1x PBS | Gibco | ||
| Aluminium Hydroxide | Imject alum Adjuvant | 77161 | 40 mg/ mL Final Concentration: in vivo: 1 mg/ 100 µL |
| C57Bl/6 mice, aged 7–9 weeks | Charles River Laboratories | ||
| Calcium | Carl roth | CN93.1 | 1 M Final Concentration: 5 mM |
| Curved forceps | FST by Dumont SWITZERLAND | 5/45 11251-35 | |
| Fine sharp scissor | FST Stainless steel, Germany | 15001-08 | |
| Laminar safety cabinet | Herasafe | ||
| Macrophotography Camera | Canon | EOS6D | |
| Macrophotography Camera (without IR filter) | Nikon | D5300 | |
| Mnase | New England biolabs | M0247S | 2 x 106 gel U/mL |
| Multi-analyte flow assay kit (Custom mouse 13-plex panel) | Biolegend | CLPX-200421AM-UERLAN | |
| NaCl 0,9% (Saline) | B.Braun | ||
| Ovalbumin (OVA) | Endofit, Invivogen | 9006-59-1 | 10 mg/200 µL in saline |
| Pertussis toxin | ThermoFisher Scientific | PHZ1174 | 50 µg/ 500 µL in saline Final Concentration: in vivo: 100 µg/ 100 µL |
| Petridish | Greiner bio-one | 628160 | |
| Scalpel | Feather disposable scalpel | No. 21 | Final Concentration: in vivo: 300 ng/ 100 µL |
| Stereomicroscope | Zaiss | Stemi508 | |
| Syringe (corneal/iris washing) | BD Microlane | 27 G x 3/4 – Nr.20 0,4 x 19 mm | |
| Syringe (i.p immunization) | BD Microlane | 24 G1"-Nr 17, 055* 25 mm |
Referências
- Gilbard, J. P., Rossi, S. R., Heyda, K. G. Tear film and ocular surface changes after closure of the meibomian gland orifices in the rabbit. Ophthalmology. 96 (8), 1180-1186 (1989).
- Mishima, S., Maurice, D. M. The oily layer of the tear film and evaporation from the corneal surface. Experimental Eye Research. 1, 39-45 (1961).
- Gipson, I. K. The ocular surface: The challenge to enable and protect vision: The Friedenwald lecture. Investigative Ophthalmology and Visual Science. 48 (10), 4391-4398 (2007).
- Hahn, J., et al. Aggregated neutrophil extracellular traps resolve inflammation by proteolysis of cytokines and chemokines and protection from antiproteases. The FASEB Journal. 33 (1), 1401-1414 (2019).
- Leppkes, M., et al. Vascular occlusion by neutrophil extracellular traps in COVID-19. EBioMedicine. 58, 102925 (2020).
- Munoz, L. E., et al. Neutrophil extracellular traps initiate gallstone formation. Immunity. 51 (3), 443-450 (2019).
- Schapher, M., et al. Neutrophil extracellular traps promote the development and growth of human salivary stones. Cells. 9 (9), 2139 (2020).
- Mahajan, A., et al. Frontline science: Aggregated neutrophil extracellular traps prevent inflammation on the neutrophil-rich ocular surface. Journal of Leukocyte Biology. 105 (6), 1087-1098 (2019).
- DEWS Definition and Classification Subcommittee. The definition and classification of dry eye disease: Report of the Definition and Classification Subcommittee of the International Dry Eye Workshop. The Ocular Surface. 5 (2), 75-92 (2007).
- Nichols, K. K., et al. The international workshop on meibomian gland dysfunction: Executive summary. Investigative Ophthalmology and Visual Science. 52 (4), 1922-1929 (2011).
- Mahajan, A., et al. Aggregated neutrophil extracellular traps occlude Meibomian glands during ocular surface inflammation. The Ocular Surface. 20, 1-12 (2021).
- Jester, B. E., Nien, C. J., Winkler, M., Brown, D. J., Jester, J. V. Volumetric reconstruction of the mouse meibomian gland using high-resolution nonlinear optical imaging. The Anatomical Record. 294 (2), 185-192 (2011).
- Nien, C. J., et al. Age-related changes in the meibomian gland. Experimental Eye Research. 89 (6), 1021-1027 (2009).
- Parfitt, G. J., Xie, Y., Geyfman, M., Brown, D. J., Jester, J. V. Absence of ductal hyper-keratinization in mouse age-related meibomian gland dysfunction (ARMGD). Aging. 5 (11), 825-834 (2013).
- Lambert, R. W., Smith, R. E. Pathogenesis of blepharoconjunctivitis complicating 13-cis-retinoic acid (isotretinoin) therapy in a laboratory model. Investigative Ophthalmology and Visual Science. 29 (10), 1559-1564 (1988).
- Jester, J. V., Nicolaides, N., Kiss-Palvolgyi, I., Smith, R. E. Meibomian gland dysfunction. II. The role of keratinization in a rabbit model of MGD. Investigative Ophthalmology and Visual Science. 30 (5), 936-945 (1989).
