Vasoproliferative retinal tumor (VPT) is a term proposed by ophthalmologists in relation to the totality of manifestations of an intraocular volumetric process with involvement of the inner lining of the eye, an integral part of which is the active growth of blood vessels. The available literature data on the morphology of this process are very contradictory and ambiguous. The article presents two clinical cases of vasoproliferative retinal tumor with own illustration of morphological studies.

Objective: to study in vitro survival and preservation of the proliferative activity of limbal stem cells (LSCs) in femtosecond laser-cut limbal tissue fragments. Materials and methods. Limbal fragments were formed from donor cadaver eyes (n = 8) in the upper and lower limbus containing the highest number of limbal stem cells, using a Z8 femtosecond laser (FSL) (Ziemer, Switzerland). The limbal fragments were fragmented into 4 mini-grafts using different energy levels (100, 110, 120%). Mini-grafts from symmetrical sections of the cadaver eyes, which were manually isolated using a microsurgical blade, served as controls. The mini-grafts were cultured for two weeks in culture media intended for limbal epithelial stem cells (LESCs) (Epilife (0.06 mM Ca++) and for multipotent mesenchymal stem cells (MMSCs) (DMEM/F12), with the addition of specific growth factors to selectively stimulate LESCs or MMSCs, respectively. The phenotype of the obtained cultured cells in the «laser» and «knife» groups was determined by flow cytometry using a set of markers (CD166, CD105, CD90, CD29, CD34) for the membrane proteins of LESCs and MMSCs. The ability of cultured cells to adhesion and proliferation in the «laser» and «knife» groups was determined by seeding the third passage of the resulting cultures on Bowman’s membrane of acellular corneas.Results. Primary cell culture was obtained from mini-grafts of all donors in both groups. Cell morphology was consistent with the phenotype of corneal epithelial cells (cobblestone pattern). When cultured in the EpiLife medium (0.06 mM Ca++), we determined the presence of LSCs proliferation from 38.6% of minigrafts; in the DMEM/F12 medium (1 : 1) the presence was determined from 31.8%. Two weeks later, cell yield from mini-grafts in the «laser» and «knife» groups was 77.2% and 63.6%, respectively. Cell growth by the end of week 2 of culturing of mini-grafts obtained by FSL at 120, 110 and 100% energies was 87.5, 71.4 and 71.4%, respectively. It was found that the resulting cell cultures in the «laser» and «knife» groups and in the «120%», «110%» and «100%» subgroups were not different phenotypically. Cytofluorimetric analysis showed that cell cultures in the groups had a mixed pattern of marker expression of both LESCs (CD29+) and MMSCs (CD90+, CD105+). Seeding of the third passage of cell culture in the test groups in all cases demonstrated adhesion and formation of a cell monolayer on the Bowman’s membrane of model corneas.Conclusion. The use of FSL for cutting out limbal grafts seems to be effective and safe in comparison with the traditional mechanical (knife) technique. Cell cultures obtained from FSL-cut mini-grafts were able to grow and migrate for at least 21 days.

Purpose:The vitreous body (VB), due to the complexity of its structure, remains one of the least studied anatomical structures to this day.In literature there are attempts to describe the anatomy of the vitreous body, since the II century.The most relevant works are the studies of J. Worst., et al. in 1973, in which the authors proposed new methods of CT preparation with the introduction of dyes.Despite many years of research on the structure and functions of the vitreous body and the presence of a large number of works, and there are no methods and protocols for macromicroscopic examination of the vitreous body to develop a method and propose a protocol for macromicroscopic examination of the vitreous body (VB), allowing to obtain new data on VB topographic anatomy.The purpose of the study is to develop the algorithm of examination of the vitreous body microsurgical anatomy that would let to assess its topographic specifics. Materials and Methods:The proposed method of macromicroscopic examination was used to study the VB topographic anatomy of 38 cadaver eyeballs.In order to color transparent structures of the vitreous, poorly soluble metallic salts barium sulfate (Vitreocontrast) was used.Macroscopic examination was performed using a TopconOMS-800 operating microscope with a magnification of x8 to x21, microscopic changes were evaluated by light microscopy at x50, x100, x200, x400 x630 multiple magnification with Leica DM LB2 microscope followed by photographic recording.The algorithm for macroscopic examination performing. Results and Discussion:The result of macroscopic preparation was the compilation of individual anatomical and topographic maps of VB patients.A distinctive feature of the developed method is the ability to dissect any VB structure and to isolate each cortical layer with the possibility of studying its anatomical and topographic features and relationships with underlying tissues (internal limiting membrane, ciliary body, lens capsule).In addition, the method allows to maintain the shape and integrity of the specimens after passing through all stages of histological processing.In order to fixate VB samples, we used a method with fixing VB structures on a special adhesive-metric tablet, and placing them in a biopsy bag placed in a biopsy cassette.After that, filled in formalin, the specimens were delivered to the laboratory, where all the stages of standard processing took place. Conclusion:The developed technique of macromicroscopic examination of the vitreous allows to create an individual map of the VB topographic anatomy.After collecting of sufficient material and its statistical processing, it is possible to provide maps of the VB topographic anatomy in normal, age-related and pathological conditions.

