Ascorbic acid ameliorates corneal endothelial dysfunction and enhances cell proliferation via the noncanonical GLUT1-ERK axis.

BACKGROUND: The pumping function of corneal endothelial cells (CECs) plays a pivotal role in the maintenance of corneal water homeostasis. Corneal endothelial dysfunction (CED) leads to corneal edema and opacity, but with the exception of keratoplasty, no optimal therapeutic strategies have been established for CED. In this study, we aimed to investigate the ameliorative effect of ascorbic acid (AA) on CED and the underlying mechanism of action in the corneal endothelium. METHODS: Rabbit corneal endothelial damage was induced by anterior chamber injection of benzalkonium chloride (BAK). AA was topically administered to the corneal surface, and the transparency and thickness of the cornea were assessed by external eye photography, slit-lamp photography, and ultrasonic pachymetry. To further analyze the mechanism, rabbit CECs and immortalized human CECs (B4G12 cells) were cultured. A ferric reducing/antioxidant and AA (FRASC) assay was performed to measure the AA concentration. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (BrdU) labeling assays, and protein expression was examined by liquid chromatography-mass spectrometry (LC/MS) and immunoblotting. The involvement of glucose transporter 1 (GLUT1) and phospho-ERK was evaluated via GLUT1-siRNA and phospho-ERK inhibitor (PD98059) treatment. INTERPRETATION: We observed that topical AA ameliorates BAK-induced rabbit corneal endothelial damage. Furthermore, we demonstrated that AA is transported into B4G12 cells via GLUT1, and afterward, AA increases ERK phosphorylation and promotes cell proliferation. Our findings indicate that CEC proliferation stimulated via the noncanonical AA-GLUT1-ERK axis contributes to AA-enhanced healing of CED.

Evaluation of moxifloxacin-induced cytotoxicity on human corneal endothelial cells.

Moxifloxacin hydrochloride (MXF) is widely used for the prevention of bacterial endophthalmitis after intraocular surgeries. However, the safety issue of intracameral injection of MXF for human corneal endothelial cells (HCECs) is still debatable. In this study, we investigated concentration-dependent cytotoxicity (0.05-1 mg/ml) of MXF for immortalized HCECs (B4G12 cell) and the underlying mechanism. Reactive oxygen generation (ROS) and cell viability after MXF exposure was measured. Flow cytometric analysis and TUNEL assay was used to detect apoptotic HCECs after MXF exposure. Ultrastructure of damaged HCECs by MXF was imaged by transmission electron microscope. Western blot analysis and caspase 2, 3 and 8 analysis were used to reveal the underlying mechanism of MXF induced damage in HCECs. We found that MXF induced dose-dependent cytotoxicity in HCECs. MXF exposure increased ROS generation and induced autophagy in HCECs. Increased LDH release represented the cellular membrane damage by MXF. In addition, caspases activation, Bax/Bcl-xL-dependent apoptosis pathway and apoptosis inducing factor nuclear translocation were all involved in MXF induced HCECs' damage, especially after exposure to high dose of MXF (0.5 and 1.0 mg/ml). These findings suggest that MXF toxicity on HCECs should be thoroughly considered by ophthalmologists when intracameral injection of MXF is planned.

[Effects of Benzalkonium Chloride in Ophthalmic Eyedrop Medications on Corneal Epithelium].

