Deep anterior lamellar keratoplasty or penetrating keratoplasty are currently considered the optimal methods of surgical treatment of stromal dystrophies and corneal degeneration. Despite certain advantages and benefits of these methods, they also have significant limitations: involvement of superficial corneal layers in the surgery, need for suturing, development of post-keratoplasty astigmatism etc.PURPOSE:This study aimed to test and describe the new method of closed sutureless keratoplasty (intracorneal selective stromal transplantation), which was indicated in isolated dystrophic and degenerative pathology of the stroma.MATERIAL AND METHODS:Intracorneal selective stromal transplantation was performed in a 62-year-old patient with stromal degeneration and intact corneal layers between the altered stroma and the Descemet's membrane posteriorly, and the Bowman's layer anteriorly. The patient also had immature senile cataract. Corneal stroma was removed and replaced with a graft in the optical center of the lens, while the endothelium, the Descemet's membrane and the Bowman's layer remained intact.RESULTS:The proposed technique of intracorneal selective stromal transplantation makes it possible to replace only the pathologically altered stroma through closed surgical approach, without affecting the anterior and posterior surfaces of the cornea. Best-corrected visual acuity has increased in the patient from 0.01 to 0.6, while mean endothelial cell density has not changed in the course of 24-months follow-up.CONCLUSION:The proposed keratoplasty method can be used in patients with dystrophy or degeneration of the corneal stroma and preserved endothelial cells, intact Descemet's membrane and Bowman layer. Since the superficial corneal layers are not involved during the surgery, intracorneal selective stromal transplantation combined the advantages of both deep anterior lamellar keratoplasty and endothelial keratoplasty. The biologically favorable result in this first clinical case allows a preliminary conclusion on the technical possibility and functional effectiveness of the proposed method, but further long-term observation and more clinical cases are required.

Purpose. The aim of the work is to compare the time of epithelization and the change in the thickness of the cornea in the treatment of erosions after corneal burns of 2–3 degrees and ulcers after keratitis with preserved amnion and amnion saturated with autologous PRP lysate. Materials and methods. To compare the effectiveness of preserved amnion and lysate-soaked PRP, 2 groups of patients were identified: the main group (amnion saturated with lysate PRP) and the comparison group (amnion without lysate PRP). Each group included patients with erosions after recent burns with an average area of 60 % of the cornea and ulcers after keratitis with a corneal thickness at the bottom of the ulcer of 382 microns in the experimental group and 393 microns in the comparison group. In the comparison group, ulcers were treated by covering the cornea with an amniotic membrane for 7–14 days, after which the amnion was removed and the condition of the cornea was monitored. An amniotic membrane saturated with PRP lysate was used in the experimental group. PRP lysate was made from autologous blood of patients in the laboratory of cell transplantation and immunotyping of the N.V. Sklifosovsky Research Institute of Emergency Medicine using a patented technology. To do this, platelet-rich plasma (PRP) with a platelet content of over 1000 thousand/ml was isolated from the blood of donors, which was then frozen at -80 °C and thawed at 0–4 °C in order to destroy cell membranes. Assessment of the condition of the cornea was carried out using clinical and instrumental studies, including biomicroscopy with fluorescein staining, photoregistration and OCT on the Heidelberg Engineering “OCT Spectralis” apparatus. Results. The use of an amniotic membrane saturated with PRP lysate led to epithelialization of erosions after recent burns in 1.3 months and a decrease in corneal edema by 247 microns, epithelialization of ulcers after keratitis in 1.3 months and an increase in the thickness of the cornea at the bottom of the ulcer by 62.5 microns. Treatment with conventional preserved amnion took 1.8 months before complete epithelization in recent burns with a decrease in corneal stroma edema by 193 microns, and epithelization of ulcers after keratitis required 1.6 months and ended with an increase in the thickness of the cornea at the site of the ulcer by 42 microns. Conclusion. The study showed that the presented method of treating erosions and ulcers of the cornea of various genesis using an amniotic membrane saturated with autologous PRP lysate allows achieving complete and persistent epithelialization in a shorter time and with fewer amnion coatings, unlike the comparison group. At the same time, patients do not need additional conservative treatment of epitheliopathies.

