Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers.

The cryopreservation of endothelial cell monolayers is an important step that bridges the cryopreservation of cells in suspension to that of tissues. Previous studies have identified clear distinctions in freezing mechanisms between cells in suspension and in monolayers, as well as developed novel protocols for monolayer cryopreservation. Recently, our group has shown that human umbilical vein endothelial cell (HUVEC) and porcine corneal endothelial cell (PCEC) monolayers grown on Rinzl plastic substrate can be cryopreserved in 5% dimethyl sulfoxide, 6% hydroxyethyl starch, and 2% chondroitin sulfate, following a slow-cooling protocol (-1 °C/min) with rapid plunge into liquid nitrogen from -40 °C. However, membrane integrity assessments were done immediately post thaw, which may result in an overestimation of cell viability due to possible delayed injury responses. Here, we show that for the optimal protocol condition of plunge at the -40 °C interrupt temperature, HUVEC and PCEC monolayers exhibited no significant immediate post-thaw injuries nor delayed injury responses during the 24-h post-thaw overnight culture period. HUVEC monolayers experienced no significant impact to their natural growth rate during the post-thaw culture, while PCEC monolayers experienced significantly higher growth than the unfrozen controls. The difference in the low-temperature responses between HUVEC and PCEC monolayers was further shown under high temperature plunge conditions. At these suboptimal plunge temperatures, HUVEC monolayers exhibited moderate immediate membrane injury but a pronounced delayed injury response during the 24-h post-thaw culture, while PCEC monolayers showed significant immediate membrane injury but no additional delayed injury response during the same period. Therefore, we provide further validation of our group's previously designed endothelial monolayer cryopreservation protocol for HUVEC and PCEC monolayers, and we identify several cell-type-specific responses to the freezing process.

Factors associated with severe corneal endothelial damage following acute primary angle closure in Chinese subjects.

PURPOSE: To investigate the corneal endothelial damage caused by acute primary angle closure (APAC) and related risk factors for severe corneal endothelial cell damage in Chinese subjects. METHODS: In this multicentre retrospective study, 160 Chinese patients (171 eyes) diagnosed with APAC were recruited. Endothelial cell density (ECD) and morphological changes short after APAC were studied. Univariate regression and multivariate regression were used to identify risk factors associated with the extent of ECD reduction, including age, gender, education level, patients' location, systemic diseases, APAC duration (hours), highest recorded intraocular pressure (IOP), and presenting IOP. Factors associated with the probability of severe corneal damage (ECD lower than 1000/mm2) were analysed based on a linear function. RESULTS: After one APAC episode, 12.28% eyes had ECD lower than 1000/mm2, 30.41% had ECD between 1000 and 2000/mm2, and 57.31% had ECD more than 2000/mm2. Attack duration was the only factor associated with severe endothelial damage (p < 0.0001). If the attack were to be subsided within 15.0 h, possibility of ECD lower than 1000/mm2 could be controlled under 1%. CONCLUSION: Shortly after the abortion of APAC, 12.28% patients experienced severe endothelial cell damage with ECD less than 1000/mm2. The only factor associated with severe ECD decrease was attack duration. Immediate and effective treatment is pivotal for preserving corneal endothelial function in APAC patients.

Endotelitis por citomegalovirus y su diagnóstico mediante la microscopia confocal / Cytomegalovirus endotheliitis and its diagnosis by confocal microscopy

Objetivo: Describir las características del endotelio corneal por microscopia confocal en pacientes con lesiones basofílicas de inclusión como patognomónicas de endotelitis por citomegalovirus. Métodos: Se realizó un estudio observacional de casos clínicos en pacientes con diagnóstico de endotelitis por citomegalovirus, atendidos en la Consulta de Córnea del Instituto Cubano de Oftalmología "Ramón Pando Ferrer", entre febrero del año 2010 y junio del 2018. La muestra incluyó 39 pacientes con diagnóstico clínico de endotelitis, a quienes se les realizó microscopia confocal con el equipo Confoscan 4 (Nidek Technologies). Resultados: De los 39 pacientes, 29 fueron del sexo masculino y 10 del femenino. Todos tenían entre 41 y 60 años de edad. En el 97,4 por ciento de los casos existió el antecedente de una conjuntivitis viral y un solo paciente fue positivo de HIV (2,6 por ciento). La mejor agudeza visual corregida de 0,3 o menos se mostró en el 100 por ciento de ellos antes del tratamiento, y después de este los 39 tenían entre 0,8 y 1,0. En el 100 por ciento de los casos se observaron cuerpos de inclusión basofílicos en el endotelio corneal, que fueron apreciables mediante la microscopia confocal. Conclusiones: Existe una relación entre la presencia de cuerpos de inclusión basofílicos en el endotelio corneal y las pruebas virológicas a citomegalovirus positivas, lo que puede permitir hacer PCR a casos que ya tienen confirmación mediante microscopia confocal de las características patognomónicas del endotelio corneal(AU)

