Corneal storage at 4 degrees Celsius in a chondroitin sulphate and dextran containing medium.

Corneas were stored at 4 degrees C in a chondroitin sulphate and dextran containing medium from one to ten days. The viability of the corneal endothelium was assessed after storage with the vital stain, trypan blue.     

Minnesota system corneal preservation.

The clinical and laboratory results with a modified Minnesota system of organ culture corneal preservation are presented. A refinement of our preservation technique using a closed system, as well as the addition of chondroitin sulphate to the medium is presented. Laboratory results show preservation of corneal endothelial integrity for at least 21 days with maintenance of normal corneal thickness. In addition, a 10-day quarantine system reduces the risk of donor contamination and secondary endophthalmitis. Preliminary results of the 34 degrees C and 4 degrees C closed Minnesota corneal preservation system using chondroitin sulphate show that it is safe and efficacious and allows intermediate to long-term maintenance of sterile thin tissue prior to corneal transplantation.     

Proteoglycans on normal and migrating human corneal endothelium

Proteoglycans are of fundamental importance to the normal functioning of the cornea. They consist of a core protein to which one or more glycosaminoglycan chains are attached. Cell surface proteoglycans are known to mediate many aspects of cell behaviour including cell adhesion, control of extracellular matrix deposition, cell proliferation, cell migration, leukocyte adhesion and modulation of growth factor activity. This paper describes the first investigation into the distribution and function of the three main classes of proteoglycans on human corneal endothelium. Immuno-gold labelling techniques were used at the light, scanning and transmission electron microscope level to localise heparan sulphate, chondroitin sulphate and keratan sulphate proteoglycans on human corneal endothelium. Human corneas were freeze-wounded and kept in organ culture for 3 days in order to study the distribution of proteoglycans on migrating corneal endothelium. An Optimas image analysis system was used to quantify the change in proteoglycan labelling during cell migration. Labelling for chondroitin sulphate and heparan sulphate was at very low levels on normal corneal endothelium while keratan sulphate labelling was at high levels. The wound healing experiments showed that migrating cells had increased labelling for heparan sulphate and chondroitin sulphate with greatly decreased labelling for keratan sulphate. Statistical analysis showed these changes were highly significant (P<0.001). Transmission electron microscopy revealed that chondroitin sulphate and keratan sulphate were present throughout Descemet's membrane while heparan sulphate was concentrated at the interface of Descemet's membrane and the migrating corneal endothelial cells. The pattern of occurrence of chondroitin sulphate, heparan sulphate and keratan sulphate on the human endothelium in normal and wounded cornea suggests that these proteoglycans are linked to the process of cell migration.     

Influence of temporary hypothermia on corneal endothelial cell density during organ culture preservation

Purpose To evaluate temporary exposure to hypothermia for its effects on endothelial cell density of porcine corneas in dextran containing organ-culture medium, with regard to possible negative influences of low temperatures during the transport of corneal grafts. Methods Two groups of central discs from pig corneas (diameter 8 mm) were first organ-cultured (MEM with 6% dextran 500) for 24 hours at 32°C. Twelve corneas were exposed to 4°C in group 1 for 12 hours and to 21°C in group 2 for 48 hours each. The paired corneal discs were not treated, and served as controls. After further organ culture of all corneas for 48 hours at 32°C to allow regenerative processes, corneal endothelium was stained with alizarin red S and examined by light microscopy. The endothelial cell densities were determined manually on three central images. Results Exposure for 12 hours to 4°C as well as for 48 hours to 21°C induced an endothelial cell loss of 0.3% and 1.8% respectively. Statistical analysis showed no significant difference (p = 0.680) of the endothelial cell density between corneas exposed to 4°C and the control corneas (4166 ± 389 cells/mm² and 4177 ± 407 cells/mm² respectively). Despite the minor cell loss, the difference of the endothelial cell density between corneas exposed to 21°C and the control corneas (4085 ± 260 cells/mm² and 4159 ± 312 cells/mm² respectively) was statistically significant (p = 0.025). Conclusions Exposure of organ-cultured porcine corneas in dextran containing medium to 4°C for 12 hours and 21°C for 48 hours does not compromise the endothelial cell density of donor corneas in a clinically relevant manner. A storage of corneal grafts at temperatures down to 4°C for 12 hours, as might be the case during transport from the cornea bank to the ocular surgeon, does not seem to damage the endothelial cell layer. Presented in part at the annual meeting of the Deutsche Opthalmologische Gesellschaft (DOG), Berlin, 2007.     