Eyedrops often contain additives other than active pharmaceutical ingredients, such as preservatives. The most frequently used preservative is benzalkonium chloride (BAC). When the ocular surface is exposed to eyedrops, the active pharmaceutical ingredients and additives can cause corneal epithelial disorder. Particularly in clinical settings, there is great interest in corneal epithelial disorders resulting from the use of glaucoma eyedrops, which is inevitable when instilled for a long period of time after the onset of disease. At the authors' institute, glaucoma is treated with consideration of reducing corneal epithelial disorder while ensuring the effect of lowering intraocular pressure by the appropriate choice of eyedrops. In this review, we show the examples of the retrospective studies. Sodium hyaluronate eyedrops are prescribed for corneal epithelial disorders such as superficial punctate keratitis associated with dry eye. Prescribable concentrations of sodium hyaluronate in Japan are 0.1% or 0.3%, and the 0.3% formulation does not contain BAC. The authors' study showed that 0.3% sodium hyaluronate pretreatment reduced the cytotoxicity of BAC in cultured corneal epithelial cells, whereas an in vivo study in mice showed that a 0.3% sodium hyaluronate instillation was suggested and that the drug may enhance the cytotoxicity of separately administered BAC. It is suggested that sodium hyaluronate prolonged the retention of BAC on the ocular surface. However, there have been no reports of this problem in the clinical setting. It is important for ophthalmologists to understand the properties of additives other than the active pharmaceutical ingredients in eyedrops.

Cannabis and the Cornea.

Purpose: While cannabis has the potential to reduce corneal pain, cannabinoids might induce side effects. This review article examines the effects of cannabinoids on the cornea. As more states and countries consider the legalization of adult cannabis use, health-care providers will need to identify ocular effects of cannabis consumption.Methods: Studies included in this review examined the connection between cannabis and the cornea, more specifically anti-nociceptive and anti-inflammatory actions of cannabinoids. NCBI Databases from 1781 up to December 2019 were consulted.Results: Five studies examined corneal dysfunctions caused by cannabis consumption (opacification, decreased endothelial cell density). Twelve studies observed a reduction in corneal pain and inflammation (less lymphocytes, decreased corneal neovascularization, increased cell proliferation and migration).Conclusion: More than half of the studies examined the therapeutic effects of cannabinoids on the cornea. As the field is still young, more studies should be conducted to develop safe cannabinoid treatments for corneal diseases.

Effect of Intracameral Cefuroxime on Graft Endothelial Cells in Descemet Membrane Endothelial Keratoplasty: A Comparative Study.

Purpose: To evaluate the effect of intracameral cefuroxime on graft endothelial cell loss after simple Descemet Membrane Endothelial Keratoplasty (DMEK) and combined DMEK and cataract surgery.Materials and Methods: Single-center retrospective comparative analysis. One hundred and three patients were included, 31 in the cefuroxime group and 72 in the non-cefuroxime (NC) group. Best Spectacle-Corrected Visual Acuity (BSCVA), endothelial cell density (ECD) of the graft measured by specular microscopy, and the recipient's pachymetry were recorded pre-operatively and at 1, 3, and 6 months after surgery.Results: In the cefuroxime group, BSCVA was 0.22 ± 0.27 LogMAR, 0.15 ± 0.24 LogMAR and 0.07 ± 0.22, respectively, at 1, 3, and 6 months after surgery with no significant differences found when compared to the NC group (p > .05). Anatomical outcomes were similar as mean pachymetry decreased from 599 ± 51 µm preoperatively to 511 ± 30 µm at 6 months after surgery in the cefuroxime group and from 607 ± 67 µm preoperatively to 519 ± 32 µm at 6 months in the NC group (p = .25). Endothelial cell loss was comparable between both groups: 33.4% versus 33.6% at 1 month (p = .97), 37.4% versus 34.9% at 3 months (p = .68) and 41.6% versus 38.3% at 6 months (p = .42) in the cefuroxime and NC groups, respectively. The rates of rebubbling, graft rejection, and cystoid macular edema were not significantly higher in the cefuroxime group.Conclusion: The use of intracameral cefuroxime during simple or combined DMEK did not lead to higher graft endothelial cell loss.

Comparative corneal endothelial cell toxicity of differing intracameral moxifloxacin doses after phacoemulsification.