Late complications of radial keratotomy (RK), are as a rule caused by a change in the biomechanical properties of the cornea. Purpose : to study the long-term stabilizing effect of the Bowman’s layer (BL) transplantation after RK. Material and methods . In a limited, prospective, monocentric pilot study we operated 2 male patients (2 eyes) aged 60 and 55. The radial incisions had been performed 34 and 38 years ago. The indications for surgery were progressive biomechanical instability of the cornea and functional refractive visual fluctuations. Within 12 months after the operation, complications, corrected visual acuity, keratometric parameters, biomicroscopic transparency of the cornea, and patient satisfaction were assessed. Results. No complications were noted during the follow-up period, the cornea and graft retained transparency. The average thickness of the BS graft was 20±2.1 µm. The average visual acuity corrected by contact lenses increased from 0.4±0.3 to 0.65±0.1, the average indicator of keratometric astigmatism changed from 1.9±1.6 to 3.0 D, these indicators remained after 6, 9 and 12 months, which indicates that biomechanical stability of the cornea was achieved and that the refractive-visual fluctuations are absent. Conclusion . In late term RK complications, BS transplantation partially restores the normal anatomy of the anterior surface of the cornea and ensures its biomechanical stability during a 12-month follow-up period. For the conclusions to be maximally objective, more clinical cases should be studied within a longer observation period.

Purpose : to compare the effectiveness of conventional preserved amnion and amnion saturated with platelet-rich (PRP) plasma lysate for the treatment of persistent corneal erosions (PCE) after high-risk keratoplasty. Materials and methods . 40 patients with persistent corneal erosions after high-risk penetrating keratoplasty, followed up for 12 months, were divided into two clinical groups of 20 people each. The main group of patients, aged 34 to 84, received Flexamer amniotic membrane + PRP lysate, while the comparison group, aged 41 to 80, received Flexamer amniotic membrane only. Amniotic membrane coating was used in persistent corneal erosions of penetrating keratoplasty after unsuccessful conservative treatment. The amnion was sewn 2 mm from the limb with a continuous suture and covered with a soft contact lens. As a source of platelets, we used the blood of healthy volunteers, from which platelet-rich plasma with platelet concentration of over 1000 thousand/µl was taken, which was then frozen at -40 °C and defrosted at 0…4 °C to obtain PRP lysate. The criteria for evaluating the effectiveness of both groups were the times of epithelialization, the number of amniotic membrane coatings, and the number of preserved transplants at the end of the follow-up. Results . Lyophilized amniotic membrane saturated with autologous PRP lysate growth factors was shown to be biocompatible. It was found to be safe for the patients, to reduce the epithelialization time, to reduce the number of operations required to cover the PÑE with the amnion, and to increase the likelihood of successful transparent and translucent engraftment of a penetrating keratograft. Conclusion . The use of a lyophilized amniotic membrane enriched with growth factors of autologous PRP lysate is a promising method for the treatment of PCE of the penetrating corneal graft after high-risk keratoplasty.

Purpose: To study the biological effect of a combination of platelet lysate and amniotic membrane, preserved by various techniques, on human buccal epithelium culture. Materials and methods. Human amnion transplants were preserved using 3 methods: silicate drying, lyophilization, cryopreservation. The blood of healthy volunteers was used as a source of platelets. Platelet-rich plasma (PRP) with a platelet content over 1000 thousand/mcl and more was isolated from the donors blood, frozen at -80 °С and defrosted at 0–4 °С to prepare platelet lysate. Growth-stimulating effect of the amnion transplants was studied in different groups: control group 1 — without amnion and without PRP lysate; control group 2 — PRP lysate without amnion; experimental group 1 — amnion without PRP lysate; experimental group 2 — amnion samples combined with PRP lysate. The study was carried out on the example of human buccal epithelium culture of 3–5 passages. The dynamics of cell growth was evaluated after 1, 2 and 3 days from the moment of seeding. The number of cells and their viability were evaluated using original methods based on vital cell staining and their examination in a fluorescent microscope. Results. All samples of preserved amnions were non-toxic and did not damage the structural and functional characteristics of the buccal epithelium. On the other hand, the use of amnion without PRP lysate did not have a growth-stimulating effect on cells. Among the amnion samples combined with PRP lysate, the combination of lyophilized amnion and PRP lysate was the most effective during the entire study period. Conclusions. Silicate drying, lyophilization and cryopreservation of the amniotic membrane makes it possible to obtain biocompatible and non-toxic transplants, based on human amnion. Lyophilized amnions are the most optimal for saturating PRP lysate. The combination of lyophilized amnion and PRP lysate stimulates cell growth in vitro without violating their structural integrity.