Clinical Outcomes of Nanothin Descemet Stripping Automated Endothelial Keratoplasty in Korean Patients with Corneal Endothelial Dysfunction

PurposeTo evaluate the clinical outcomes of nanothin Descemet stripping automated endothelial keratoplasty (DSAEK) in Korean patients with corneal endothelial dysfunction.MethodsWe retrospectively reviewed medical records of the patients who underwent nanothin DSAEK (graft thickness ≤50 μm) due to corneal endothelial dysfunction and followed up more than 1 year. We evaluated best-corrected visual acuity (BCVA), central corneal thickness, and corneal endothelial cell density at preoperative and 1, 3, 6, and 12 months postoperatively.ResultsSixteen eyes of 16 patients with the mean follow-up period of 13.00 ± 0.96 months were included. The mean graft thickness after deswelling was 45.25 ± 4.59 μm (range, 38.0–50.0 μm). The mean logarithm of the minimum angle of resolution BCVA improved from 1.37 ± 0.53 preoperatively to 0.68 ± 0.46, 0.55 ± 0.35, 0.40 ± 0.25, and 0.39 ± 0.25 at 1, 3, 6, and 12 months postoperatively (p = 0.005, p < 0.001, p < 0.001, and p < 0.001), respectively. The mean central corneal thickness improved from 752.00 ± 129.11 to 555.75 ± 54.66 μm at 12 months postoperatively (p = 0.006). The mean graft endothelial cell density decreased from 2,859.62 ± 228.34 to 1,542.25 ± 627.34 cells/mm² at 12 months postoperatively (p = 0.012). The postoperative complications included increased intraocular pressure (n = 3, 18.75%) and graft dislocation (n = 1, 6.25%), all of which were successfully managed by anterior chamber paracentesis or rebubbling. No other serious complications were encountered.ConclusionsNanothin DSAEK produced significant and stable visual improvements without severe postoperative complications in Korean patients with corneal endothelial dysfunction.     

Cryopreservation of porcine corneas with chondroitin sulfate

A new method for cryopreservation of porcine corneoscleral discs has been developed. Instead of the widely used cryoprotectant DMSO, chondroitin sulfate was applied as a cryoprotectant for the first time. Post-thawing evaluation was done after short-term tissue culture. Central cell counts showed a mean endothelial cell density of 2430 cells/mm2 (+/- 383) after freezing with a combination of 2% chondroitin sulfate and 20% fetal calf serum. Corneae which were cryopreserved with DMSO using the method established by Capella and Kaufman showed a mean endothelial cell density of 1672 cells/mm2 (+/- 617). These experiments demonstrated significant cryoprotective properties of chondroitin sulfate. In addition to its application in refrigerated corneal storage at 4 degrees C and organ culture at 32 degrees C, this substance may also be useful in cryopreservation.     

Freeze preservation of swine corneas with combinations of intra- and extracellular cryoprotective agents]

Clinically employed methods of corneal cryopreservation usually use the intracellular cryoprotectant dimethyl sulfoxide (DMSO). However, it has been demonstrated that extracellular cryoprotectants such as chondroitin sulfate (ChS) also display effective cryoprotection. The purpose of our study was to investigate the effect of combinations of intra- and extracellular cryoprotectants in corneal cryopreservation. Porcine corneas were cryopreserved in a cryopreservation medium consisting of MEM-medium containing 20% fetal calf serum and 2% chondroitin sulfate. The medium was varied by the addition of 2%, 4% and 8% DMSO. Sixty corneas were cryopreserved at -196 degrees C and thawed at 37 degrees C in a water bath. Morphometric evaluation was not performed directly after thawing but after a 24-h storage period in organ culture. Cryopreservation in medium without DMSO revealed the best results concerning endothelial cell density (2581 cells/mm2). Addition of 2% or 4% DMSO revealed no significant changes in endothelial cell density. Addition of 8% DMSO, however, resulted in a significant decrease (1312 +/- 319 cells/mm2) combined with a significantly higher amount of necrotic areas in the central corneal surface. We conclude that combining intra- and extracellular cryoprotectants does not enhance endothelial cell density after corneal cryopreservation. Higher concentrations of DMSO added to the cryopreservation medium appear to have a negative impact on endothelial cell viability.     