PURPOSE: To determine whether intracameral moxifloxacin 500 µg is noninferior to 250 µg for central endothelial cell loss (ECL) after phacoemulsification. SETTING: Aravind Eye Care System. DESIGN: Prospective masked randomized study. METHODS: Eyes with bilateral nuclear cataracts, central endothelial cell density (ECD) of more than 2000 cells/mm, and ECD not differing between eyes by more than 200 cells/mm underwent phacoemulsification at least 14 days apart. Intraoperatively, the first eye was randomized to receive either a 500 or 250 µg dose of moxifloxacin intracamerally and received the other dose for the second-eye surgery. Postoperative course was monitored at 1 day, 1 week, 1 month, and 3 months. Preoperative and 30-day and 90-day postoperative central ECD was determined by a reading center for a masked analysis of ECL at 3 months postoperatively. RESULTS: Fifty eyes of 25 patients (aged 48 to 69 years) underwent uneventful surgery and had normal postoperative courses. The point estimate (PE) and 95% CI for the mean difference in % ECL between the 500 µg and 250 µg doses at 3 months postoperatively was 0.8% (-5.8%, 7.4%). Upon identifying and removing 2 outliers, noninferiority was proven with a mean difference of the PE, -2.2% (CI, -6.5%, 2.1%). CONCLUSIONS: Clinical and corneal endothelial cell were comparable in this study population for the 250 µg and 500 µg doses of intracameral moxifloxacin. Both doses were well tolerated clinically, supporting the use of the higher dose for improved antimicrobial coverage for the prevention of postoperative endophthalmitis.

Comparison of Mean Corneal Endothelial Cell Loss after Trabeculectomy with and without Mitomycin C.

OBJECTIVE: To compare mean endothelial cell loss in patients of primary open angle glaucoma undergoing trabeculectomy with and without mitomycin C. STUDY DESIGN: Randomised control study. PLACE AND DURATION OF STUDY: Institute of Ophthalmology, Mayo Hospital, Lahore, from May 2016 to April 2017. METHODOLOGY: Patients with primary open angle glaucoma, not controlled with medication, were selected from the outpatient department. Patients with secondary glaucomas and concomitant ocular disease were excluded. Selected patients were divided into two groups of 30 each. Group A patients underwent trabeculectomy with adjunctive mitomycin C while group B patients underwent trabeculectomy alone. Pre- and post-trabeculectomy endothelial cell counts were recorded with the help of specular microsope and entered on proforma. RESULTS: Median cell loss was 283 (66.50) when trabeculectomy was done with the use of adjunctive mitomycin C in group A while median endothelial cell loss was 72.50 (19.25) when the trabeculectomy was done without the use of adjunctive mitomycin C in group B. CONCLUSION: Use of mitomycin C causes more endothelial cell loss during trabeculectomy as compared to when done without it.

Safety of Nonporous Silica Nanoparticles in Human Corneal Endothelial Cells.

Nonporous silica nanoparticles (SiNPs) are promising drug carrier platforms for intraocular drug delivery. In this study, we investigated the safety of three different sizes of SiNPs (50, 100, and 150 nm) in a human corneal endothelial cell (HCEC) line, B4G12. The HCECs were exposed to different concentrations (0, 25, 50, and 100 µg/ml) of three sizes of SiNPs for up to 48 h. Cellular viability, autophagy, lactate dehydrogenase (LDH) assay, and mammalian target of rapamycin (mTOR) pathway activation were evaluated. Intracellular distribution of the SiNPs was evaluated with transmission electron microscopy (TEM). TEM revealed that the SiNPs were up-taken by the HCECs inside cytoplasmic vacuoles. No mitochondrial structural damage was observed. Both cellular viability and LDH level remained unchanged with up to 100 µg/mL of SiNP treatment. Autophagy showed a significant dose-dependent activation with 50, 100, and 150 nm SiNPs. However, the mTOR activation remained unchanged. Human corneal tissue culture with 100 µg/ml concentrations of SiNPs for 72 h revealed no significant endothelial toxicity. In vivo corneal safety of the SiNPs (0.05 ml intracameral injection, 200 mg/ml concentration) was also verified in rabbit models. These findings suggested that 50, 100, and 150 nm SiNPs did not induce acute significant cytotoxicity in corneal endothelial cells at concentrations up to 100 µg/mL. However, long-term toxicity of SiNPs remains unknown.