Purpose: The vitreous body (CT), due to the complexity of its structure, remains one of the least studied anatomical structures to this day. In the literature there are attempts to describe the anatomy of the vitreous body, since the II century. The most relevant works are the studies of J. Worst et al. in 1973, in which the authors proposed new methods of CT preparation with the introduction of dyes. Despite many years of research on the structure and functions of the vitreous body and the presence of a large number of works, and there are no methods and protocols for macromicroscopic examination of the vitreous body to develop a method and propose a protocol for macromicroscopic examination of the vitreous body (VB), allowing to obtain new data on VB topographic anatomy.Materials and methods. The proposed method of macromicroscopic examination was used to study the VB topographic anatomy of 38 cadaver eyeballs. In order to color transparent structures of the vitreous, poorly soluble metallic salts (barium sulfate and copper acetate) were used. Macroscopic examination was performed using a Topcon OMS-800 operating microscope with a magnification of ×8 to ×21, microscopic changes were evaluated by light microscopy at ×50, ×100, ×200, ×400, ×630 multiple magnification with Leica DM LB2 microscope followed by photographic recording. The algorithm for macroscopic examination performing.Results and discussion. The result of macroscopic preparation was the compilation of individual anatomical and topographic maps of patients. A distinctive feature of the developed method is the ability to dissect any VB structure and to isolate each cortical layer with the possibility of studying its anatomical and topographic features and relationships with underlying tissues (internal limiting membrane, ciliary body, lens capsule). In addition, the method allows to maintain the shape and integrity of the specimens after passing through all stages of histological processing. In order to fixate VB samples, we used a method with fixing VB structures on a special adhesive-metric tablet, and placing them in a biopsy bag placed in a biopsy cassette. After that, filled in formalin, the specimens were delivered to the laboratory, where all the stages of standard processing took place.Conclusion. The developed technique of macromicroscopic examination of the vitreous allows to create an individual map of the VB topographic anatomy. After collecting of sufficient material and its statistical processing, it is possible to provide maps of the VB topographic anatomy in normal, age-related and pathological conditions.