Histological study of corneas preserved in two new media.

A new corneal preserving medium (K-Sol), developed by Kaufman and others, contains purified chondroitin sulphate, TC 199, HEPES buffer, and gentamicin. Another new medium (JM) containing bicarbonate-free glucose-phosphate Ringer solution and dextran 70 has been developed in Japan. New Zealand white rabbit corneas with scleral rims were stored in each medium at 4 degrees C for one or two weeks. The condition of the endothelium was evaluated histologically. Corneas preserved in both media were in good condition at the end of one week. Corneas preserved in K-Sol for two weeks showed fewer endothelial changes than similar tissue stored in JM for two weeks. Corneal swelling was also less in corneas preserved in K-Sol, than in corneas preserved in JM.     

Corneal storage at 4° Celsius in a chondroitin sulphate and dextran containing medium

Corneas were stored at 4°C in a chondroitin sulphate and dextran containing medium from one to ten days. The viability of the corneal endothelium was assessed after storage with the vital stain, trypan blue.     

Corneal Keratocyte Density and Corneal Nerves Are Reduced in Patients With Severe Obesity and Improve After Bariatric Surgery

PurposeObesity is associated with peripheral neuropathy, which bariatric surgery may ameliorate. The aim of this study was to assess whether corneal confocal microscopy can show a change in corneal nerve morphology and keratocyte density in subjects with severe obesity after bariatric surgery.MethodsTwenty obese patients with diabetes (n = 13) and without diabetes (n = 7) underwent assessment of hemoglobin A1c (HbA1c), lipids, IL-6, highly sensitive C-reactive protein (hsCRP), and corneal confocal microscopy before and 12 months after bariatric surgery. Corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), and keratocyte density (KD) from the anterior, middle, and posterior stroma were quantified. Twenty-two controls underwent assessment at baseline only.ResultsCNFL (P < 0.001), CNBD (P < 0.05), and anterior (P < 0.001), middle (P < 0.001), and posterior (P < 0.001) keratocyte densities were significantly lower in obese patients compared to controls, and anterior keratocyte density (AKD) correlated with CNFL. Twelve months after bariatric surgery, there were significant improvements in body mass index (BMI; P < 0.001), HDL cholesterol (P < 0.05), hsCRP (P < 0.001), and IL-6 (P < 0.01). There were significant increases in CNFD (P < 0.05), CNBD (P < 0.05), CNFL (P < 0.05), and anterior (P < 0.05) and middle (P < 0.001) keratocyte densities. The increase in AKD correlated with a decrease in BMI (r = –0.55, P < 0.05) and triglycerides (r = –0.85, P < 0.001). There were no significant correlations between the change in keratocyte densities and corneal nerve fiber or other neuropathy measures.ConclusionsCorneal confocal microscopy demonstrates early small fiber damage and reduced keratocyte density in obese patients. Bariatric surgery leads to weight reduction and improvement in lipids and inflammation and an improvement in keratocyte density and corneal nerve regeneration.     

Donor organ cultured corneal tissue selection before penetrating keratoplasty

AIMS—Donor organ cultured corneal tissue selection before penetrating keratoplasty is carried out by taking into account different variables. The objective was to identify preoperative variables which are significantly and independently associated with transplant outcome and should effectively be taken into account before transplantation.
METHODS—231 consecutive penetrating keratoplasties were prospectively studied using organ cultured tissue. Morphometric analysis of the donor corneal endothelium was performed before transplantation. Graft survival and endothelial cell density, during the second year following transplantation, were studied both at a univariate and multivariate level.
RESULTS—Recipient age, recipient rejection status, and preoperative diagnosis significantly influenced graft survival. Graft survival was higher when using corneal tissue from donors older than 80 years. Postoperative endothelial density decreased with preservation time and coefficient of variation after preservation. It increased with endothelial cell density after preservation and deswelling time, and correlated with preoperative diagnosis.
CONCLUSION—Organ cultured corneas with endothelial cell density after preservation <2000 cells/mm², and high coefficient of variation, may be discarded before transplantation. Corneas should be preserved for less than 3 weeks, and allowed to deswell before transplantation for 2 or 3 days rather than 1 day.

Keywords: corneal transplantation; endothelium; graft survival; morphometry     

Quantification of allospecific and nonspecific corneal endothelial cell damage after corneal transplantation

Purpose

To investigate the effect of host immunity (allospecific) and surgical manipulation (non-allospecific) on corneal endothelial cells (CECs) in corneal transplantation.

