Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions.

PURPOSE: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers. MATERIALS AND METHODS: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence. RESULTS: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished. CONCLUSION: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.

Clinical study of pain sensation during phacoemulsification with and without cryoanalgesia.

PURPOSE: To compare the analgesic efficacy of 2 types of anesthetic techniques-topical and topical associated with cryoanalgesia-during cataract surgery. SETTING: Department of Ophthalmology, University of São Paulo, Ribeirão Preto, Brazil. DESIGN: Prospective randomized study. METHODS: Patients with symmetrical bilateral cataract had phacoemulsification with the use of an intraocular irrigation solution at room temperature in 1 eye and a cold (4°C) solution in the contralateral eye (Group 2). The pain related to the procedure was assessed using a visual analog scale ranging from 0 to 100, with 0 representing no pain and 100 the worst pain. In addition, endothelial cell loss, the change in corneal thickness, and visual acuity were evaluated 30 days ± 2 (SD) after each surgery. The surgery duration, total irrigation volume, phacoemulsification time, and ultrasound power used were analyzed. RESULTS: The study enrolled 25 patients (50 eyes). There was no statistically significant difference in the mean pain score between Group 1 (26.0 ± 3.7) and Group 2 (21.3 ± 3.6) (P = .2016, paired t test). No significant difference was found in the mean corneal endothelial cell loss (Group 1: 10.0% ± 0.4%; Group 2: 9.9% ± 0.3%; P = .7576), corneal thickness increase (Group 1: 1.5 ± 1.0 µm; Group 2: 1.4 ± 0.9 µm; P = .9340), or visual acuity gain (Group 1: 0.54 ± 0.06 logMAR; Group 2: 0.55 ± 0.09 logMAR; P = .8208). CONCLUSION: There is no difference in the intensity of pain during phacoemulsification with the use of topical anesthesia or topical anesthesia associated with cryoanalgesia. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Corneal endothelial cell loss and corneal thickness in conventional compared with femtosecond laser-assisted cataract surgery: three-month follow-up.

PURPOSE: To quantify changes in endothelial cell counts and corneal thickness measurements in patients having standard phacoemulsification compared with femtosecond laser-assisted cataract removal. SETTING: Ruhr University Eye Clinic, Bochum, Germany. DESIGN: Prospective randomized intraindividual cohort study. METHODS: One eye of each patient had standard phacoemulsification (control group) and the other eye had femtosecond laser-assisted phacoemulsification (study group), both with intraocular lens implantation. Pulsed ultrasound energy was used for phacoemulsification. Noncontact endothelial cell microscopy and corneal pachymetry were performed preoperatively and 1 day, 3 to 4 days, 7 to 10 days, 50 to 60 days, and 90 to 100 days postoperatively. RESULTS: The mean endothelial cell loss was 7.9% ± 7.8% (SD) 1 week postoperatively and 8.1% ± 8.1% 3 months postoperatively in the study group and 12.1% ± 7.3% and 13.7% ± 8.4%, respectively, in the control group. The mean relative change in corneal thickness from the preoperative values was -0.0% ± 1.9% at 1 day, 2.8% ± 1.8% at 1 week, and 3.3% ± 1.7% at 3 months in the study group and -0.9% ± 2.3%, 2.4% ± 1.5%, and 3.2% ± 1.4%, respectively, in the control group. CONCLUSION: The femtosecond laser did not add to the endothelial damage caused by cataract surgery and might be beneficial in eyes with low preoperative endothelial cell values (eg, cornea guttata cases).

Protective effects of dispersive viscoelastics on corneal endothelial damage in a toxic anterior segment syndrome animal model.