В связи с увеличением числа заболеваний, в основе которых лежит развитие новообразованных сосудов, в последние десятилетия возрос интерес к проблеме патологического ангиогенеза в различных тканях глаза. Традиционные консервативные методы лечения неоваскуляризации роговицы на данный момент недостаточно эффективны. Возможным путем решения проблемы неоваскуляризации роговицы может быть использование паракринных факторов, выделяемых мезенхимальными стволовыми клетками в процессе культивирования в кондиционированную среду (КС). Целью исследования является изучение влияния пептидов КС на сосудистое бельмо, которое сформировалось после химического ожога глаз крыс в эксперименте, и оценка влияния КС на функциональную активность нейтрофилов. Методы исследования. Исследование было выполнено на 48 крысах (48 глаз) в несколько этапов. На первом этапе всем животным был смоделирован химический ожог роговицы. На втором этапе проводилось лечение ожогов роговицы. В зависимости от способа лечения на втором этапе была сформирована контрольная группа 22 крысы (22 глаза) и опытная группа 22 крыс (22 глаза). Опытная группа была разделена на 2 подгруппы. В первой подгруппе было 11 крыс (11 глаз), которым в качестве лечения применялись пептиды КС общей фракции. Во второй подгруппе также было 11 крыс (11 глаз), в качестве лечения применялся пептиды КС фракцией до 30 кДа. В контрольной группе для лечения ожогов мы применяли культуральную среду. Результаты. По результатам офтальмоскопии и гистологических исследований на 14-е и 30-е сутки в контрольной группе животных динамики не наблюдалось. Выявлялись множественные новообразованные сосуды роговицы (преимущественно крупные сосудистые стволы) со стромальными помутнениями в их проекции. Эпителий роговицы был не изменен, однако, сама роговица была истончена. Наблюдалось сформированное васкуляризированное бельмо роговицы: субэпителиальная фиброплазия и неоваскуляризация роговицы. В опытной группе животных к 14-м суткам инстилляций в конъюнктивальную полость пептидов КС офтальмоскопически мы наблюдали уменьшение количества новообразованных сосудов, помутнения роговицы исчезли в проекции запустевших сосудов. На 30-е сутки лечения в опытных группах животных у большинства крыс новообразованных сосудов обнаружено не было, сохранялись лишь отдельные сосудистые стволы, которые ранее образовывали мощные анастамозы с другими новообразованными сосудами. По результатам хемолюминесцентного анализа показано, что фракция до 30 кДа угнетает функциональную активность нейтрофилов, в том числе за счет изменения активности внутриклеточных мембранных ферментов, отвечающих за передачу рецепторных сигналов внутрь клетки. Заключение. Полученные результаты свидетельствуют о перспективности применения препарата раствора пептидов для лечения такой сложной патологии, как неоваскуляризация роговицы. In recent decades, due to increasing number of diseases associated with the development of newly formed vessels, the interest to pathological angiogenesis in eye tissues has been growing. Traditional, conservative methods for treatment of corneal neovascularization are not sufficiently effective. A possible solution may be the use of paracrine factors secreted by mesenchymal stem cells during cultivation in a conditioned medium (CW). The aim of this study was to assess the effect of CW peptides on vascular leukoma induced by chemical burn of rat eyes and to evaluate the effect of CW on the functional activity of neutrophils. Methods. The study was performed on 48 rats (48 eyes) and included several stages. At the first stage, chemical corneal burn was modeled in all animals. At the second stage, corneal burns were treated. Based on the method of treatment at the second stage, a control group (22 rats, 22 eyes) and an experimental group (22 rats, 22 eyes) were formed. The experimental group was divided into 2 subgroups. The first subgroup rats (n = 11, 11 eyes) were treated with CW peptides of the entire fraction. The second subgroup rats (n = 11, 11 eyes) received a CW peptide fraction of up to 30 kDa. In the control group, the culture medium was used as a treatment. Results. According to the results of ophthalmoscopy and histological studies on days 14 and 30, no changes were observed in the control group. Multiple newly formed corneal vessels (mainly large vascular trunks) with stromal opacities in their projection were detected. The corneal epithelium was unchanged; however, the cornea itself was thinned. A formed vascularized corneal leukoma was observed that was evident as subepithelial fibroplasia and corneal neovascularization. In the experimental group by the 14th day of CW peptide instillations into the conjunctival cavity, ophthalmoscopy showed a decrease in the number of newly formed vessels, and corneal opacities disappeared in the projection of the deserted vessels. In most of rats in the experimental groups on the 30th day of the treatment, newly formed vessels were not found; only individual vascular trunks remained, which had previously formed powerful anastomoses with other newly formed vessels. According to the results of chemiluminescence analysis, a fraction of up to 30 kDa inhibited the functional activity of neutrophils, including by changing the activity of intracellular membrane enzymes responsible for the transmission of receptor signals into the cell. Conclusion. The results showed that the peptide solution is promising for the treatment of such a complex pathology as corneal neovascularization.

OBJECTIVE:To determine the prognostic value of a uveal melanoma (UM) cell type for the development of metastases (MTS).SUBJECTS AND METHODS:The investigation enrolled 96 patients (96 eyes) with UM after enucleation. Forty-one patients without signs of MTS were included in this group, who were followed up for more than 36 months (mean, 70.5 months (36 to 105 months)), 55 patients with MTS who were followed up for an average of 21 months (2 to 44 months). The MTS and non-MTS groups were statistically homogeneous in age, gender, tumor size, location, and ciliary body involvement in the process, as well as in extrabulbar growth.RESULTS:There were spindle cell, mixed cell, and epithelioid cell UMs in 44, 35, and 21% of patents, respectively. The tumors in patients without MTS were noted to be significantly more likely to have spindle cell-type UM (p<0.0001). Mixed cell and epithelioid cell UMs were more frequently detected in patients with MTS (p<0.0001), which was believed to be due to the presence of epithelioid cells in both cell types. A survival analysis showed that the 3- and 5-year survival rates for patients with spindle cell UM were significantly higher than that for those with epithelioid cell or mixed cell UM (p<0.001); the 3-and 5-year survival rates for spindle cell UM were 78 and 70%, respectively; those for mixed cell UM were 37 and 24%; and those for epithelioid cell UM were 50 and 31%.CONCLUSION:The similar survival rates for patients with mixed cell or epithelioid cell type UM could conclude that it is advisable to use the binary principle - the presence or absence of epithelioid cells in the tumor, when assessing a UM cell type as a prognostic factor.