Purpose: to study the clinical signs and effectiveness of laboratory diagnostics of fungal corneal ulcer complicated by endophthalmitis aimed at treatment tactics optimization.Materials and methods. 15 patients aged 20–55 with severe monocular fungal ulcer complicated by endophthalmitis were examined by microscopy and culture of corneal scraping on Sabouraud Agar; microscopy and culture of intraocular fluid and corneal fragments, obtained during the surgical treatment. Corneal scrapings from the corneal ulcer and corneal fragments removed during keratoplasty were tested in polymerase chain reaction (PCR) for the presence of deoxyribonucleic acid (DNA) of Candida albicans and total Fungi DNA.Results. Before treatment start, 2.5 hours after the biomaterial was taken, PCR revealed Fungi DNA in all 15 cases. Symptoms of the severe fungal corneal ulcer complicated by endophthalmitis and caused by Aspergillus spp have been identified and described. Conservative therapy and total penetrating keratoplasty (PKP) helped retain the eye in 13 out of 15 patients (86.7 %) and partially restore the visual functions.Conclusion. Rapid laboratory diagnosis of ophthalmomycosis using PCR offers an advantage over the culturing technique: the results are ready within 2.5 hours instead of 10-14 days (culture method); it is highly sensitive and allows identifying the fungi not only in the tissue of the removed cornea, but also in the сorneal scrapings. The effective treatment tactics of fungal corneal ulcer complicated by endophthalmitis combining PKP with pre-operative and long-term postoperative antifungal therapy was proposed.

Secondary decompensation of corneal endothelium, including transplanted, is a frequent long-term complication of glaucoma drainage surgery. According to literature data, after implantation of a glaucoma drainage device into the anterior chamber, the speed of endothelial cells density (ECD) loss increases significantly.PURPOSE:To study the possibility of performing modified Descemet membrane endothelial keratoplasty (DMEK) with maximum graft diameter, and to assess its short-term effectiveness in the treatment of bullous keratopathy (BK) in the presence of a glaucoma drainage device in the anterior chamber.MATERIAL AND METHODS:Modified surgery 3/4-DMEK was performed in 9 eyes of 7 patients with BK and Ahmed glaucoma drainage using graft of maximum diameter (11±1 mm). Average follow-up time was 15.3±2.5 months (12 to 18 months).RESULTS:No intraoperative or postoperative complications were observed. Mean ECD after 12 months amounted to 1664±346 cells/mm2, which corresponds to ECD reduction of 34±9.6% (24 to 49%) in comparison to preoperative values. Mean best corrected visual acuity 12 months after surgery increased from 0.04±0.03 (0.01 to 0.1) to 0.3±0.2 (0.04 to 0.7). The last follow-up examination revealed all corneas to have preserved transparency and stable pachymetry measurements (from 488 to 548 µm).CONCLUSION:Performing 3/4-DMEK with a mega graft for treatment of BK after drainage surgery is technically possible. Absence of Descemet membrane (DM) donor and preservation of DM recipient in the drainage tube plane eliminates its contact with the endothelium. Maximum graft diameter should in the long term help prolong the effectiveness of the surgery.