- Jester, J. V., et al. In vivo biomicroscopy and photography of meibomian glands in a rabbit model of meibomian gland dysfunction. Investigative Ophthalmology and Visual Science. 22 (5), 660-667 (1982).
- Lambert, R., Smith, R. E. Hyperkeratinization in a rabbit model of meibomian gland dysfunction. American Journal of Ophthalmology. 105 (6), 703-705 (1988).
- Knop, E., Knop, N., Millar, T., Obata, H., Sullivan, D. A. The international workshop on meibomian gland dysfunction: Report of the subcommittee on anatomy, physiology, and pathophysiology of the meibomian gland. Investigative Ophthalmology and Visual Science. 52 (4), 1938-1978 (2011).
- Huang, W., Tourmouzis, K., Perry, H., Honkanen, R. A., Rigas, B. Animal models of dry eye disease: Useful, varied and evolving (Review). Experimental and Therapeutic Medicine. 22 (6), 1394 (2021).
- Reyes, N. J., et al. Neutrophils cause obstruction of eyelid sebaceous glands in inflammatory eye disease in mice. Science Translational Medicine. 10 (451), (2018).
- Knop, E., Korb, D. R., Blackie, C. A., Knop, N. The lid margin is an underestimated structure for preservation of ocular surface health and development of dry eye disease. Developments in Ophthalmology. 45, 108-122 (2010).
- Knop, N., Knop, E. Meibomian glands. Part I: anatomy, embryology and histology of the Meibomian glands. Ophthalmologe. 106 (10), 872-883 (2009).
- Nien, C. J., et al. Effects of age and dysfunction on human meibomian glands. Archives of Ophthalmology. 129 (4), 462-469 (2011).
- Lio, C. T., Dhanda, S. K., Bose, T. Cluster analysis of dry eye disease models based on immune cell parameters – New insight into therapeutic perspective. Frontiers in Immunology. 11, 1930 (2020).
- Nguyen, D. D., Luo, L. J., Lai, J. Y. Thermogels containing sulfated hyaluronan as novel topical therapeutics for treatment of ocular surface inflammation. Materials Today Bio. 13, 100183 (2022).
- Lin, P. H., et al. Alleviation of dry eye syndrome with one dose of antioxidant, anti-inflammatory, and mucoadhesive lysine-carbonized nanogels. Acta Biomaterialia. 141, 140-150 (2022).
- Yu, D., et al. Loss of beta epithelial sodium channel function in meibomian glands produces pseudohypoaldosteronism 1-like ocular disease in mice. American Journal of Pathology. 188 (1), 95-110 (2018).
- Mauris, J., et al. Loss of CD147 results in impaired epithelial cell differentiation and malformation of the meibomian gland. Cell Death & Disease. 6 (4), 1726 (2015).
- Ibrahim, O. M., et al. Oxidative stress induced age dependent meibomian gland dysfunction in Cu, Zn-superoxide dismutase-1 (Sod1) knockout mice. PloS One. 9 (7), 99328 (2014).
- McMahon, A., Lu, H., Butovich, I. A. A role for ELOVL4 in the mouse meibomian gland and sebocyte cell biology. Investigative Ophthalmology and Visual Science. 55 (5), 2832-2840 (2014).
- Miyake, H., Oda, T., Katsuta, O., Seno, M., Nakamura, M. Meibomian gland dysfunction model in hairless mice fed a special diet with limited lipid content. Investigative Ophthalmology and Visual Science. 57 (7), 3268-3275 (2016).
- Schaumberg, D. A., et al. The international workshop on meibomian gland dysfunction: Report of the subcommittee on the epidemiology of, and associated risk factors for, MGD. Investigative Ophthalmology and Visual Science. 52 (4), 1994-2005 (2011).
- Lee, S. Y., et al. Analysis of tear cytokines and clinical correlations in Sjogren syndrome dry eye patients and non-Sjogren syndrome dry eye patients. American Journal of Ophthalmology. 156 (2), 247-253 (2013).
- Nakae, S., et al. Antigen-specific T cell sensitization is impaired in IL-17-deficient mice, causing suppression of allergic cellular and humoral responses. Immunity. 17 (3), 375-387 (2002).
- von Vietinghoff, S., Ley, K. IL-17A controls IL-17F production and maintains blood neutrophil counts in mice. Journal of Immunology. 183 (2), 865-873 (2009).
- Langrish, C. L., et al. IL-23 drives a pathogenic T cell population that induces autoimmune inflammation. Journal of Experimental Medicine. 201 (2), 233-240 (2005).
- Chen, Y., et al. Anti-IL-23 therapy inhibits multiple inflammatory pathways and ameliorates autoimmune encephalomyelitis. Journal of Clinical Investigation. 116 (5), 1317-1326 (2006).
Citar este artigo
Singh, J., Shan, X., Mahajan, A., Herrmann, M., Schauer, C., Knopf, J., Muñoz, L. E. Induction of Ocular Surface Inflammation and Collection of Involved Tissues. J. Vis. Exp. (186), e63890, doi:10.3791/63890 (2022).