Purpose. The key to understanding the development of any vitreoretinal pathology is a presence of a clear conception to possible variants of changes in the anatomical and topographic relationships of the posterior cortical layers of the vitreous body and the retinal internal limiting membrane (ILM) in a formation of posterior vitreous detachment (PVD). An original method of macro-microscopic examinations of anatomical objects proposed by professor V.P. Vorobyov, which includes investigations of morphological objects whose dimensions lie in the boundary area between macroscopic parameters studied by anatomists and microstructures studied by histologists, discover a new boundary area for the study of anatomical structures. However, it has not been practically used in ophthalmology until now. The study of a process for changes in the vitreoretinal interface during the PVD occurrence is difficult due to a complexity of simulation, a possibility of visualization, and an absence of approved protocols for macro-microscopic research. Purpose. To study variants of anatomical and topographical changes in the vitreoretinal interface during the formation of induced PVD in ex vivo experiments using the method of macro-microscopic examination. Material and methods. The study was performed in 24 eyeballs of cadaver donors. To assess changes in the vitreoretinal interface in the process of PVD induction, an original method of dissection was used, that allows to isolate layer-by-layer the cortical layers of the vitreous body and the retinal ILM, with a possibility of further evaluation of changes in the vitreoretinal interface by the macro-microscopic examination. Salt barium sulfate suspension Video-contrast was used with the purpose to contrast vitreous fibers. Macroscopic investigation was carried out using the Topcon OMS-800 operating microscope with an indication from x8 to x21 magnification, microscopic changes were evaluated by the light microscopy method with x50, x100, x200, x400 x630 magnification using the Leica DM LB2 microscope with the subsequent photo registration. Results. During the macro-microscopic study, three variants of possible changes in the vitreoretinal interface during the development of induced PVD were revealed, which were interpreted as the normal (true) PVD and abnormal PVD, which occurred both with the formation (the variant A) of vitreoschisis zones (exfoliation of the cortical layers of the vitreous body) and with the zones of retinal ILM exfoliation (the variant B). Conclusion. The proposed method of macro-microscopic investigation of the vitreoretinal interface allows to dissect isolated sequentially layer-by-layer the cortical layers of the vitreous body and the retinal ILM with subsequent fixation on a special substrate according to the original method and the possibility to carry out a histological section and its evaluation by the light microscopy method. The application of the developed method allows to accurately assess changes in the vitreoretinal interface on any isolated site of the vitreoretinal surface. The presented work demonstrates possibilities for the application of the method to detect variants of changes in the vitreoretinal interface within the formation of induced PVD. Keywords: posterior vitreous detachment, internal limiting membrane, dissection of the vitreous body, vitreoretinal interface.

Introduction. Fine needle aspiration biopsy (FNAB) of uveal melanoma (UM) is an invasive procedure. Many authors consider UM FNAB to be safe procedure, however extraocular extension of the tumor through the scleral tracts may occur. Current studies of scleral tracts after FNAB do not compare different biopsy techniques (cannula-assisted and without cannula) in terms of UM cells. Purpose. To assess morphology of scleral tracts for the presence of malignant cells when different biopsy techniques are approached. Material and methods. Forty-four scleral samples were analyzed after transvitreal FNAB performed in 22 enucleated eyes with UM. Cannula-assisted FNAB was performed in 22 cases. In the same eyes FNAB (n=22) was performed without cannula. Results. FNAB material was adequate for cytological examination in all cases. UM was confirmed in 22 eyes pathologically. UM cell implantation was detected in 5 scleral samples after FNAB without cannula. No signs of cell implantation were seen after cannula-assisted FNAB. The risk of scleral tract UM cell implantation was statistically lower in cannula-assisted FNAB technique (p=0.018, Pearson Chi-square test). Conclusions. UM FNAB performed directly through the sclera without cannula is associated with tumor cells implantation in scleral tract. Cannula-assisted FNAB significantly reduces the incidence of UM implantation in scleral tract. Key words: uveal melanoma, fine-needle aspiration biopsy, enucleation, morphology, ophthalmology.

