Evaluation of the effects of ethanol and mitomycin on survival of rat limbal stem cells: an in vitro study.

PURPOSE: Ethanol and mitomycin C (MMC) are clinically used to treat corneal diseases such as LASEK and LASIK surgery. In this study, we investigated the effects of time-dependent alcohol and MMC in cultured rat limbal stem cells (LSCs) to determine the appropriate time for the use of this compound in the clinical setting. METHODS: LSCs (N = 10 eyes) isolated from male Wistar rats were cultured and characterized; then, isolates were divided into three groups. One group was exposed to a 20% concentration of ethanol for 5, 10, 15, 20, 25, and 30 s, and cell viability was assessed one, three, and five days following ethanol exposure using an MTT assay. To investigate the effect of MMC, cells in the second group were treated with 0.02% MMC in various periods (i.e., 15 s, 30 s, 60 s, 90 s, and 120 s) and time-dependent responses of cultured LSCs were recorded. Cells in the third group were co-treated with ethanol and MMC; then, dose and time dependency was evaluated. RESULTS: In comparison with the viable cells in the control group, ethanol markedly decreased the viability of cells in a time-dependent manner in days one and three. On day five, the viability of LSCs was improved significantly (p < 0.05) in comparison with day one. The number of viable progenitor cells was significantly decreased after MMC treatment in a time-dependent manner, as determined by the MTT assay (p < 0.001). The use of mitomycin, along with alcohol, decreased cell viability in all groups treated with ethanol + MMC compared to the control on days one, three, and five (p < 0.0001). CONCLUSIONS: Our findings suggest that ethanol and MMC reduced cell viability in cultured LSCs in a time-dependent manner. In addition, when LSCs were exposed to alcohol alone, they had a better recovery process within 5 days in comparison to when exposed to mitomycin alone or mitomycin + alcohol.

Evaluation of effects of riboflavin and/or ultraviolet-A on survival of rat limbal epithelial stem cells in ex-vivo.

Purpose: To investigate the effects of riboflavin and/or ultraviolet-A (UV-A) irradiation on the cell viability of ex-vivo-cultured rat limbal stem cells (LSCs). Methods: LSCs of male Wistar rats (N = 12 eyes) were cultured, and immunofluorescence staining was performed to evaluate them. After characterization, these cells were assigned to four groups of control (C), a group that was exposed to UV-A radiation (UV), a group that was treated with riboflavin (R), and a group that cotreated with both UV-A and riboflavin (UV+R). To determine the cell viability of LSCs, these cells were subjected to MTT assay on days 1, 3, and 7 after exposure to UV-A and/or riboflavin. The duration of exposure to UV-A and riboflavin was similar to levels used during the conventional corneal collagen cross-linking procedure. Results: Compared with the viable cells in the control group, there was a significant decrease (P < 0.0001) in the number of LSCs in the UV group during all study days. In the R group, the level of viable LSCs was as same as the level of viable LSCs in the C group. Combined treatment with UV-A plus riboflavin significantly decreased the survival of LSCs on days 1 and 3 (P < 0.0001, P < 0.001, respectively) compared with the control group. Interestingly, in the UV+R group, the photosensitizing effect of riboflavin significantly decreased the cytotoxic effect of UV irradiation 7 days after exposure. Conclusion: These results suggest that the administered UV energy in the presence or absence of riboflavin can damage LSCs. Likewise, riboflavin could decrease the toxic effect of UVA on LSCs.

Corneal Collagen Cross-linking for Treatment of Non-healing Corneal Ulcers.