Methods

Draining lymph nodes and grafted C57BL/6 corneas were harvested from syngeneic recipients, allograft acceptors, and allograft rejectors (BALB/c) 1, 3, and 8 weeks after transplantation. We analyzed CEC apoptosis using an ex vivo cornea-in-the-cup assay, and visualized cell-to-cell junctions using immunohistochemical staining (ZO-1). Automatic cell analysis using Confoscan software was used to measure CEC density as well as changes in CEC morphology by quantifying the coefficient of variation in cell size (polymegethism) and shape (pleomorphism).

Results

The cornea-in-the-cup assay showed that allogeneic acceptor T cells and to an even greater extent rejector T cells (but not syngeneic T cells) induced CEC apoptosis. CEC density after corneal transplantation was significantly reduced in allogeneic acceptors compared with syngeneic grafts (P<0.001), and CEC density was even further reduced in the allo-rejector group compared with the allo-acceptor group. Allogeneic grafts showed a greater increase in the coefficient of variation in cell size (polymegethism) when compared with syngeneic grafts 1 week after transplantation (P=P<0.001). However, pleomorphism was not significantly different between syngeneic and allo-acceptor grafts, indicating that polymegethism (but not pleomorphism or cell density) is a sensitive indicator of the effect of alloimmunity on CECs.

Conclusions

Our data demonstrate that host alloimmunity rather than surgical manipulation alone is the major cause of CEC damage in corneal transplantation, and such morphologic changes of CECs can be detected before the clinically visible onset of allograft rejection.     

Cryopreservation of human donor corneas with dextran

PURPOSE: To assess freeze-thaw-induced endothelial cell loss by using phase-contrast microscopy and early morphologic changes within each layer of human donor corneas by using confocal microscopy. METHODS: Twenty-eight human corneas were cryopreserved in minimum essential medium containing 10% dextran with a molecular weight (MW) of 500,000 as an extracellular cryoprotectant, at a cooling rate of 1 degrees C/min and stored in liquid nitrogen at -196 degrees C. After thawing, the tissue was organ cultured to detect latent cell damage. In 22 of the corneas, the endothelial layer was subjected to routine phase-contrast microscopy after 24 hours of organ culturing. The other six specimens were evaluated layer by layer in a scanning slit confocal microscope after 6, 24, and 48 hours of organ culturing. RESULTS: Before cryopreservation, the mean +/- SD numerical density of endothelial cells was 1940 +/- 220 cells/mm(2). After cryopreservation and subsequent organ culturing, the endothelial cell density decreased to 1300 +/- 360 cells/mm(2), and two of the corneas had a completely necrotic endothelium (P = 0.001). Confocal microscopy revealed all corneal layers in each of the six specimens examined to be structurally integral after 48 hours of organ culturing. Although the reflectivity of some of the keratocytes was enhanced, there were no signs of keratolysis. CONCLUSIONS: The present study demonstrates that each corneal layer is capable of regaining its structural integrity after cryopreservation in the presence of dextran. Because the freeze-thaw-induced endothelial cell loss is still highly variable, the technique must be further refined before it can be applied clinically.     

Corneal endothelial morphology and central thickness in patients with type II diabetes mellitus

Purpose: To investigate corneal endothelial cell density and morphology in type II diabetic and non‐diabetic patients and to relate potential differences to the glycaemic status. Methods: A prospective clinical study including 107 patients with type II diabetes and 128 non‐diabetic patients. Sample size was based on a power calculation (power = 0.90; p = 0.05). The diabetic patients had on average more than four HbA1c tests performed (mean 4.1; range 2–14) with intervals of at least 3 months as a reflection of the long‐term glycaemic status. The controls had no diabetes confirmed by two causal blood tests. The endothelial cell density, the variation in endothelial cell size (CV), the percentage of hexagonal cells, and the central corneal thickness (CCT) were recorded. Results: Type II diabetic subjects did not differ from the non‐diabetic control subjects with regards to endothelial cell density, hexagonality or variation in CV, but showed a significant increase in CCT (538 versus 546 μm, p < 0.05). In the diabetic group, lower cell counts were associated with higher HbA1_c values (p < 0.05). The HbA1c did not, however, have any impact on the CCT. Conclusion: Type II diabetes has no impact on corneal cell density or morphology in subjects with good glycaemic status. However, higher HbA1c was associated with lower endothelial cell density. CCT was significantly increased in the diabetic group_.     