Objective: Describe the characteristics of the corneal endothelium by confocal microscopy in patients with inclusion basophilic lesions as pathognomonic signs of cytomegalovirus endotheliitis. Methods: An observational study was conducted of clinical cases of patients diagnosed with cytomegalovirus endotheliitis attending the Cornea Service at Ramón Pando Ferrer Cuban Institute of Ophthalmology from February 2010 to June 2018. The study sample was 39 patients with a clinical diagnosis of endotheliitis who underwent confocal microscopy with a Confoscan 4 device (Nidek Technologies). Results: Of the 39 patients examined, 29 were male and 10 were female. All were aged 41-60 years. 97.4 percent had a history of viral conjunctivitis and only one was HIV positive (2.6 percent). Best corrected visual acuity was 0.3 or less in 100 percent before treatment, and 0.8 to 1.0 after treatment. Inclusion basophilic bodies visible by confocal microscopy were observed in the corneal endothelium of all patients. Conclusions: A relationship exists between the presence of inclusion basophilic bodies in the corneal endothelium and virological tests positive for cytomegalovirus, making it possible to perform PCR testing in cases with confocal microscopy confirmation of the pathognomonic characteristics of the corneal endothelium(AU)

Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing.

PURPOSE: To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. METHODS: UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 µL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 µL Opti-MEM I reduced serum medium (EPC group), 100 µM Y-27632 diluted in 100 µL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 µM Y-27632 (final volume 100 µL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. RESULTS: Culturing UCB EPCs in CM supplemented with 10 µM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase ß1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. CONCLUSIONS: Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.

Endothelial keratoplasty: The relationship between recipient anterior chamber depth and endothelial cell loss.

PURPOSE: To determine the relationship between anterior chamber depth (ACD) and percent endothelial cell loss (ECL) after Descemet Stripping Automated Endothelial Keratoplasty (DSAEK). METHOD: In 78 eyes receiving triple procedure (DSAEK combined with cataract extraction and posterior chamber intraocular lens (PCIOL) implantation), ACD was measured preoperatively with an intraocular lens (IOL) Master and ECL was calculated with specular microscopy at 6 months, 1, 2, 3, and 4 years postoperatively. ACD and ECL from all 78 eyes were compared using correlation analysis and students t test. Eyes were then separated into 2 groups based on ACD, group 1 with ACD < 3mm and group 2 with ACD ≥ 3mm. Students t test was then performed to compare group 1 and group 2 ECL at 6 months, 1, 2, 3, and 4 years postoperative. RESULTS: Mean ACD for all 78 eyes was 2.93 ±â€Š0.43 mm. Mean ECL was 32.7%, 27.6%, 29.6%, 32.5%, and 37.2% at 6 months, 1, 2, 3, and 4 years. No significant correlation between ACD and ECL was observed at any time point for the combined analysis of 78 eyes (P > .05). At 2 and 4 years postoperative, mean ECL was 32.6% ±â€Š16.1% and 43.0% ±â€Š23.2% in eyes with ACD < 3mm and 25.3% ±â€Š13.0% and 29.6% ±â€Š18.2% in eyes with ACD ≥ 3 mm (P = .041 at 2 years and .008 at 4 years). CONCLUSION: ACD and ECL were not directly correlated; however, there may be a threshold ACD in which shallower anterior chambers preoperatively result in greater donor ECL over time.