PURPOSE: To evaluate the efficacy of corneal collagen cross-linking (CXL) for treatment of corneal ulcers not responding to antimicrobial therapy. METHODS: Eight patients with corneal ulcers associated with corneal melting, not responding to conventional antibiotic therapy, were treated with CXL. The procedure was performed according to the standardized protocol for keratoconus. Preoperative medications were continued after CXL in all cases. Microbiological exams revealed Pseudomonas aeruginosa in 3 cases. Follow up continued from 1 to 10 months. RESULTS: In 6 of 8 eyes, progression of corneal melting was halted and complete epithelialization occurred. In one eye emergency keratoplasty was needed due to corneal perforation. A conjunctival flap was performed to treat severe localized corneal thinning in one of the patients a few days after CXL. Significant clinical improvement occurred in all cases of Pseudomonas aeruginosa keratitis. CONCLUSION: CXL can be considered as a promising new treatment in the management of refractory non-healing corneal ulcers, including Pseudomonas aeruginosa keratitis.

Oxygen Tension in the Aqueous Humor of Human Eyes under Different Oxygenation Conditions.

PURPOSE: To measure oxygen tension in the aqueous humor of human eyes under different oxygenation conditions. METHODS: This prospective comparative interventional case series consisted of two parts. In the first part, 120 consecutive patients scheduled for cataract surgery were randomized into group I (control group) in which surgery was performed under local anesthesia inhaling 21% oxygen; group II in whom general anesthesia using 50% oxygen was employed; and group III receiving general anesthesia with 100% oxygen. After aspirating 0.2 ml aqueous humor under sterile conditions, the aqueous sample and a simultaneously drawn arterial blood sample were immediately analyzed using a blood gas analyzer. In part II the same procedures were performed in 10 patients after fitting a contact lens and patching the eye for 20 minutes (group IV) and in 10 patients after transcorneal delivery of oxygen at a flow rate of 5 L/min (group V). RESULTS: Mean aqueous PO2 in groups I, II and III was 112.3±6.2, 141.1±20.4, and 170.1±27 mmHg, respectively (P values <0.001) and mean arterial PO2 was 85.7±7.9, 184.6±46, and379.1±75.9 mmHg, respectively (P values <0.001). Aqueous PO2 was 77.2±9.2 mmHg in group IV and 152.3±10.9 mmHg in group V (P values <0.001). There was a significant correlation between aqueous and blood PO2 (r=0.537, P<0.001). The contribution of atmospheric oxygen to aqueous PO2 was 23.7%. CONCLUSION: Aqueous oxygen tension is mostly dependent on the systemic circulation and in part on the atmosphere. Increasing inspiratory oxygen and transcorneal oxygen delivery both increase aqueous PO2 levels.

Early changes in intraocular pressure following phacoemulsification.

PURPOSE: To evaluate early postoperative changes in intraocular pressure (IOP) following phacoemulsification and intraocular lens (IOL) implantation. METHODS: This prospective study included 129 eyes with open angles and normal or high IOP undergoing phacoemulsification and IOL implantation for senile cataracts. The patients were divided into 3 groups (Gs) based on preoperative IOP: ≤15 mmHg (G1, n=76); from 16 to 20 mmHg (G2, n=43) and; from 21 to 30 mmHg (G3, n=10). IOP was measured by Goldmann applanation tonometry one day before surgery, and 1 and 6 weeks postoperatively. RESULTS: IOP was decreased postoperatively in all study groups 1 and 6 weeks after surgery as follows: 2.8±1.5 and 1.8±1.7 mmHg respectively in G1 (P<0.001); 4.2±1.9 and 4.3±2.9 mmHg respectively in G2 (P<0.001), and 8.3±4.3 and 9.3±4.1 mmHg respectively in G3 (P<0.001). At the end of the sixth postoperative week, the percentage of IOP change for G1, G2 and G3 was 13.5%±12.7, 24.5%±11.7 and 38.3%±16.2, respectively. CONCLUSION: IOP significantly decreased after phacoemulsification and IOL implantation in normal subjects with open angles and those with ocular hypertension. IOP reduction was greater in eyes with higher preoperative IOP.

Oxygen therapy for acute ocular chemical or thermal burns: a pilot study.