PURPOSETo evaluate biocompatibility of the new keratoprosthesis supporting plates (KSP) in rabbits in vivo.MATERIAL AND METHODSThe study included 15 chinchilla rabbits. In the first group (5 rabbit eyes) KSP made of hydrophobic acryl with square penetrating holes of 220×220 micron (model 1) were inserted into rabbits' corneas. In the second group (5 eyes), KSP made of hydrophobic acryl were used that had trapezoidal fenestrations with size (from 170×130 micron to 180×70 microns) gradually changing from periphery to the center of KSP (model 2). The control group rabbits (5 eyes) had 1/2 of Fyodorov-Zuev KSP made of titanium implanted. All animals were observed for up to 3 months with biomicroscopy and optical coherence tomography of the anterior segment. The animals were then euthanized and had their corneo-scleral discs excised and then examined with optical microscopy and scanning electron microscopy (SEM).RESULTSAfter 3 months, there was only one case of KSP protrusion in the first group. In the second group, thinning of the corneal layers above the central part of KSP occurred in one case. The presence of polymer KSP (of both models) in the corneal stroma was found not to cause formation of rough fibrotic tissue. At the same time, adhered cellular and fibrous elements were discovered on the surface and inside the holes of the polymer KSP, while on the surface of the titanium plate cellular elements were absent.CONCLUSIONSupporting plates made of hydrophobic acrylic material can potentially serve as a foundation for the new keratoprosthesis design.

Актуальность. Стекловидное тело, ввиду сложности своего строения до настоящего времени остается наименее изученной структурой в анатомии человека. В литературе встречаются попытки описания анатомии стекловидного тела начиная с II века до сегодняшних дней. Наиболее актуальными работами являются исследования J. Worst с соавторами, в которых он предложил новые методы препарирования СТ с введением красителей. Но несмотря на многолетние исследования строения и функций стекловидного тела и наличия большого количества работ, посвященных исследованиям глаза, стекловидное тело ввиду своей сложной микроструктуры, все еще остается наименее изученной структурой глазного яблока. Цель. Разработать метод препарирования стекловидного тела, позволяющий получить новые данные об анатомии и топографо-анатомическом взаимоотношении между структурами СТ, и не нарушив их целостность, провести в гистологическое исследование. Материал и методы. По данной методике было исследовано 28 глазных яблок доноров-трупов. С целью контрастирования структур СТ была применена суспензия из солей малорастворимых металлов. Для фиксации отдельных структур СТ использовали специальный адгезивно-метрический планшет и биопсийный мешочек, закладываемый в биопсийную кассету. Из всех структур изготавливали стеклопрепараты и проводили световую микроскопию. Результаты. Используя оригинальную технику препарирования, включающую в себя метод макро-микроскопического исследования и последовательный алгоритм препарирования. Применив для фиксации отдельных структур СТ специальный адгезивно-метрический планшет, и биопсийный мешочек, закладываемый в биопсийную кассету, нам удалось пошагово выделить изолированные структуры СТ, морфологически изучить их, при этом сохранить форму и целостность препаратов после прохождения всех этапов гистологической обработки, вплоть до изготовления срезов и окрашивания. У всех пациентов были составлены топографические карты. Выводы. Разработанный метод позволит получить новые данные о пространственной анатомиии гистологии СТ, а также выявить характер патологических изменений стекловидного тела при различных патологиях.