PURPOSE: We evaluated whether viscoelastics have protective effects on the corneal endothelial cell damage in a toxic anterior segment syndrome (TASS) animal model depending on the types of viscoelastics. METHODS: A TASS animal model was established with an injection of 0.1 mL o-phthaldehyde solution (0.14%) into the anterior chamber of New Zealand white rabbits. One of two different viscoelastics, 1% sodium hyaluronate (cohesive group) or a 1:3 mixture of 4% chondroitin sulfate and 3% sodium hyaluronate (dispersive group), was injected into the anterior chamber. After five minutes, it was removed using a manual I/A instrument, and then 0.1 mL of o-phthaldehyde solution (0.14%) was injected into the anterior chamber. Damage to corneal endothelial cells was compared between the two groups. RESULTS: The corneal thickness increased quickly in both groups after the disinfectant injection. However, the dispersive group showed relatively mild corneal edema compared to the cohesive group. The mean corneal haze score in the dispersive group also was lower than that of the cohesive group. These partial protective effects of the dispersive viscoelastic were demonstrated by the different findings of a live/dead cell assay, TUNEL staining, and scanning electron microscopy between the two groups. CONCLUSIONS: The TASS animal model seems to be a useful means to evaluate corneal endothelial cell damage caused by toxic substances to find ways to protect or reduce endothelial cell damage. Dispersive viscoelastics were shown to have partial protective effects against corneal endothelial cell damage caused by a toxic disinfectant.

Protective effect of different ophthalmic viscosurgical devices on corneal endothelial cells during phacoemulsification: rabbit model.

PURPOSE: To evaluate the protective effect of different ophthalmic viscosurgical devices on corneal endothelial cells during phacoemulsification in a rabbit model. SETTING: Harlan Biotech Israel and Ophthalmology Department, Kaplan Medical Center, Rehovot, Israel. DESIGN: Experimental study. METHODS: Rabbit eyes were randomly assigned to 3 equally sized groups. Endothelial cell counts were performed in all eyes before initiation of the study. The aqueous humor was completely replaced by Biolon (sodium hyaluronate 1.0%) in Group A, by a combination of Viscoat (sodium chondroitin sulfate 4.0%-sodium hyaluronate 3.0%) and Provisc (sodium chondroitin sulfate 1.0%) using the soft-shell technique in Group B, and by a combination of Visiol (sodium hyaluronate 2.0%-mannitol 0.5%) and Biolon using the soft-shell technique in Group C. The eyes were exposed to alternating 10 seconds of phacoemulsification and a 10-second pause until a total exposure time of 2.5 minutes elapsed. Endothelial cell counts were repeated 3 days after surgery. RESULTS: The study used 18 rabbit eyes, 6 in each group. Group A had the highest endothelial cell loss (13%) followed by Group B (7%), and Group C (4%). The difference in cell loss between Group C and Group A was statistically significant (P = .037). CONCLUSION: The study showed the efficiency and advantages of the soft-shell technique using the combination of Visiol and Biolon over Biolon alone.

Protection of thymosin beta-4 on corneal endothelial cells from UVB-induced apoptosis.

Cornea absorbs most of daily ultraviolet (UV) light. An excess of UV damages results in not only keratopathy and cataract but also maculopathy. It has been reported that thymosin beta-4 (Tbeta4) promotes wound healing, decreases inflammatory response and prevents apoptosis of corneal epithelial cells. However, it is not clear whether Tbeta4 protects UVB-induced corneal injury, particularly in corneal endothelial cells because of its non-proliferation in nature. The purpose of this study is to compare the protective effects of Tbeta4 on bovine corneal endothelial (BCE) cells from low- and high-dose UVB damage. In this study, 1 microg/ml of Tbeta4 was added to BCE cells 2 h before low (12.5 mj/cm2) or high dosage (100 mj/cm2) UVB exposure. Using a fluorogenic substrate cleavage assay, we found that Tbeta4 diminished the reactive oxygen species level in BCE cells elicited by UVB. However, the protection of viability by Tbeta4 could only be detected under low-dose UVB exposure. Moreover, both caspase-9 activity and annexin V/propidium iodine staining demonstrated that Tbeta4 only protected BCE cells from low-dose UVB-induced apoptosis but not high-dose UVB-induced necrosis. Together, Tbeta4 protected corneal endothelial cells from UVB-induced oxidative stress and apoptosis after low-dose UVB exposure. The results support further investigation towards topical use or anterior chamber injection of this small hydrophilic peptide in treating and preventing UVB-induced corneal endothelial damage.