The Role of Biologically Active Aqueous Humor Molecules of the Anterior Chamber and Lacrimal Fluid in the Implementation of the Hypotensive Effect of Non-Penetrating Deep Sclerectomy

To date, the factors affecting the course of the reparative process after non-penetrating deep sclerectomy (NPDS) have not been fully determined. There is no systematic information about the regulatory role of the cytokines TGF-β, IL-6, IL-8 and MMP-9, VEGF A 121 and 165 in the formation mechanisms of the newly created pathways consistency of intraocular aqueous humor outflow.Purpose. To determine possible ways of impact of biologically active aqueous humor molecules of the anterior chamber and lacrimal fluid on the hypotensive effect of non-penetrating deep sclerectomy.Methods. A prospective study of 65 patients with open-angle glaucoma before and 12 months after NPDS and 22 patients without eye hydrodynamic disorders with the determination of the initial concentrations of biologically active molecules in the lacrimal fluid and aqueous humor of the anterior chamber. Twelve months after NPDS all patients were divided into three groups, depending on the hypotensive effect of the operation, according to the criteria.Results. Multivariate discriminant analysis showed the greatest inter-group differences, calculated by the square of the Mahalanobis distance, between group 3 with no hypotensive effect of NPDS and the control group (R2 = 8.48, p = 0.001). The most informative features that determine the differences between the 4 groups in the total population, calculated according to the Fischer F-test, were MMP-9 (F = 14.7, p = 0.001) and TGF-β (F = 7.08, p = 0.001) in the aqueous humor of the anterior chamber. In pairwise comparison of groups 1 and 2, the maximum level of significance according to the F-criterion was characteristic of the level of tear IL-6 (F = 21.25, p = 0.001), with approximately equal degree – IL-8 (F = 7.85, p = 0.001) and VEGF (F = 7.12, p = 0.001), to a lesser extent TGF of aqueous humor (F = 4.43, p = 0.001) and MMR-9 (F = 2.23, p = 0.001). Between groups 1–3, the maximum differences according to the Fisher criterion were observed in the IL-8 (F = 20.99, p = 0.001), TGF (F = 8.75, p = 0.001) and to a lesser extent – TGF (F = 5.83, p = 0.001).Conclusion. The analysis of the obtained data showed the decisive role of the imbalance of proinflammatory cytokines, signaling proteins with prolymphoangiogenic activity, and MMP-9 in the aqueous humor of the anterior chamber, as well as in the initial state of the lacrimal fluid in the postoperative healing processes after NPDS

Некоторые аспекты формирования фильтрационных подушек у больных с первичной открытоугольной глаукомой после непроникающей глубокой склерэктомии