Topical Rho-Associated Kinase Inhibitor, Y27632, Accelerates Corneal Endothelial Regeneration in a Canine Cryoinjury Model.

PURPOSE: Corneal endothelial cell regeneration varies by species, with nonhuman primates (NHPs) and rabbits displaying low and high proliferative capacities, respectively. Recent studies report that topical application of rho-associated kinase (ROCK) inhibitors accelerates corneal endothelial wound healing in animal models and human patients with endothelial dysfunction. This study determines the regenerative capacity of canine corneal endothelial cells in vivo and their response to a topical ROCK inhibitor, Y27632, after transcorneal freezing. METHODS: Right eyes of 6 beagles underwent transcorneal freezing; 10 mM ROCK inhibitor Y27632 or vehicle control was applied topically to both eyes at least 4 times daily for 56 days. Endothelial cell density was evaluated by in vivo confocal microscopy, and corneal thickness was measured by Fourier-domain optical coherence tomography (FD-OCT) and ultrasound pachymetry. RESULTS: Transcorneal freezing induced severe central corneal edema in dogs, with restoration of transparency occurring within 4 weeks. Y27632 significantly decreased corneal thickness by FD-OCT and ultrasound pachymetry in the acute phase and significantly increased endothelial cell density at days 28 and 42 post-cryoinjury, suggesting faster restoration of endothelial cell recovery. CONCLUSIONS: Canine corneal endothelial function recovers at a similar rate as NHPs but more slowly than rabbits after cryoinjury. Faster corneal endothelial wound healing was observed by in vivo confocal microscopy and FD-OCT in dogs treated with Y27632 versus vehicle controls. Thus, a canine cryoinjury model may be a useful alternative to NHPs in detecting a response to therapies directed at endothelial regeneration.

New ex vivo model of corneal endothelial phacoemulsification injury and rescue therapy with mesenchymal stromal cell secretome.

PURPOSE: To develop a reproducible ex vivo model of corneal endothelial cell injury using phacoemulsification in porcine eyes and to evaluate the effects of mesenchymal stromal cell secretome in this injury model. SETTING: Department of Ophthalmology, University of Illinois at Chicago, Illinois, USA. DESIGN: Experimental study. METHODS: A corneal endothelial injury model was optimized using different powers and durations of ultrasound energy inside ex vivo porcine eyes. Conditioned media from corneal mesenchymal stem cells was collected under serum-free conditions from passages 4 to 6. Immediately after the phacoemulsification injury, the anterior chamber fluid was replaced with unconditioned media or conditioned media and incubated at 37°C for 4 hours. At the end, endothelial cell viability was evaluated using trypan blue staining and analyzed with ImageJ software. RESULTS: Using specific parameters (50% power for 30 seconds), phacoemulsification inside fresh porcine eyes led to a consistent level of endothelial cell injury. Incubation with corneal mesenchymal stromal cell-conditioned media after the injury significantly reduced endothelial cells loss compared with unconditioned media (mean 1.29% ± 0.91% [SD] and 5.33% ± 3.24%, respectively, P < .05). CONCLUSIONS: Phacoemulsification inside fresh porcine eyes provided a reproducible model to study endothelial cell injury. Treatment with corneal mesenchymal stromal cell secretome after injury appeared to significantly enhance the survival of corneal endothelial cells. This might provide a new strategy for preventing corneal endothelial cell loss after phacoemulsification or other endothelial injuries. Further in vivo studies are necessary to determine the therapeutic potential.

Corneal endothelial decompensation due to airbag injury.

PURPOSE: We report a case of localized endothelial decompensation due to airbag deployment during a motor vehicle accident. CASE REPORT: A middle-aged woman involved in a motor vehicle accident presented with diminution of vision in left eye. Initial ocular examination revealed corneal abrasion, localized central corneal edema and mild anterior chamber reaction. An anterior-segment ocular coherence tomography (AS-OCT) revealed focal paracentral corneal edema. Patient was managed with lubricating eye drops and antibiotic steroid combination. Significant endothelial cell loss compared to right eye was noted on specular examination. At one- month follow-up, visual acuity recovered to 6/6 but the pleomorphism and polymegathism persisted. CONCLUSION: Airbag-related localized corneal endothelial decompensation is a less known occurrence. This case emphasizes on the fact that serial monitoring of endothelial counts and conservative management can prove beneficial in such scenarios.