PURPOSE: To evaluate the effect of systemic oxygen therapy in the management of acute ocular chemical and thermal burns. DESIGN: Prospective, nonrandomized, comparative, interventional case series. METHODS: Twenty-four eyes of 22 patients with grade III to IV acute ocular chemical and thermal burns received conventional medical therapy. The oxygen therapy group (13 eyes) additionally received 100% oxygen using a simple mask at a flow rate of 10 L/minute for 1 hour twice daily. Main outcome measures were time for healing of the corneal epithelial defect and improvement in perilimbal ischemia. Secondary outcome measures included visual acuity, corneal transparency and vascularization, and complications. RESULTS: Corneal epithelial defects healed within 15.23 ± 3.94 days (range, 10 to 21 days) in the oxygen group versus 59.9 ± 23.33 days (range, 28 to 95 days) in controls (P < .001). Vascularization of ischemic areas was complete in 14.54 ± 2.70 days (range, 10 to 21 days) in the oxygen group versus 45.09 ± 22.20 days (range, 25 to 105 days) in controls (P = .001). In the oxygen group, the cornea was more transparent and less vascularized 3 and 6 months after injury. Mean final visual acuity (logarithm of the minimal angle of resolution) was 0.40 ± 0.52 (range, 0 to 1.3) versus 1.11 ± 0.83 (range, 0.1 to 3) in the oxygen and control groups, respectively (P = .018). In the oxygen group, symblepharon or corneoscleral melting did not develop in any patient; however, in the control group, symblepharon developed in 3 eyes and corneoscleral melting developed in 1 patient. CONCLUSIONS: In the acute phase of ocular chemical or thermal burns, oxygen therapy improves limbal ischemia, accelerates epithelialization, increases corneal transparency, and decreases corneal vascularization. It also may improve visual acuity and reduce complications.

The effect of mitomycin-C on corneal endothelial cells after photorefractive keratectomy.

PURPOSE: To evaluate short-term changes in central corneal endothelial cell density and morphology after photorefractive keratectomy (PRK) with mitomycin-C (MMC) 0.02% in patients with moderate myopia. METHODS: In this prospective interventional case series, patients with moderate myopia (spherical equivalent refractive error from -4.0 to -8.0 D) underwent PRK with a single intraoperative application of MMC 0.02% for 40 seconds. Specular microscopy was performed preoperatively and repeated 6 months after surgery to determine changes in central corneal endothelial cell density (ECD), mean cell area (MCA) and coefficient of variation in cell size (CV). RESULTS: Overall, 42 eyes of 21 participants with mean age of 26.2±6.3 years underwent surgery. Mean preoperative spherical equivalent refractive error was -5.2±1.2 D which was reduced to -0.4±0.5 D postoperatively (P < 0.001). Mean ECD was reduced insignificantly from 2,920±363 cells/mm(2) preoperatively to 2,802±339 cells/mm(2) postoperatively (P = 0.59). Similarly, there was no significant change in MCA or CV at six months (P = 0.76 and 0.52, respectively). CONCLUSION: Application of MMC 0.02% for 40 seconds during PRK in patients with moderate myopia did not significantly affect central corneal endothelial cell density and morphology after a 6 month follow up period.

Hydrophilic Acrylic versus PMMA Intraocular Lens Implantation in Pediatric Cataract Surgery.

PURPOSE: To compare primary implantation of foldable hydrophilic acrylic with polymethylmethacrylate (PMMA) intraocular lenses (IOLs) in pediatric cataract surgery in terms of short-term complications and visual outcomes. METHODS: This randomized clinical trial included 40 eyes of 31 consecutive pediatric patients aged 1 to 6 years with unilateral or bilateral congenital cataracts undergoing cataract surgery with primary IOL implantation. Two types of IOLs including foldable hydrophilic acrylic and rigid PMMA were randomly implanted in the capsular bag during surgery. Primary posterior capsulotomy and anterior vitrectomy were performed in all eyes. Patients were followed for at least 1 year. Intra- and postoperative complications, visual outcomes and refractive errors were compared between the study groups. RESULTS: Mean age was 3.2±1.8 years in the hydrophilic acrylic group and 3.7±1.3 years in the PMMA group. Mean follow-up period was 19.6±5 (12-29) months. No intraoperative complication occurred in any group. Postoperative uveitis was seen in 2 (10%) eyes in the acrylic group versus 5 (25%) eyes in the PMMA group (P=0.40). Other postoperative complications including pigment deposition (30%), iridocorneal adhesions (10%) and posterior synechiae formation (10%), were seen only in the PMMA group. The visual axis remained completely clear and visual outcomes were generally favorable and comparable in the study groups. CONCLUSION: In pediatric eyes undergoing lensectomy with primary posterior capsulotomy and anterior vitrectomy, hydrophilic acrylic IOLs are comparable to PMMA IOLs in terms of biocompatibility and visual axis clarity, and seem to entail less frequent postoperative complications.