Purpose: to evaluate the results of spontaneous non-penetrating keratoprosthesis with a Fedorov — Zuev prosthesis in a patient with severe chemical burns in both eyes. Material and methods.Patient K., 38, who had sustained a severe burn injury in the past and numerous reconstructive plastic operations on both eyes (amniotic tissue implantation, allolymbal transplantation, layer-by-layer and penetrative keratoplasty, cataract extraction with IOL implantation, total auto-conjunctival corneal plastic surgery) with no functional effect, was subjected to a multi-stage keratoprosthesis of the left eye according to the method practiced by the Helmholtz National Medical Research Center of Eye Diseases. Results.After one of the stages (implantation of the haptic part of the keratoprosthesis with a temporary cylindrical plug), an aseptic necrosis of the tissue above the cylinder occurred. As a result, an unexpected functional effect was revealed: visual acuity of the operated eye 0.02 sph -20.0 D = 0.2. During a dynamic follow-up that lasts 2.5 years, visual acuity remains stable, and the corneallayers behind the cylinder retain transparency. During this time, all stages of keratoprosthesis were performed on the fellow eye with a functional result of 1.0. Conclusion. The long-term result of spontaneous non-penetrating keratoprosthesis indicates the need to study the prospects and develop a method of non-penetrating keratoprosthesis with a Fedorov — Zuyev prosthesis.

Fungi implicated in mycotic keratitis include different species. Conventional methods for the diagnosis of fungal keratitis include staining of corneal scarpings, culture medium (Sabouraud agar) for isolating fungi.Purpose. To evaluate the effectiveness of polymerase chain reaction (PCR) for the detection of fungal etiology in comparison with the conventional diagnostic methods in cases with suspected fungal corneal ulcer.Patients and methods. Seven patients with severe corneal ulcers with more than 3 weeks duration. Corneal scarpings and corneal buttons from seven patients who had undergone therapeutic keratoplasty were used for microbiological and PCR analysis. PCR diagnostic kits for the differential detection of Candida albicans DNA and total fungi DNA (DNA Fungi), which allows to identify most pathogenic fungi without determining their species were used. Microbiological methods: microscopy of gramstained smears, culture techniques, including selective for fungi agar Saburo with chloramphenicol.Results. PCR: Fragments of all corneas removed from keratoplasty (6 patients) revealed fungal-common DNA (Fungi DNA) and did not detect Candida albicans DNA, which correlated with sowing results on Saburo medium (mold fungi found in 5 of 6 corneas). Fungi DNA was also detected in the corneal scraps taken prior to surgery; however, growth of fungi during sowing on various nutrient media was not found.Conclusion. Corneal fungal ulcers are a serious disease, often leading to visual disability. The rapid determination of etiology and the correct choice of therapy determines the outcomes of the disease. The advantage of PCR over the culture method: the speed of obtaining results (4 hours instead of 3–7 days); high sensitivity, which allows detecting fungi not only in the tissue of the removed cornea, but also in scrapes from the cornea ulcer of patients who previously received antifungal therapy. The presence of commercial kits for differential detection of fungal-common DNA and DNA of Candida albicans extends the possibilities of PCR in the screening diagnosis of fungal keratitis and the selection of drugs before determining the type of pathogen.

Purpose. The aim is to estimate the content of proinflammatory chemokines (CXCL1/GRO-α CXCL8/IL-8, CXCL10/IP-10, CXCL12/ SDF-1α, CCL2/MCP-1, CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL11/Eotaxin) in blood serum in corneal ulcers and their relationship to the cellular immune response to corneal and uveoretinal tissues. Material and methods. 96 patients with corneal ulcer and 38 apparently healthy subjects, who made up the control group, were examined. Chemokines were determined by multiplex analysis using xMAP technology (Luminex, USA) in the xPONENT3.1 program using magnetic fluorescent microspheres and a ProcartaPlex kit (eBioscience, USA). To identify the autoimmune component, a micromodification of the leukocyte migration inhibition reaction (MIRL) with extracts of the corneal and the uveoretinal tissue of bovine eyes were used. Based on MIRL results, the patients were divided into two groups: 1 group sensitized patients (positive response to tissue antigens) — 33 (34 %) patients, 2 group — nonsensitized patients (negative response to tissue antigens) — 63 (66 %) patients. Results. The group of patients showed 2–10 times higher concentrations of the following chemokines, with respect to the control group: CXCL8/IL-8, CXCL12/SDF-1α, CXCL10/IP-10, ССL2/MCP-1, CCL4/MIP-1β, CCL5/RANTES. Shifts of CXC-chemokines (CXCL8/IL-8, CXCL12/SDF-1α, CXCL1/GRO-α, CXCL10/IP-10) in patients with central corneal ulcer were associated with increased systemic cellular immune response to eye tissue antigens; the most expressed dynamics was noted for chemokine CXCL8/IL8. Сonclusions. The revealed changes in CXC- and CC-classes chemokine levels in patients’ blood characterize the corneal ulcers an immopathologic process and validate the necessity of further research into diagnostic informativeness of the presented mediator spectrum as biological markers for the prognosis of corneal ulcers and their complications in wider clinical samples. Concentration of serum CXC chemokines, and most directly CXCL8/IL-8 can serve as an additional marker for the development of a cellular immune response to eye tissue antigens in patients with a central corneal ulcer.