Purpose. Experimental study the morphological changes of the retina and choroid, providing adhesion of the detached retina in the first 72 hours after laser coagulation.Material and methods. A clinical and morphological study of 12 eyes of 12 Chinchilla rabbits with experimentally simulated retinal detachment was performed 12, 24, 48 and 72 hours after the operation of transciliary vitrectomy with laser coagulation.Results. Due to local destruction and exudative effects in the field of laser coagulation of the retina (increasing to 24 hours and decreasing to 72 hours) detected the formation of a tight adhesive contact between the shells resulting from the destruction of exudative fibrinogen and deposition fibrin. Histological changes in the retina and choroid in the area of laser coagulation in the first two days corresponded to the growing destructive-exudative phase of fibrinoid’s inflammation moderate with maximum cases in the period of 48 hours with the attenuation of exudative effects and proliferative phase after 72 hours.Conclusion. In the period from 24 to 48 hours of the adhesive after laser coagulation is formed of a dense chorioretinal contact by deposits of fibrin, which serves as a fibrinogen serum, part extravasal exudate area of laser coagulation. Based on the studied morphological processes, it can be assumed that chorioretinal adhesion in the LC area becomes strong enough to independently hold the retina in place 48 hours after surgery.

Purpose: to study the histological features of the Muller muscle of the upper eyelid in patients with congenital and acquired ptosis to understand the mechanism of ptosis. Material and methods. Retrospective analysis of 27 intraoperatively obtained biopsies of the Muller muscle of the upper eyelid. For the study, the biopsies were fixed in a formalin and prepared by paraffinization. After staining with hematoxylin and eosin and Van Gieson, the preparations were studied in a light microscope. Results. In congenital ptosis of the upper eyelid histologically revealed atrophy of smooth muscle tissue, which manifested itself in a decrease in the number of muscle fibers on the background of fibrous transformation. In micro specimens of patients with acquired ptosis discovered fat cells located among the bundles of smooth muscle fibers, the dispersion of the myocytes. Fat infiltration of muscle ranged from 29 to 51.6 %.Conclusion The presence of dystrophic changes in Muller muscle in congenital and acquired ptosis was proved by histological method. The revealed features explain the pathogenetic mechanisms of the formation of congenital and acquired ptosis. In the case of acquired ptosis, fat dystrophy is an independent etiological factor, and therefore requires non-standard tactics of surgical treatment in order to neutralize the risk of recurrence in the early and late postoperative periods.

Introduction. Available methods in the treatment of age-related macular degeneration (AMD) do not always lead to significant vision improvement. A new advanced method of AMD treatment is transplantation of retinal pigment epithelium (RPE) in the form of cell suspension or choroidal pigment complex. In our opinion, the most modern form of RPE transplant is a multicellular spheroid – the form of 3D cell culture in which cells are close to the conditions of native tissue. However, transplantation of 3D spheroids of RPE requires preclinical studies. Purpose. This research is aimed to devise the technique for transplantation of RPE 3D spheroids in the eyes of experimental animals (rabbits). Material and methods. 1. In vitro research phase. For immunocytochemical tests the 3D spheroids were explored on the 3rd, 7th, and 11th day of steroidogenesis the laser scanning confocal microscope detachment in the surgery area. As we observed, the retina was attaching and retinal edema was decreasing. Also, on day 3, 7 and 10 we revealed subretinal round conglomerates with a diameter of 60 to 80 µm – presumably RPE 3D spheroids. No morphological changes of the retina were seen on day 14 and day 20. According to histological findings, there was found adhesion of the RPE spheroids to the choroid with subsequent spreading and formation of a new cell layer with an increase of follow-up periods. Conclusion. 1. The proposed technology of cultivation of rabbit RPE with subsequent construction of 3D spheroids allows to preserve the epithelial phenotype of cells, that is confirmed by immunocytochemical tests. 2. The developed surgical technique of RPE transplantation is acceptable, that is confirmed by the OCT and histological investigation. 3. The proposed surgical technique of subretinal transplantation of 3D spheroids of RPE is promising for further experimental studies to be implemented in clinical practice.