Vasoactive Intestinal Peptide Promotes Corneal Allograft Survival.

Corneal transplantation is the most prevalent form of tissue transplantation. The success of corneal transplantation mainly relies on the integrity of corneal endothelial cells (CEnCs), which maintain graft transparency. CEnC density decreases significantly after corneal transplantation even in the absence of graft rejection. To date, different strategies have been used to enhance CEnC survival. The neuropeptide vasoactive intestinal peptide (VIP) improves CEnC integrity during donor cornea tissue storage and protects CEnCs against oxidative stress-induced apoptosis. However, little is known about the effect of exogenous administration of VIP on corneal transplant outcomes. We found that VIP significantly accelerates endothelial wound closure and suppresses interferon-γ- and tumor necrosis factor-α-induced CEnC apoptosis in vitro in a dose-dependent manner. In addition, we found that intracameral administration of VIP to mice undergoing syngeneic corneal transplantation with endothelial injury increases CEnC density and decreases graft opacity scores. Finally, using a mouse model of allogeneic corneal transplantation, we found for the first time that treatment with VIP significantly suppresses posttransplantation CEnC loss and improves corneal allograft survival.

Vulnerability of corneal endothelial cells to mechanical trauma from indentation forces assessed using contact mechanics and fluorescence microscopy.

Corneal endothelial cell (CEC) loss occurs from tissue manipulation during anterior segment surgery and corneal transplantation as well as from contact with synthetic materials like intraocular lenses and tube shunts. While several studies have quantified CEC loss for specific surgical steps, the vulnerability of CECs to isolated, controllable and measurable mechanical forces has not been assessed previously. The purpose of this study was to develop an experimental testing platform where the susceptibility of CECs to controlled mechanical trauma could be measured. The corneal endothelial surfaces of freshly dissected porcine corneas were subjected to a range of indentation forces via a spherical stainless steel bead. A cell viability assay in combination with high-resolution fluorescence microscopy was used to visualize and quantify injured/dead CEC densities before and after mechanical loading. In specimens subjected to an indentation force of 9 mN, the mean ±â€¯SD peak contact pressure P0 was 18.64 ±â€¯3.59 kPa (139.81 ±â€¯26.93 mmHg) in the center of indentation and decreased radially outward. Injured/dead CEC densities were significantly greater (p ≤ 0.001) after mechanical indentation of 9 mN (167 ±â€¯97 cells/mm2) compared to before indentation (39 ±â€¯52 cells/mm2) and compared to the sham group (34 ±â€¯31 cells/mm2). In specimens subjected to "contact only" - defined as an applied indentation force of 0.65 mN - the peak contact pressure P0 was 7.31 ±â€¯1.5 kPa (54.83 ±â€¯11.25 mmHg). In regions where the contact pressures was below 78% of P0 (<5.7 kPa or 42.75 mmHg), injured/dead CEC densities were within the range of CEC loss observed in the sham group, suggesting negligible cell death. These findings indicate that CECs are highly susceptible to mechanical trauma via indentation, supporting the established "no-touch" policy for ophthalmological procedures. While CECs can potentially remain viable below contact pressures of 5.7 kPa (42.75 mmHg), this low threshold suggests that prevention of indentation-associated CEC loss may be challenging.

Endothelial Wound Repair of the Organ-Cultured Porcine Corneas.

