ophthalmologyОфтальмологияOphthalmology in Russia1816-50952500-0845Ophthalmology10.18008/1816-5095-2014-3-38-44ophthalmology-187Research ArticleКЛИНИЧЕСКИЕ И ЭКСПЕРИМЕНТАЛЬНЫЕ ИССЛЕДОВАНИЯCLINICAL & EXPERIMENTAL RESEARCHИммунное картирование периферического отдела зрительного анализатора и зрительного нерваImmune mapping of the peripheral part of the visual analyzer and optic nerveЛихванцеваВ. Г.LikhvantsevaV. G.КузьминК. А.KuzminK. A.СоломатинаМ. В.SolomatinaM. V.maria.vikto@gmail.comКоростелёваЕ. В.KorostelevaE. V.Бен РежебА.Ben RegebA.ФГ БОУ ВПО МГ У им. М. В. Ломоносова, кафедра офтальмологииРоссияThe Department of Fundamental medicine of Moscow State University of M. V. Lomonosov, The Department of ophthalmologyRussian FederationГБОУ ВПО «Рязанский государственный медицинский университет имени академика И. П. Павлова», кафедра глазных и ЛОР болезнейРоссияRyazan State Medical University, The Department of eye and ENT diseasesRussian Federation2014121020141133844Copyright © Лихванцева В.Г., Кузьмин К.А., Соломатина М.В., Коростелёва Е.В., Бен Режеб А., 20142014Лихванцева В.Г., Кузьмин К.А., Соломатина М.В., Коростелёва Е.В., Бен Режеб А.Likhvantseva V.G., Kuzmin K.A., Solomatina M.V., Korosteleva E.V., Ben Regeb A.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.ophthalmojournal.com/opht/article/view/187Цель

Цель: проведение иммунного картирования периферического отдела зрительного анализатора и зрительного нерва для выявления потенциальных антигенных мишеней аутоиммунной агрессии.

Методы

Методы. Исследовали энуклеированные глаза пациентов с терминальной болящей открытоугольной глаукомой (n = 30). Проводили иммуногистохимическое исследование (ИГХ) на парафиновых срезах препаратов изолированной сетчатки и зрительного нерва с использованием широкого спектра антител: моноклональных мышиных антител Myelin Basic Protein (MBP), поликлональных кроличьих антител AntiAlpha Fodrin (α-fodrin), моноклональных мышиных антител NSE2 (NSE), моноклональных мышиных антител Anti-GFAP (GFAP), поликлональных кроличьих антител S-100p (S100). Для визуализации ИГХ реакции использовали специальную систему детекции (Mouse and Rabbit Specific HRP / AEC (ABC) Detection IHC Kit). Негативным контролем служила ИГХ реакция без добавления первичных антител (АТ). Отрицательной считали реакцию при отсутствии специфического окрашивания клеток или при наличии менее 10 % окрашенных клеток от всей площади в зоне просмотра. Слабо положительной реакцию считали в случае окрашивания от 10 до 30 % клеток. Умеренно положительной считали реакцию в случае экспрессии маркера в 30‑75 % клеток. Экспрессию маркера более 75 % клеток расценивали как выраженную реакцию. Наряду с этим, отмечали интенсивность окраски: +1 (слабая окраска), +2 (умеренная окраска), +3 (выраженная окраска), +4 (интенсивное окрашивание).

Результаты

Результаты. Иммунное картирование с широкой панелью моноклональных антител позволило выявить структуры глаза, окрашенные ИГХ-маркерами. Сетчатка окрашивалась практически всеми используемыми маркерами нейрональной дифференцировки (антителами к NSE, GFAP и S100), а также антителами к α-fodrin. Исключение составили антитела к MBP. Интенсивность ИГХ-реакции в различных ее слоях и структурах варьировала и зависела от маркера. Цитоплазма отростков глиальных элементов зрительного нерва умеренно (2+) окрашивалась антителами к МBP, NSE, GFAP, S100 и более выражено (3+) — антителами к α-fodrin.

Заключение

Заключение. Проведена полноценная маркировка структур сетчатки, в которой три вида зрительных нейронов, зрительные аксоны, интернейроны, клетки микроглии получили ИГХ-профиль. Выявленные особенности ИГХ-профиля слоев сетчатки и структур зрительного нерва могут быть полезны в качестве маркеров серологической иммунодиагностики глазных болезней.

Aim

Aim. To perform immune mapping of the peripheral part of visual analyzer and optic nerve in order to identify potential antigenic targets of autoimmune attack.

Methods

Methods. Eyes enucleated for terminal painful glaucoma (n = 30) were studied. Immunohistochemistry (IHC) was performed on paraffin-embedded sections of isolated retina and optic nerve using a broad panel of antibodies, i.e., monoclonal murine anti-MBP (myelin basic protein) antibodies, polyclonal rabbit anti-alpha fodrin antibodies, monoclonal murine anti-NSE2 (neuron-specific enolase) antibodies, monoclonal murine anti-GFAP (glial fibrillary acidic protein), and polyclonal rabbit anti-S100 antibodies. IHC reaction was visualized using Mouse and Rabbit Specific HRP / AEC Detection IHC Kit. IHC reaction without primary antibodies included was a negative control. IHC reaction was considered as follows: negative — no specific cellular staining or less than 10 % of cells are stained; mild — 10‑30 % of cells are stained (+); moderate — 30‑75 % of cells are stained (++); marked — more than 75 % of cells are stained (+++); overexpression — 100 % of cells intensively express markers. Additionally, staining intensity was considered as mild (+1), moderate (+2), strong (+3) and intense (+4).

Results

Results. Immune mapping with a broad panel of monoclonal antibodies identified ocular structures which were stained with IHC markers. Retina was stained with almost all markers of neural differentiation (i.e., antibodies against NSE, GFAP, S100, and α-fodrin) excepting anti-MBP autoantibodies. IHC reaction intensity in retinal layers and structures varied and depended on markers. Moderate (2+) staining with antibodies against MBP, NSE, GFAP, and S100 and marked (3+) staining with antibodies against alpha-fodrin was detected in the cytoplasm of optic nerve glia.

Conclusion

Conclusion. Complete labelling of retina structures was performed. As a result, IHC profiles of retinal neurons, optic nerve axons, interneurons, and microglial cells were described. IHC profiles of retinal layers and optic nerve are useful markers which can be applied in serological diagnostics of various ocular disorders.

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The authors declare that there are no conflicts of interest present.