Introduction. Available methods in the treatment of age-related macular degeneration (AMD) do not always lead to significant vision improvement.A new advanced method of AMD treatment is transplantation of retinal pigment epithelium (RPE) in the form of cell suspension or choroidal pigment complex.In our opinion, the most modern form of RPE transplant is a multicellular spheroid - the form of 3D cell culture in which cells are close to the conditions of native tissue.However, transplantation of 3D spheroids of RPE requires preclinical studies.Purpose. This research is aimed to devise the technique for transplantation of RPE 3D spheroids in the eyes of experimental animals (rabbits).Material and methods. 1. In vitro research phase. For immunocytochemical tests the 3D spheroids were explored on the 3rd, 7th, and 11th day of steroidogenesis (using the laser scanning confocal microscope «Fluo View FV10i», Olympus, Japan). The expression of epithelial markers (Alexa Fluor, Great Britain), such as: RPE65, ZO-1, Cytokeratin 8, 18, and Vimentin (the mesenchymal marker) was analyzed.2. In vivo research phase. Vitrectomy (2500 cuts per minute, vacuum 600 mmHg), (Alcon, Accurus, USA) was performed on all rabbits (n=10). Then, a sharp cannula 39G was used to make a retinotomy above the central zone of retina, and spheroids (n=81) were injected (MicroDose injection kit 1 ml, Med One, USA) in subretinal space. The operation ended with the replacement of fluid into air and suturing scleral incision and the conjunctiva. The following methods of control were used: ultrasound B-scan (Ultrasonic UD-6000, Tomey, Japan) and optical coherence tomography (OCT) - (Askin Spectralis, Heidelberg engineering, Germany).Animals were taken out of the experiment on days 7, 10, 14 and 20 by air embolism. The eyeballs were enucleated for a subsequent histological examination.Results. 1. In vitro research phase. During immunocytochemical tests on the obtained 3D cultures, the presence of high expression of specific marker of retinal pigment epithelium RPE-65, also epithelial markers Cytokeratin 8, 18 and ZO-1 was noted. The expression of mesenchymal marker Vimentin was weak - that indicates the advantage of 3D cultivation of RPE cells to keep their phenotype. This indicates the advantage of 3D cultivation of epithelial cells to preserve their epithelial phenotype.2. In vivo research phase. On day 1 during ultrasonic B-scanning in 6 rabbits there was observed a flat retinal detachment in the area of surgical intervention height up to 1 mm; in 4 rabbits there was detected adhesion of the membranes, detachment of retina was not visible.The picture of the morphological state during retinal OCT was similar in all experimental animals - during the first 7 days after surgery cystic edema was noted and also a flat retinal detachment in the surgery area. As we observed, the retina was attaching and retinal edema was decreasing. Also, on day 3, 7 and 10 we revealed subretinal round conglomerates with a diameter of 60 to 80 pm - presumably RPE 3D spheroids. No morphological changes of the retina were seen on day 14 and day 20.According to histological findings, there was found adhesion of the RPE spheroids to the choroid with subsequent spreading and formation of a new cell layer with an increase of follow-up periods.Conclusion. 1. The proposed technology of cultivation of rabbit RPE with subsequent construction of 3D spheroids allows to preserve the epithelial phenotype of cells, that is confirmed by immunocytochemical tests.2. The developed surgical technique of RPE transplantation is acceptable, that is confirmed by the OCT and histological investigation.3. The proposed surgical technique of subretinal transplantation of 3D spheroids of RPE is promising for further experimental studies to be implemented in clinical practice.

Aim. This research is aimed to devise the technique for transplantation of 3D spheroids retinal pigment epithelium (RPE) in the experimental animal’s eyes (rabbits).Materials and methods. 3D spheroids of RPE for subsequent transplantation were created using agarose tablets (3D Petri Dishes, Microtissue, USA). The phenotype of the obtained cell cultures was studied by immunocytochemical tests (laser scanning confocal microscope “Fluo View FV10i”, Olympus, Japan). Vitrectomy - 2500 cuts per minute, vacuum 600 mm Hg (Alcon, Accurus, USA) was performed on all rabbits (n = 10). Then, we made retinotomy and injected spheroids in subretinal space (MicroDose injection kit 1 ml, Med One, USA). The following methods of control: ultrasound B-scan (Ultrasonic UD-6000, Tomey, Japan) and optical coherence tomography (OCT), (Askin Spectralis, Heidelberg engineering, Germany). Eyes were enucleated for histological examination on 7, 10, 14 and 20 days.Results. Immunocytochemical tests revealed preservation of the RPE epithelial phenotype in 3D spheroids. Clinical map was similar in all experimental animals - during the first 7 days after surgery we saw cystic edema and flat retinal detachment in the surgery area. As we observed, the retina was adjoining and retinal edema was decreasing. Also, on day 3, 7 and 10 on OCT we saw subretinal round conglomerates with a diameter of 60 to 80 µm - presumably RPE 3D spheroids. According to histological findings, there was observed adhesion of the RPE spheroids to the choroid with subsequent spreading and formation of new cell layer with the increase of observation periods.Conclusion. The proposed technology of cultivation of rabbit RPE with subsequent construction of 3D spheroids allows to preserve the epithelial phenotype of cells. The developed surgical technique of RPE transplantation is acceptable and can be used for further experimental studies to be implemented in clinical practice.

