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Immunology and Infection

诱导眼表炎症和受累组织收集

Published: August 04, 2022
doi:
10.3791/63890
, , , , , ,
1Department of Internal Medicine 3-Rheumatology and Immunology,Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen, 2Deutsches Zentrum für Immuntherapie,Friedrich-Alexander-University Erlangen-Nürnberg and Universitätsklinikum Erlangen

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?…

Disclosures

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
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Tags

Ocular Surface InflammationMeibomian Gland DysfunctionNeutrophil Extracellular TrapsImmunogenOvalbuminOcular ExudatesConjunctivaCornea
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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).
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