PURPOSE. To determine the role of extracellular matrix (ECM), transforming growth factor β (TGF-β) and matrix metalloproteinase 9 (MMP-9) in the mechanisms of filtering bleb formation after non-penetrating deep sclerectomy (NPDS) in patients with primary open-angle glaucoma (POAG).METHODS. The study consisted of prospective examination of 65 patients with POAG operated by the NPDS method. Three groups were formed: group 1 — optimal hypotensive effect of NPDS (21 patients); group 2 — relative hypotensive effect (23 people); group 3 — absence of hypotensive effect (21 patients). During the postoperative period, the condition of the intraocular fluid outflow pathways was evaluated with ultra biomicroscopy, optical coherence tomography, level of intraocular pressure, as well as the concentrations of TGF-β and MMR-9 in the tear and aqueous humor.RESULTS. The anterior chamber aqueous humor was found to contain TGF-β in all patients with glaucoma. Group 1 was noted to have minimal initial TGF-β concentrations in the tear and aqueous humor, high MMR-9 level in the pre- and postoperative period. Sparse extracellular matrix was the substrate for the filtering blebs. Group 2 was revealed to have elevated initial concentrations of TGF-β in the tear and aqueous humor, extremely high level of MMP-9 in the aqueous humor, accumulation of MMP-9 in the tear after 2 weeks, and inhibition of TGF-β and MMP-9 two months after NPDS. In the early period, loci of rigid ECM were visualized in the filtering blebs, but functional outflow pathways were successfully formed by month two after additional therapy. Group 3 was established to have maximum TGF-β concentrations and low MMP-9 concentrations in the anterior chamber aqueous humor, a decrease in tear MMP-9 after two weeks, and inhibition of TGF-β and MMP-9 two months after NPDS. In the early postoperative period, the filtering blebs were based on rigid ECM, by month two there was scar deformation of the outflow pathways.CONCLUSION. The important conditions for the formation of functional filtering blebs after NPDS are the structural organization of temporary ECM of the filtering blebs in the early postoperative period and the timely degradation of ECM components with predominant concentration of MMR-9 over TGF-β.ЦЕЛЬ. Определить роль внеклеточного матрикса (ВКМ), трансформирующего фактора роста β (TGF-β) и матриксной металлопротеиназы 9 (ММР-9) в механизмах формирования фильтрационной подушки (ФП) после непроникающей глубокой склерэктомии (НГСЭ) у пациентов   с первичной открытоугольной глаукомой (ПОУГ).МЕТОДЫ. Проведено проспективное обследование 65 пациентов с ПОУГ, прооперированных методом НГСЭ. Сформированы три группы: группа 1 — оптимальный гипотензивный эффект (21 пациент); группа 2 — условный гипотензивный эффект (23 человека); группа 3 — отсутствие гипотензивного эффекта (21 пациент). В послеоперационном периоде оценивали состояние путей оттока внутриглазной жидкости по ультразвуковой биомикроскопии, оптической когерентной томографии, уровню внутриглазного давления и концентрации TGF-β и ММР-9 в слезе и влаге передней камеры.РЕЗУЛЬТАТЫ. У всех больных установлено наличие TGF-β во влаге передней камеры. Первую группу отличали минимальные исходные концентрации TGF-β в слезе и влаге, высокий уровень ММР-9 в пред- и послеоперационном периоде. Субстратом ФП был разреженный ВКМ. Во второй группе выявлены повышенные исходные концентрации TGF-β влаги и слезы, экстремально высокий уровень ММР-9 влаги, нарастание ММР-9 в слезе через 2 недели и подавление TGF-β и ММР-9 через 2 месяца после НГСЭ. В раннем периоде в ФП визуализировались локусы жёсткого ВКМ, но к двум месяцам после дополнительной терапии сформированы функциональные пути оттока. В третьей группе установлены максимальные концентрации TGF-β и низкие ММР-9 во влаге передней камеры, снижение ММР-9 слезы через 2 недели и подавление TGF-β и ММР-9 через 2 месяца после НГСЭ. В раннем послеоперационном периоде основу ФП составлял жёсткий ВКМ, к двум месяцам отмечалась рубцовая деформация путей оттока.ЗАКЛЮЧЕНИЕ. Структура временного ВКМ ФП в раннем послеоперационном периоде, а также своевременная деградация компонентов ВКМ на фоне преобладающей концентрации ММР-9 над TGF-β является важным условием формирования функциональной ФП после НГСЭ

Immunohistochemical identification of lymphatic outflow in filtering blebs after non-penetrating deep sclerectomy (NPDS)

Relevance. One of the conditions for the functional outflow pathways’formation after filtering glaucoma surgeries is the uniform moisture absorption from the filtering blebs, which is provided by the blood and, possibly, lymphatic vessels of the conjunctiva. Purpose. To conduct a structural and molecular assessment of filtering blebs in patients with open-angle glaucoma (OAG) after non-penetrating deep sclerectomy (NPDS) according to OCT data, as well as ultrastructural and immunohistochemical studies. Material and methods. A clinical study of 12 patients with primary open-angle glaucoma (POAG) after NPDS was conducted. In the long-term postoperative period (12–18 months), the level of intraocular pressure (IOP) was determined, the structure of the filtering blebs using biomicroscopy and OCT of the anterior segment of the eye was evaluated, the immunohistochemical study of the filtering blebs’ tissue for the expression of podoplanin and ultrastructural analysis of the samples obtained using a laser confocal microscope LSM 710 (Zeiss) was performed. In 8 cases (group 1), these were non-functional scarred filtering blebs, IOP 24.12±2.24 mm Hg, in 4 cases (group 2) – functional filtering blebs, IOP 15.31±4.08 mm Hg. Results. During the immunohistochemical examination of the first group patients’ conjunctival samples, no vessels with characteristics of the lymphatic system were detected in any case. In each sample of the second group patients’ filtering blebs, from 5 to 7 vessels with different variants of podoplanin expression were determined. Conclusion. The aqueous outflow entering the subconjunctival space through the newly created outflow pathway during glaucoma surgery is carried out at the expense of conjunctival lymphatic structures, the condition of which to a certain extent determines the success or failure of the surgery