Трансмиссионная электронная микроскопия является одним из ключевых маркёров при рассмотрении патогенеза коллагенового кросслинкинга. Цель работы: оценить эффект кросслинкинга при фоторефракционной кератоабляции с рибофлавином по данным трансмиссионной электронной микроскопии. Материал и методы. Экспериментальное исследование было выполнено in vivo на 16 глазах 16 кроликов породы Шиншилла, с массой тела от 2,5 до 3,5кг, в возрасте от 1 до 1,5 лет. Животные были разделены на контрольную и опытную группы по 8 животных на каждый из способов проведения трансэпителиальной фоторефракционной кератоэктомии (ТрансФРК), без и с предварительным насыщением стромы 0,25% изотоническим раствором рибофлавина. На светооптическом уровне исследовали эпителий, наружные и глубокие слои стромы в проекции оперативного вмешательства. По данным трансмиссионной электронной микроскопии оценивали волокна и пучки коллагена, и их концентрацию на единицу площади, а также клеточный компонент стромы роговицы в проекции оперативного вмешательства. Результаты. На светооптическом уровне роговица кролика по завершению эпителизации сохраняла нормальную морфологию плоского многослойного неороговевающего эпителия, с практически полным восстановлением стратификации слоев. В случае образцов после абляции с предварительным насыщением стромы раствором рибофлавина наблюдались единичные вакуоли в клетках базального слоя и появление псевдомногорядности. Комплексной морфологической оценкой установлено, что роговицы из экспериментальной группы (с предварительным насыщением стромы рибофлавином) были подвержены изменениям соответствующим крослинкингу стромы с формированием стабильных поперечных сшивок коллагеновых волокон. В роговицах контрольной группы без насыщения стромы рибофлавином морфологически выявлено полное восстановление структуры после лазерного воздействия без признаков кросслинкинга стромы. Заключение. После проведения эксимерлазерной абляции с рибофлавином обнаруженные при трансмиссионной электронной микроскопии стабильные сшивки в коллагеновых структурах стромы роговицы подтверждают наличие эффекта крослинкинга при таком способе фоторефракционной кератоэктомии. Transmission electron microscopy is one of the key methods in studying the pathogenesis of collagen crosslinking. Aim. To evaluate the effect of crosslinking induced by photorefractive ablation of the cornea with riboflavin using transmission electron microscopy. Material and methods. This experimental in vivo study was performed on 16 eyes of 16 Chinchilla rabbits weighing from 2.5 to 3.5 kg, aged from 1 to 1.5 years. Animals were divided into control and experimental groups, including 8 animals for each of the transepithelial photorefractive keratoectomy (TransFRK) methods, with and without prior saturation of the stroma with a 0.25% isotonic riboflavin solution., The epithelium and outer and deep layers of the stroma were studied in the projection of surgery at the light-optical level. Based on data of the transmission electron microscopy, collagen fibers and bundles, and their concentration per unit area, and the cellular component of corneal stroma were evaluated. Results. The rabbit cornea studied at the light-optical level upon completion of epithelialization maintained a normal morphology of the flat multi-layered non-squamous epithelium with almost complete recovery of the layer stratification. In post-ablation samples with the prior saturation of stroma with riboflavin solution, isolated vacuoles were observed in cells of the basal layer along with emergence of pseudostratified epithelium. According to results of the complex morphological evaluation, corneas of the experimental group (with riboflavin pre-saturation of the stroma) showed changes corresponding to the stroma crosslinking with formation of stable cross-links of collagen fibers. In the cornea of the control group (without riboflavin saturation of the stroma), the stromal structure completely recovered after the laser treatment without any signs of stromal crosslinking. Conclusion. As detected by transmission electron microscopy, the stable cross-links in collagen structures of the corneal stroma induced by excimer laser ablation with riboflavin confirm the presence of the crosslinking effect in using the given method of the photorefractive keratoectomy.

«Российская офтальмология онлайн» - электронное информационное издание, создается с 1 марта 2010 года под эгидой Российского общества офтальмологов. Основная задача проекта - научно-информационная поддержка специалистов в области офтальмологии.