Oxygen therapy for severe corneal alkali burn in rabbits.

PURPOSE: To evaluate the efficacy of oxygen therapy in the treatment of severe corneal alkali injury in rabbits. METHODS: In a double-blind experiment, 28 white New Zealand rabbits were randomized into an oxygen treatment group (n = 14) and a control group (n = 14). Under general anesthesia, severe corneal alkali injuries were induced by application of 1 N sodium hydroxide to the right eye of each rabbit. The oxygen treatment group was treated with oxygen 100% at a flow of 5 L/min for 1 hour daily for 1 month. Daily photographs were taken of the rabbits' eyes, and the sizes of the epithelial defect in the 2 groups were compared. The principal endpoint was descemetocele and perforation of the cornea. The animals were euthanized at the end of the study or earlier if corneal perforation had occurred, and the corneas were excised and fixed in 10% neutral-buffered formalin for histologic examination. RESULTS: Experimentally induced severe eye burns gave similar opacity of the cornea in both groups. Three eyes in the oxygen group and 9 eyes in the control group developed descemetocele and perforation (P = 0.022). Mean time to beginning of ulceration was 13.45 days in the control group and 18.11 days in the oxygen treatment group (P = 0.032). There was no other significant difference between the 2 groups. CONCLUSIONS: Oxygen therapy at a flow of 5 L/min for 1 hour daily reduces the possibility of corneal perforation in rabbits and may delay ulceration of the cornea compared with the control group.

Penetrating keratoplasty in young children with congenital hereditary endothelial dystrophy.

PURPOSE: To report the visual results and success rate of penetrating keratoplasty (PKP) in a series of young children with congenital hereditary endothelial dystrophy (CHED). METHODS: This is a retrospective study on twenty-four eyes of 15 patients (seven male and eight female) operated on for CHED. Children less than 12 years of age at the time of surgery who were followed for at least 6 months were recalled. Characteristics of the patients, indications for PKP, final visual outcome, and graft clarity were evaluated. The following tests were employed: McNemmar test for evaluating visual results, Kaplan-Meyer analysis for determination of graft survival, and Mann-Whitney U test for evaluating the relationship between visual outcome and age at PKP. RESULTS: Patients' age at diagnosis and at initial PKP was 6.5 +/- 3.6 and 8.1 +/- 2.5 years, respectively. Follow-up period was 35.5 +/- 36.2 months. Visual acuity could be evaluated by Snellen chart in 19 eyes. Preoperative visual acuity was less than 20/80 in all of these. Postoperatively, visual acuity was less than 20/80 in nine eyes (47.4%) (P < 0.002). Visual acuity improved in 18 (94.7%) of 19 eyes. There was no relationship between age at initial PKP and final visual outcome (P = 0.35). At the last examination (24 grafts), 19 were clear (79.1%), two were hazy (8.3%), and three were opaque (12.5%). Allograft rejection was seen in 10 eyes (43.4%), seven of which were endothelial. Excluding one case of trauma, all graft failures resulted from endothelial rejection. The probability of primary graft survival was 88% at 3 years and 74% at 5 years. CONCLUSION: Regarding the difficulties in pediatric keratoplasty and the absence of a relationship between postoperative visual outcome and age at keratoplasty, a conservative approach and careful risk-benefit ratio evaluation are recommended in patients with CHED.