Purpose:to study the efficiency of Bowman layer transplantation (BLT) in corneal stroma for the treatment of progressive keratoconus (KC).Material and methods.30 patients (30 eyes) with progressive KC stages III to IV (according to Amsler — Krumeich classification), underwent BLT into a mid-stromal pocket. At the time of surgery, the patients were 14 to 37 (averagely 26.6 ± 6.2 years). Before and after the surgery, the maximum keratometry index (Kmax) and the corneal thinnest point (CTP) values were determined. Also, we evaluated visual acuity before and after surgery in scleral contact lenses, endothelial cells density (ECD) and the depth of graft location. The indication for surgery was the refusal of corneal transplantation, progression of KC, contraindications for ultraviolet crosslinking or implantation of intracorneal ring segments based on the CTP and Kmax values. The follow-up continued 6 to 36 months (averagely, 26.6 ± 6.0 months).Results.Throughout the observation period, no intra or postoperative complications associated with BLT or deterioration of CTP, Kmax or best corrected visual acuity (BCVA) in scleral lenses were noted.Conclusions.Kmax, CTP and ECD values remaining stable during the follow-up is the evidence of a stabilizing BLT effect on the KC progression. The absence of BCVA decrease in scleral lenses indicates a functional safety of BLT.

To study the role of HHV-6 in the development of eye diseases, 193 patients with various eye diseases were examined. Using polymerase chain reaction (PCR) tests DNA of HHV-6 (n = 146) in peripheral blood was detected, using immunoferment analysis markers of chronic (IgG antibodies, n = 142) and active (IgM antibodies, n = 74) HHV-6 infection were detected in blood serum. HHV-6 could be detected in patients with different ophthalmic pathology forms both in the chronic (66.9 %) and the active (43.2 %) form, and DNA of the virus could be found in the blood (20.5 %). A parallel simultaneous study of the blood (n = 40) revealed markers of chronic and/or active infection and DNA of the virus in patients of all nosological groups surveyed (uveitis, corneal ulcers of various etiologies, post-inflammatory cataracts, corneoscleral eye injuries and corneal graft disease). For the first time serological tests (IgG and IgM antibodies) were performed on patients with age-related macular degeneration, where 82.4 % of the patients turned out to be seropositive. The obtained data suggest that HHV-6, along with other viruses generally known to be ophthalmic, may be an etiologic or pathogenetic factor of eye diseases. In this regard, a comprehensive laboratory testing (serological diagnostics and PCR) for HHV-6 should become the object of further targeted study // Russian Ophthalmological Journal, 2016; 1: 73-77.

This article presents a detailed description of shifts in subsets of blood lymphocyte subpopulations associated with development of the corneal ulcerative lesions as well as during penetrating keratoplasty.We have revealed some general changes typical for patients with corneal ulcer and patients with keratoplasty, reflecting inflammatory activity, i.e., a significant increase in the numbers of lymphocytes (CD45+), relative and absolute contents of the T-cell population (CD3+), CD3+ CD4+ subpopulation, and B-lymphocytes (CD19+), as compared with the control group. A statistically significant increase in natural killer cells (СD16+СD56+) proved to be a distinctive feature of the group with keratoplasty.When analyzing mean values of CD4+/CD8+ cells from the patients with corneal ulcer, there was no statistically significant difference between the clinical groups studied. However, individual comparison of nature and frequency of CD4+/CD8+ shifts among the patients with «high» index allowed qualitative separation of the third group (severe corneal ulcer): a severe imbalance of main T-cell subpopulations was detected 2.4 times significantly more often than in the group of patients with mild forms of corneal ulcers.The obtained data are important for understanding pathogenesis of centrally localized corneal ulcer, and should be taken into account when predicting and developing a differentiated approach to the treatment of the disease.