PURPOSE: To assess whether injured porcine endothelium of small and large corneoscleral disc differ in its reparative/regenerative capacity under various conditions of organ culture storage. MATERIAL AND METHODS: 166 paired porcine corneas were trephined to obtain tissues with diameter 12.0 mm and 17.5 mm (with area neighboring endothelial periphery). In tested discs, central endothelium was mechanically wounded. Density of live endothelial cells (LECD), percentage of dead cells (%DC), coefficient of variation and cell hexagonality were assessed in central and paracentral endothelium following 5- or 9-day incubation in medium with 2% or 10% fetal bovine serum. The parameters were assessed also in fresh and intact cultured discs. Dead endothelial cells (EC) were visualized by trypan blue, cell borders by Alizarin Red S dye. Endothelial imprints were immunoassayed for the proliferation marker Ki-67 and the nucleolar marker fibrillarin. RESULTS: In fresh corneas, the LECD/mm2 (mean ± standard deviation) were 3998.0 ± 215.4 (central area) and 3888.2 ± 363.1 (paracentral area). Only the length of storage had significant effect on wound repair. Lesion was repaired partially after 5-day and fully after 9-day cultivation. After 9-day storage in medium with 10% serum, the mean LECD detected in small discs were 2409.4 ± 881.8 (central area) and 3949.5 ± 275.5 (paracentral area) and in large discs the mean LECD were 2555.0 ± 347.0 (central area) and 4007.5 ± 261.2 (paracentral area). Ki-67 showed cell proliferation associated with healing of EC of both large and small corneas. CONCLUSIONS: The lesions were completely repaired within 9 days of storage. Presence of the area, where stem cells appear to be located, contributes to stimulation of endothelial reparation less than serum concentration and time of culture. Both cell migration and proliferation contribute to the wound repair.

Posterior stromal cell apoptosis triggered by mechanical endothelial injury and basement membrane component nidogen-1 production in the cornea.

This study was performed to determine whether cells in the posterior stroma undergo apoptosis in response to endothelial cell injury and to determine whether basement membrane component nidogen-1 was present in the cornea. New Zealand White rabbits had an olive tip cannula inserted into the anterior chamber to mechanically injure corneal endothelial cells over an 8 mm diameter area of central cornea with minimal injury to Descemet's membrane. At 1 h (6 rabbits) and 4 h (6 rabbits) after injury, three corneas at each time point were cryopreserved in OCT for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry (IHC) for vimentin and nidogen-1, and three corneas at each time point were fixed for transmission electron microscopy (TEM). Uninjured corneas were controls. Stromal cells over approximately the posterior 25% of the stroma overlying to the site of corneal endothelial injury underwent apoptosis detected by the TUNEL assay. Many of these apoptotic cells were vimentin+, suggesting they were likely keratocytes or corneal fibroblasts. Stromal cells peripheral to the site of endothelial injury and more anterior stromal cells overlying the site of endothelial injury did not undergo apoptosis. Stromal cell death was confirmed to be apoptosis by TEM. No apoptosis of stromal cells was detected in control, uninjured corneas. Nidogen-1 was detected in the stroma of unwounded corneas, with higher nidogen-1 in the posterior stroma than the anterior stroma. After endothelial scrape injury, concentrations of nidogen-1 appeared to be in the extracellular matrix of the posterior stroma and, possibly, within apoptotic bodies of stromal cells. Thus, posterior stromal cells, likely including keratocytes, undergo apoptosis in response to corneal endothelial injury, analogous to anterior keratocytes undergoing apoptosis in response to epithelial injury.

Trophic effect of PACAP on human corneal endothelium.

Cornea's posterior surface includes endothelium maintaining stromal hydration and clarity. Due to their limited proliferative capability, the loss of endothelial cells can outcome in permanent opacity. In the last years, different studies have demonstrated the protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in different ocular diseases. However, its role on human corneal endothelial cells (HCECs) has not been investigated, yet. Here, we have developed a culture protocol to differentiate HCECs from donor's cornea. PACAP treatment prevented damage induced by growth factors deprivation of cells grown on transwell supports as revealed by TERR measurements. Moreover, this peptide significantly increased tight junction proteins expression by conferring resistance to endothelial barrier. This effect is also related to promotion of cell viability as demonstrated by MTT assay. Furthermore, PACAP stimulated repairing of corneal endothelium lesion as shown by wound healing analysis. In conclusion, our data suggest that this peptide could represent an important trophic factor in maintaining functionality of human corneal endothelium.

Characterization of Corneal Endothelial Injury During Penetrating Keratoplasty.