Corneal Endothelial Cell Loss after Phacoemulsification in Eyes with a Prior Acute Angle-closure Attack

PurposeTo evaluate endothelial damage after cataract surgery in eyes affected by an angle-closure attack (ACA) and compare it to that in the unaffected fellow eyes (FEs) of patients with ACA and normal eyes (NEs).MethodsThe medical data of eyes affected by ACA, FEs (with no history of acute glaucoma attack), and NEs of patients who underwent cataract surgery with simultaneous intraocular lens implantation were retrospectively reviewed. Endothelial cell density (ECD) and central corneal thickness (CCT) measured before surgery and at 1 week, 1 month, and 3 months after surgery were analyzed, and the percentages of loss in ECD and increase in CCT of the three groups were compared.ResultsThe study enrolled 140 eyes from 100 patients (50 eyes in the ACA group, 40 eyes in the FE group, and 50 eyes in the NE group). The mean ECD was significantly lower in the ACA group than in the other groups (p < 0.001). However, the percentage of ECD reduction was not significantly greater in the ACA group than in the other groups (p > 0.05). None of the eyes developed corneal edema at 3 months postoperatively. Moreover, the CCTs of the three groups were similar throughout the follow-up period (p > 0.05).ConclusionsPhacoemulsification was not associated with greater endothelial cell loss in the ACA group than in the NE and FE groups. This finding shows that ACA history may not contribute to the exacerbation of corneal endothelial damage in cataract surgery.     

K-Sol corneal preservation at room temperature.

K-Sol, a recently developed corneal storage medium that contains purified chondroitin sulphate in tissue culture medium (TC 199), is capable of preserving corneal tissue for 14 days at 4 degrees C. To study the effect of tissue storage in K-Sol at room temperature we preserved rabbit corneas in K-Sol and M-K medium for three or seven days at 25 degrees C. All of the corneal endothelial sheets were intact after three days. At seven days the change in pH of the K-Sol medium was less than that of M-K medium. Corneas preserved in M-K medium showed swelling of mitochondria and a decrease in the number of cytoplasmic organelles. Corneas preserved in K-Sol had organised cytoplasmic organelles and nuclei. Scanning electron micrographs revealed well preserved endothelial sheets. Corneas stored in the two media showed no significant difference in thickness. A pair of human corneas preserved in K-Sol at room temperature for six days maintained about 95% of the endothelial sheet in good condition. Small separations were observed between some of the endothelial cells. However, even in these areas, the cytoplasmic organelles were well preserved. It appeared that K-Sol is more stable than M-K medium at room temperature, and that both rabbit and human corneas can be preserved in good condition in K-Sol for at least six days at 25 degrees C.     

Effect of corneal stromal pocket irrigation in small-incision lenticule extraction

ObjectivesTo investigate the effect of corneal stromal pocket irrigation after small-incision lenticule extraction (SMILE) on visual acuity, intraocular pressure (IOP), corneal parameters and complications after surgery.MethodsA total of 242 eyes of 121 patients undergoing SMILE were enrolled in this prospective controlled study, and it was designed for one eye to randomly undergo SMILE with balanced salt solution irrigation of the corneal stromal pocket, while the other eye was not. The uncorrected distance visual acuity (UDVA) and slit lamp examination were recorded at 1 hour, 1 day, 1 week, and 1 month. Postoperative corneal density, corneal biomechanical, corneal endothelial cell number, and anterior OCT images were compared at 1 day, 1 week, and 1 month.ResultsCompared with the nonirrigation group, the irrigation group showed significantly higher UDVA at 1 day postoperatively (P < 0.05), but there was no significant difference during the rest of the postoperative period (1 hour, 1 week, and 1 month). In addition, no significant differences were found in IOP, corneal density, corneal biomechanics, corneal endothelial cells, and corneal morphology. No visual decline or severe postoperative complications were found in the patients in this study.ConclusionsInterlamellar irrigation did not affect IOP, corneal parameters, morphology, complications, or UDVA at 1 hour, 1 week, and 1 month after the operation, but it may promote UDVA 1 day after the operation.Subject terms: Refractive errors, Outcomes research, Surgery     