Purpose: to report the first case of the same day use of a single donor cornea tissue for 5 consecutive transplantations in patients with endothelial dystrophy and keratoconus. Material and methods. The Descemet membrane (DM) and the endothelium were fully detached from the stroma of the donor on the endothelial side, using first a circular and then a direct corneal cutter, forming four partial triangle-shaped Descemet grafts (each constituted a 1/4 Descemet graft). The stroma of the donor's cornea was separated by a circular cutter of a preset diameter. One patient with keratoconus underwent deep anterior lamellar keratoplasty (DALK) using the big bubble technique. Then, each of the four Descemet grafts was sequentially transplanted by the Descemet membrane endothelial keratoplasty (DMEK) method to 4 patients with Fuchs dystrophy and pseudophakia. Results. During and after the surgery, there were no complications requiring additional intervention. Six months after DALK, the best corrected visual acuity (BCVA) was 0.66. Six months after four operations each using ¼ DMEK the average BCVA was 0.95 ± 0.1 (from 0.8 to 1.0) and the endothelial cell density was, respectively, 2839 after DALK and 1680 ± 254 cells/mm2 (from 1492 to 2039 cells/mm2) after ¼ DMEK. Conclusion. One donor cornea can be successfully transplanted to at least 5 patients. Despite the fact that the implementation of DALK and several operations using the ¼ DMEK technique within one day is technically difficult, this approach can contribute to a drastic reduction in the deficit of donor tissue. This approach requires a thorough selection of patients and an extensive surgical experience. For citation: Oganesyan O.G., Makarov P.V., Grdikanyan А.А., Getadaryan V.R. A new strategy of keratoplasty: laminating and splitting the donor cornea. Russian ophthalmological journal. 2018; 11 (3): 11-8 (In Russian). doi: 10.21516/2072-0076-2018-11-3-11-18. The full English version is available online at http://roj.igb.ru/eng/issues

To study the role of the HHV-6 type in the development of eye diseases PCR tests of blood (152), cornea biopsies (61), and intraocular fluids (11) for HHV-6 and other viruses of the herpes group (HSV type 1 and 2, CMV, EBV) were conducted. It was found that the HHV-6, along with other representatives of the Herpesviridae, can be detected in patients with different clinical forms of ophthalmopathology (174 patients were surveyed). Viral DNA was detected in blood, cornea, and in the anterior chamber fluid. The obtained data allow that the HHV-6 to be suggested as a possible cause of the ophthalmic herpes along with the other viruses of this group. It makes finding the virus DNA an essential step towards setting the etiologic diagnosis of the ophthalmological patients.

To study the role of the HHV-6 type in the development of eye diseases PCR tests of blood (152), cornea biopsies (61), and intraocular fluids (11) for HHV-6 and other viruses of the herpes group (HSV type 1 and 2, CMV, EBV) were conducted. It was found that the HHV-6, along with other representatives of the Herpesviridae, can be detected in patients with different clinical forms of ophthalmopathology (174 patients were surveyed). Viral DNA was detected in blood, cornea, and in the anterior chamber fluid. The obtained data allow that the HHV-6 to be suggested as a possible cause of the ophthalmic herpes along with the other viruses of this group. It makes finding the virus DNA an essential step towards setting the etiologic diagnosis of the ophthalmological patients.

The work is dedicated to studies of interrelations between the cytokine contents in the liquid from anterior eye chamber (AEC), and development of systemic immune response to ocular tissue antigens, described in terms of an anterior eye chamber-associated immune disturbance (ACAID phenomenon). The immune assays were conducted in parallel, using multiplex cytokine analysis (flow cytometry technique), and leukocyte inhibition migration reaction. Twenty-six patients with different forms of ophthalmic pathology were examined, including cases of uveitis (18), keratouveitis (3), glaucoma (5). It is shown that the reduced TGF-β1 levels and increased concentrations of proinflammatory, chemotactic cytokines in AEC liquid are associated with development of organ-specific immune sensitization.