[Influence of 20 % sulfur hexafluoride (SF6) on human corneal endothelial cells : An in vitro study]. / Einfluss von 20 % Schwefelhexafluorid (SF6) auf humane korneale Endothelzellen : Eine In-vitro-Studie.

BACKGROUND: In posterior lamellar keratoplasties, such as Descemet membrane endothelial keratoplasty (DMEK) and Descemet's stripping automated endothelial keratoplasty (DSAEK) an air bubble is left inside the anterior chamber to promote graft attachment during the early postoperative period. In the case of insufficient graft adhesion a renewed intracameral air injection is often necessary. The use of sulfur hexafluoride diluted with air (SF6 20 %) as an alternative to pure air may further enhance graft attachment and reduce the rebubbling rate. The effect of SF6 20 % on corneal endothelium is currently unclear and was therefore examined in vitro. MATERIAL AND METHODS: For this study 12 human corneoscleral discs were mounted in artificial anterior chambers, the systems were continuously filled with culture medium and the anterior chambers with air (n = 5) or SF6 20 % (n = 7) as tamponade. After 6 days of storage in the incubator endothelial cell density, toxicity on endothelial cells and corneal thickness were evaluated. RESULTS: There were no significant differences in endothelial cell loss (p = 1.000), endothelial cell count (p = 0.648), toxicity on endothelial cells (p = 0.048) and central corneal thickness (p = 0.905) between the two groups after 1 week. The level of significance was defined as p ≤ 0.05 and adjusted to p ≤ 0.0056 according to the Bonferroni correction for multiple testing. CONCLUSION: The use of SF6 20 % as tamponade in the anterior chamber for posterior lamellar keratoplasty can be proposed as a safe alternative to pure air filling related to endothelial cell loss. Increased toxic effects on the corneal endothelium by SF6 20 % were not detected in this study; however, further prospective clinical trials are needed to examine the long-term effects in humans.

Effect of Intravitreal Injection of Methotrexate on Human Corneal Endothelial Cells.

PURPOSE: To evaluate the toxic effect of intravitreal methotrexate on human corneal endothelium. METHODS: In this prospective noncomparative interventional case series, intravitreal injection of 400 µg methotrexate was performed in eyes with persistent diabetic macular edema. Corneal endothelial cell analyses and central corneal thickness measurements were performed before and 1, 3, and 6 months after injections, using noncontact specular microscopy. RESULTS: Twenty one eyes of 18 patients with a mean age of 60.5 ± 6.8 years were evaluated. No statistically significant difference was found in endothelial cell density, coefficient of variation, average cell area, hexagonality, and central corneal thickness measurements before and after injections (P = 0.70, 0.39, 0.43, 0.64, and 0.67, respectively). CONCLUSIONS: In this 6-month follow-up study, intravitreal injection of 400 µg methotrexate had no significant effect on corneal endothelial cell measurements performed by specular microscopy.

[The influence of VEGF inhibitors on corneal endothelium after injection into the anterior chamber in a porcine eye model]. / Einfluss von VEGF-Inhibitoren auf das Hornhautendothel bei Injektion in die Vorderkammer am Modell des Schweineauges.

BACKGROUND: The injection of antiangiogenic agents, such as ranibizumab (Lucentis®) and bevacizumab (Avastin®) into the anterior chamber of the eye represents a suitable alternative for treating neovascular glaucoma by reducing intraocular pressure. OBJECTIVES: As the antiangiogenic substances are in direct contact with the sensitive corneal endothelium, the aim of this study was to show the effects of intracameral injection of ranibizumab and bevacizumab on this cell layer. METHODS: Each injection consisted of 50 µl containing either ranibizumab (0.5 mg/0.05 ml), bevacizumab (1.25 mg/0.05 ml) or triamcinolone containing benzyl alcohol (2 mg/0.05 ml) which was used as the control group. These compounds were injected into the anterior chamber of pig eyes. Afterwards the corneas were dissected, fixed, examined by a scanning electron microscopy and evaluated according to a specified score. Assessment of the endothelium was carried out by evaluating the condition of microvilli, cell borders, cell surface and cell pattern. The findings were compared to untreated corneas and those injected with 50 µl of balanced salt solution (BSS). RESULTS: The corneal endothelium exposed to the antiangiogenic substances showed only minor changes in comparison to the controls treated only with BSS. Also seen during this research was the irreversible cell damage in the control group using triamcinolone. CONCLUSION: Ranibizumab and bevacizumab have no damaging effects on the corneal endothelium when used in the anterior chamber. They can be administered as an intracameral injection for the treatment of rubeotic secondary glaucoma. Triamcinolon containing benzyl alcohol causes severe damage to the endothelial cells of the cornea by direct contact.