Oxidative Stress Levels in Aqueous Humor from High Myopic Patients

PurposeTo compare oxidative stress status in the aqueous humor of highly myopic eyes and control eyes.MethodsAqueous humor samples were collected from 15 highly myopic eyes (high myopia group) and 23 cataractous eyes (control group) during cataract surgery. Central corneal thickness, corneal endothelial cell density, hexagonality of corneal endothelial cells, and cell area of corneal endothelial cells were measured using specular microscopy. Axial length was measured using ultrasound biometry. 8-Hydroxydeoxyguanosine (8-OHdG) and malondialdehyde levels were measured using enzyme-linked immunosorbent assay.Results8-OHdG level was lower in the aqueous humor of myopic patients than in that of control group (p = 0.014) and was positively correlated with central corneal thickness and negatively correlated with axial length (r = 0.511, p = 0.02; r = -0.382, p < 0.001). There was no correlation between 8-OHdG level and corneal endothelial cell density, hexagonality, or cell area. Malondialdehyde level did not show any correlation with any parameters evaluated.Conclusions8-OHdG might be a sensitive biomarker for evaluating oxidative stress status in the eye. Oxidative stress level was lower in the aqueous humor of highly myopic eyes compared to that in control eyes, which indicates lower metabolic activity in these eyes.     

Effect of timolol on central corneal thickness and endothelial cell density

BACKGROUND: The measurement of corneal thickness plays an increasing role in glaucoma screening and diagnosis. The influence of a variety of drugs on corneal thickness is well established. Especially for antiglaucomateous drugs this effect seems to be important. However, little is known about the influence of beta receptor antagonists on corneal thickness. The aim of this study was to provide evidence of the effect of timolol on central corneal thickness and endothelial cell density. MATERIALS AND METHODS: Ten healthy volunteers (five women and five men) with a mean age of 29 years (range 25 to 56 years) were examined in a double-blind, prospective and randomised pilot study. Intraocular pressure, corneal thickness and endothelial cell density was estimated before as well as fifteen minutes, 24, 48, 72 and 96 hours after application of timolol 0.5 % eye drops twice daily. The partner eye received sodium hyaluronate eye drops twice daily and served as a control. RESULTS: The application of timolol showed a decrease of intraocular pressure from initially 12 mmHg to 9 mmHg after four days (p = 0,0188) as well as an increase of corneal thickness from 537 microm to 557 microm after four days (p = 0,0659). There was no change of intraocular pressure (p = 0,9935) or corneal thickness (p = 0,9998) in the control eyes. There was also no effect of timolol (p = 0,2782) or sodium hyaluronate (p = 0,1940) on endothelial cell density. CONCLUSIONS: The study provides evidence of the influence of beta receptor antagonists on corneal thickness. This effect may be caused by receptor mediated influences on corneal ion and fluid transport. Further studies are needed to show if the increase of corneal thickness after application of topical timolol has clinical importance.     

Effects of Argon Laser Iridotomy on the Corneal Endothelium of Pigmented Rabbit Eyes

Purpose

In Asian countries, laser iridotomy for the treatment of angle-closure glaucoma is a common cause of bullous keratopathy, which may be associated with a shallow anterior chamber and dark iris pigmentation in Asians. Several cases of corneal decompensation after argon laser iridotomy have been reported. In the present study, we evaluated the harmful effects of argon laser iridotomy on the corneal endothelium.

Methods

Argon laser iridotomy was performed on the right eyes of pigmented rabbits. Changes in corneal thickness and endothelial cell density after laser iridotomy were evaluated. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed for assessment of corneal endothelial cell apoptosis. Combined staining with alizarin red and trypan blue, as well as a live/dead cell assay, were performed for evaluation of damage to the corneal endothelium induced by laser iridotomy.

Results

Corneal thickness did not change immediately after laser iridotomy; however, a significant increase was observed 24 hours after iridotomy (p = 0.001). The endothelial cell density of laser-treated eyes four days after laser iridotomy was significantly decreased compared with control eyes (p < 0.001). TUNEL staining showed many TUNEL-positive cells in the corneal endothelium and corneal stroma. No endothelial trypan blue-stained cell nuclei were observed after laser iridotomy; however, several large endothelial cells with damaged membrane integrity were observed. The live/dead cell assay clearly showed a large number of dead cells stained red in several areas throughout the entire corneal button 24 hours after iridotomy.

Conclusions

Argon laser iridotomy induces corneal endothelial cell apoptosis in pigmented rabbit eyes, resulting in decreased endothelial cell density.