PURPOSE: To characterize corneal endothelial injury during penetrating keratoplasty in a controlled wet laboratory environment using human tissue. To identify potential areas or steps within surgery in which insult to the corneal endothelium may be most affected by trauma during routine penetrating keratoplasty. METHODS: Human donor corneas (n = 12) with intact endothelium underwent experimental penetrating keratoplasty. Endothelial injury was evaluated after each suture quartile using trypan staining, a validated modality for assessing endothelial injury. Insult was quantified using high-resolution photography and computer software. RESULTS: Statistical significance was found in the change in staining between quartiles as determined by repeated-measures analysis of variance (F3,11 = 5.83, P < 0.0044). A post hoc Tukey test indicated that the change in staining during the first quartile (3.38% ± 0.5%) was significantly lower than the remaining quartiles at P < 0.021. The change in staining did not differ significantly between the second (8.36% ± 1.2%), third (7.88% ± 1.2%), and fourth (7.73% ± 0.9%) quartiles at P > 0.97. CONCLUSIONS: Evidence from this investigation suggests that injury to the endothelium occurs most during the second quartile. This may be a promising area in which clinicians could target their efforts to avoid injury to this vital tissue layer for best surgical outcomes and graft longevity.

Morfología y morfometría del endotelio corneal en adultos sin alteraciones corneales según cantidad de células evaluadas / Morphology and morphometry of the corneal endothelium in adults without corneal alterations according to the number of evaluated cells

Objetivo: describir las características morfológicas y morfométricas del endotelio corneal en adultos sin alteraciones corneales según cantidad de células evaluadas atendidos en el Servicio de Cirugía Refractiva del Instituto Cubano de Oftalmología Ramón Pando Ferrer en el período comprendido de enero a febrero del año 2016. Métodos: se realizó una investigación descriptiva de corte transversal de serie de casos atendidos en el Servicio de Cirugía Refractiva. Después de aplicar los criterios de exclusión, la muestra quedó conformada por 90 ojos de 45 pacientes adultos sanos. Se realizó microscopia endotelial de no contacto SP-3000P, para identificar los valores morfológicos (hexagonalidad y coeficiente de variabilidad) y morfométricos (densidad celular y paquimetría), así como el promedio del tamaño celular corneal según cantidad de células evaluadas. Resultados: según la cantidad de células endoteliales evaluadas no hubo diferencias en cuanto a la paquimetría en ambos ojos (p> 0,05). En relación con la densidad no existieron diferencias significativas entre la cantidad de células evaluadas en ambos ojos (p> 0,05). Los valores de hexagonalidad, coeficiente de variabilidad y tamaño celular promedio no mostraron diferencias significativas (p> 0,05) en ambos ojos según la cantidad de células evaluadas. Conclusiones: el estudio del endotelio corneal mediante la utilización del microscopio especular constituye un método efectivo que permite conocer las características de las células endoteliales sin existir variaciones según la cantidad de células evaluadas(AU)

Objective: to describe the morphological and morphometric characteristics of the corneal endothelium in adults without corneal alterations according to the number of evaluated cells, who had been seen at the refractive survey service of Ramón Pando Ferrer Cuban Institute of Ophthalmology from January to February, 2016. Methods: descriptive, cross-sectional case-series research study of patients seen at the refractive surgery service. After applying the exclusion criteria, the sample was finally made up of 90 eyes from 45 healthy adult patients. SP-3000P non-contact endothelial microscopy was performed to determine the morphological (hexagonality and variability coefficient) and morphometric (cell density and pachymetry) values and the average of the average cellular corneal size according to the number of evaluated cells. Results: according to the number of evaluated endothelial cells, there were no differences as for the pachymetry in both eyes (p> 0.05). Regarding the cell density, there were no significant differences among the number of evaluated cells in both eyes (p> 0.05). The hexagonality, variability coefficient and average cell size did not show significant differences (p> 0.05) in both eyes according to the number of evaluated cells. Conclusions: the study of the corneal endothelium using the specular microscope is an effective method that allows to know the characteristics of the endothelial cells without no previous variations, according to the number of evaluated cells(AU)

Rabbit Model of Corneal Endothelial Injury Established Using the Nd: YAG Laser.