Corneal endothelial cell changes after cataract surgery in patients on systemic sympathetic α-1a antagonist medication (tamsulosin).

PURPOSE: The purpose of this study was to assess the incidence of intraoperative floppy iris syndrome (IFIS) and the morphology of the corneal endothelium after cataract extraction in Caucasian male patients exposed to the α-1a adrenergic receptor antagonist tamsulosin. METHODS: In a clinical prospective study, 23 male patients (23 eyes) treated with tamsulosin due to benign prostatic hyperplasia and 25 male patients (25 eyes) with no tamsulosin treatment had cataract surgery. The divide-and-conquer technique was used with the Infinity OZil(®) machine. A combination of Healon and Healon5 was used in all patients, but the use of additional Vision Blue, iris retractors or intracameral phenylephrine in the tamsulosin group was at the discretion of the surgeon. The endothelial cell density, variation in endothelial cell size (CV), percentage of hexagonal cells and central corneal thickness (CCT) were recorded at baseline and at 3 months postoperatively. RESULTS: In the tamsulosin-treated group, 19 of 23 eyes (83%) developed IFIS, compared with no IFIS in the control group. Compared with the control group, the tamsulosin group showed significantly less dilatation at the start of the operation, significant miosis during surgery and significantly greater corneal endothelial cell loss 3 months postoperatively (12% versus 3%; p< 0.001). CONCLUSION: Intraoperative floppy iris syndrome during cataract surgery is significantly associated with tamsulosin-treated male patients. Patients on tamsulosin showed less preoperative dilatation, significant miosis during surgery, and had significantly greater postoperative endothelial cell loss compared with nontreated patients despite recommended precautions.

Randomized dose-response analysis of mitomycin-C to prevent haze after photorefractive keratectomy for high myopia.

PURPOSE: To evaluate the safety and efficacy of mitomycin-C (MMC) 0.01% (0.1 mg/mL) in preventing haze formation after wavefront-guided photorefractive keratectomy (PRK) for higher myopia at 3 exposures. SETTING: United States Navy Refractive Surgery Center, San Diego, California, USA. DESIGN: Double-masked randomized prospective trial. METHODS: Sixty-, 30-, and 15-second exposures of MMC 0.01% were compared in wavefront-guided PRK for higher myopia. One eye received MMC (surgical sponge) and the other a placebo. All eyes received a 4-month tapering postoperative topical steroid regimen. Endothelial cell densities, haze scores, high- and low-contrast acuities, and manifest refraction were measured preoperatively and 1, 3, 6, and 12 months postoperatively. Outcomes were analyzed as repeated measures over time. RESULTS: The mean preoperative manifest refraction spherical equivalent was -5.98 diopters (D) (range -4.4 to -8.0 D). No eye developed more than trace haze. There was a significant difference in haze scores between MMC-treated eyes and untreated eyes at 1 and 3 months (P=.034) but no difference at 6 and 12 months. Endothelial cell densities decreased in the treated eyes and untreated eyes at all 3 exposures at 1 month but returned to baseline by 6 months. There was no difference in acuities or refractions with or without MMC. CONCLUSIONS: Mitomycin-C may not be needed to prevent haze after modern PRK with a 4-month steroid taper. There was no clinically significant difference in haze formation between MMC eyes and control eyes at the concentration and exposures used.

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.

[Corneal toxicity due to amantadine]. / Toxicidad corneal por amantadina.

CASE REPORT: A 64 year-old female with Parkinson disease treated with amantadine for two years who suddenly suffered bilateral corneal oedema. It was initially treated as herpetic endotheliitis without improvement as we lacked information on her chronic treatment. The corneal oedema finally resolved after withdrawing the drug. DISCUSSION: Amantadine hydrochloride may produce endothelial dysfunction. Once the amantadine treatment is stopped, the corneal oedema may be reversible but endothelial density remains low. An ophthalmologist examination should be performed before the initiation of amantadine treatment in order to establish a risk: benefit ratio, especially in those patients with low endothelial density or any endothelial anomaly.

Toxicity comparison of intraocular azithromycin with and without a bioadhesive delivery system in rabbit eyes.

PURPOSE: To determine whether the addition of a bioadhesive drug-delivery system to topical azithromycin induces intraocular inflammation and damage when introduced intraocularly by different approaches and in varying doses. SETTING: John A. Moran Eye Center, Salt Lake City, Utah, USA. DESIGN: Experimental study. METHODS: Commercial topical azithromycin 1.0% was duplicated, including the benzalkonium chloride, but without inclusion of the Durasite bioadhesive drug-delivery system. Injections of 50 µL, 25 µL, and 10 µL of the antibiotic solutions were administered in a masked fashion to 2 rabbits; 1 eye (study eye) in each rabbit was randomized to receive azithromycin with the delivery system and the fellow eye (control eye) to receive azithromycin without the delivery system. Two rabbits had topical drops of each solution placed after a 2.8 mm incision was created. Masked slitlamp examinations, pachymetry, and intraocular pressure (IOP) were determined 1 day and 2 days postoperatively. The animals were humanely killed, and the endothelial density and histopathology were examined. RESULTS: The IOP (P<.001), pachymetry (P<.001), and signs of inflammation (P=.38 to .003) were consistently higher in the study eye, especially at the 50 µL dose, than in the control eye. This was confirmed by histopathology. CONCLUSION: If the drug-delivery system gains access to the anterior chamber, it may cause substantial corneal edema and inflammation, even at low doses and after topical administration.

Effects of mitomycin C on corneal endothelial cell counts in pterygium surgery: role of application location.

PURPOSE: To evaluate changes in corneal endothelial cell counts after pterygium surgery with application of mitomycin C (MMC) either on the perilimbal sclera or in the subconjunctival space. DESIGN: Prospective, randomized interventional study. METHODS: Fifty-six eyes of 56 patients with primary pterygium underwent excision followed by removal of subconjunctival fibrovascular tissue, 0.02% MMC application, and amniotic membrane transplantation. These were stratified randomly into 2 groups. In 1 group (n = 28), MMC was applied on the perilimbal bare sclera (sclera group), and in other group (n = 28), MMC was applied under conjunctiva, where subconjunctival fibrovascular tissue was removed (subconjunctiva group). Based on severity of pterygium fleshiness, MMC was used for 1, 3, or 5 minutes in 8, 13, and 7 eyes, respectively, in the sclera group and in 9, 13, and 6 eyes, respectively, in the subconjunctiva group. Central corneal endothelial cell counts were evaluated before and during 6 months of follow-up after surgery. RESULTS: Mean preoperative endothelial cell count was 2810 ± 278 cells/mm(2) in the sclera group and 2857 ± 332 cells/mm(2) in the subconjunctiva group. Mean endothelial cell losses in sclera and subconjunctiva groups were 9.7% and 9.0% at 1 week, 6.5% and 6.5% at 1 month, 4.0% and 5.0% at 3 months, and 3.4% and 4.8% at 6 months, respectively, with no statistically significant difference between the 2 groups. Longer durations of MMC application were associated with significantly greater endothelial losses in both groups. CONCLUSIONS: Regardless of application location, MMC use during pterygium surgery can cause a significant decrease in central endothelial cell count.