PURPOSE: To develop a new rabbit model of corneal endothelial injury using a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. METHODS: The corneal endothelia of 2 groups of New Zealand white rabbits were treated with an Nd:YAG laser in a uniformly scattered fashion. Rabbits in group A underwent laser burns on the whole corneal endothelium, including the limbus area, whereas rabbits in group B were subjected to laser burns in the central 9-mm diameter zone of the endothelium. Slit-lamp biomicroscopy, optical coherence tomography, applanation tonometry, confocal microscopy, scanning electron microscopy, and histological examinations were performed during 4 weeks of follow-up. RESULTS: In both groups, dotted or focal corneal endothelium defects were directly observed. The stroma was intact. Right after laser application, a series of clinical manifestations appeared, including subepithelial and stromal edema, increased central corneal thickness, and corneal opacity. Laser burn had more notable effects in group A than in group B. In both groups, we observed no damage to the intraocular structures, and intraocular pressure was normal after laser treatment. CONCLUSIONS: Nd:YAG laser treatment in a 9-mm diameter zone of the endothelium can effectively induce bullous keratopathy in a rabbit, whereas treatment for the entire corneal endothelium maintains bullous keratopathy for a longer period. The procedure is simple and reproducible, and it retains normal intraocular structures. This study provided a promising model for future research into endothelial cell damage and for the development of new therapies.

All laser cataract surgery compared to femtosecond laser phacoemulsification surgery: corneal trauma.

The aim of this study was to evaluate corneal tissue trauma after femtosecond laser-assisted cataract surgery (FLACS) and phacoemulsification (femtophaco surgery) compared to FLACS and nanolaser emulsification (all laser surgery). This is a prospective nonrandomized clinical study conducted at the Ophthalmology Clinic, University "G. d'Annunzio" of Chieti-Pescara, Italy, involving forty-two eyes of 42 patients candidates to cataract surgery. Patients were enrolled in two groups: femtophaco surgery (group 1 with 21 eyes) and all laser surgery (group 2 with 21 eyes). Main outcome measures included uncorrected visual acuity and distance corrected visual acuity, corneal endothelial cell count, and corneal thickness at the tunnel site and at the center of the cornea. Best correct visual acuity was not significantly different between the two groups. Postoperatively, a significant decrement of endothelial cell count at the center of the cornea was observed in group 1 compared with preoperative values at 90 days (p < 0.001) while t remained stable in group 2. The central corneal thickness showed a statistically significant increase for both groups that reached a maximum thickness at 7 days and then returned to presurgery levels after 90 days for group 1 and after 60 days for group 2. The tunnel corneal thickness showed a statistically significant increase for both groups that reached a maximum thickness at 7 days, which did not return to presurgery level for group 1 but did return to presurgery levels after 60 days for group 2. All laser surgery induced lower central endothelial cell loss and lower increase of corneal thickness compared to femtophaco surgery.

[A new technique to facilitate donor preparation for DMEK surgery]. / Eine neue Technik zur Optimierung der Spenderpräparation bei der DMEK.

BACKGROUND: Descemet membrane endothelial keratoplasty (DMEK) is becoming more and more the method of choice to treat corneal endothelial diseases in specialized centers. The reasons that prevent this technique from becoming widespread are the delicate donor tissue preparation. By inverting the curvature of the cornea from convex to concave after mounting onto an artificial anterior chamber, we developed a combined manual delamination and hydrodissection technique, which allows a rapid and endothelium-preserving method of separating donor Descemet membranes from the underlying stroma. MATERIAL AND METHODS: Experiments were perfomed with 60 donor corneas that were not suitable for transplantation. Donor age ranged between 42 and 94 years. Two experimental groups were formed: 1 inverse manual delamination (n = 16) and 2 combined manual delamination and hydrodissection (n = 44). All experiments were undertaken by an experienced surgeon who was, however, not experienced with these techniques. We examined the frequency of Descemet membrane rupture as well as the amount of induced endothelial damage (trypan blue staining with quantitative image analysis). RESULTS: Significant lesions of Descemet's membrane that would have led to a loss of the graft occurred in 25% of the manual delamination cases and in 4.5% using the combined technique. Endothelial damage induced by both techniques was low (6 and 5.2%, respectively). CONCLUSION: For DMEK donor preparation, a combination of manual delamination and hydrodissection was shown to be a safe and endothelium-protective technique to separate Descemet membranes from the underlying stroma. A very rapid learning curve for the combination technique is of specific additional interest for beginners in DMEK surgery.