New simulation software to predict postoperative corneal stiffness before laser vision correction.

PURPOSE: To develop a new virtual surgery simulation platform to predict postoperative corneal stiffness (Kc mean ) after laser vision correction (LVC) surgery. SETTING: Narayana Nethralaya Eye Hospital and Sankara Nethralaya, India; Humanitas Clinical and Research Center, Italy. DESIGN: Retrospective observational case series. METHODS: 529 eyes from 529 patients from 3 eye centers and 10 post-small-incision lenticule extraction (SMILE) ectasia eyes were included. The software (called AcuSimX) derived the anisotropic, fibril, and extracellular matrix biomechanical properties (using finite element calculation) of the cornea using the preoperative Corvis-ST, Pentacam measurement, and inverse finite element method assuming published healthy collagen fibril orientations. Then, the software-computed postoperative Kc mean was adjusted with an artificial intelligence (AI) model (Orange AI) for measurement uncertainties. A decision tree was developed to classify ectasia from normal eyes using the software-computed and preoperative parameters. RESULTS: In the training cohort (n = 371 eyes from 371 patients), the mean absolute error and intraclass correlation coefficient were 6.24 N/m and 0.84 (95% CI, 0.80-0.87), respectively. Similarly, in the test cohort (n = 158 eyes from 158 patients), these were 6.47 N/m and 0.84 (0.78-0.89), respectively. In the 10 ectasia eyes, the measured in vivo (74.01 [70.01-78.01]) and software-computed (74.1 [69.03-79.17]) Kc mean were not statistically different ( P = .96). Although no statistically significant differences in these values were observed between the stable and ectasia groups ( P ≥ .14), the decision tree classification had an area under the receiver operating characteristic curve of 1.0. CONCLUSIONS: The new software provided an easy-to-use virtual surgery simulation platform for post-LVC corneal stiffness prediction by clinicians and was assessed in post-SMILE ectasia eyes. Further assessments with ectasia after surgeries are required.

Surgical technique for the management of corneal perforation in brittle cornea.

Brittle cornea syndrome is among the few special scenarios in ophthalmology that are a nightmare not only for the operating surgeon but also for the patient. Here, the thin and fragile corneas are unable to maintain the shape and structural integrity of the globe and are more prone to minor traumatic or spontaneous corneal perforations. Suturing a brittle cornea and closure of the corneal perforation in a brittle cornea are very challenging requiring the utmost care and special precautions. If proper measures are not taken during the surgery, it may be difficult to salvage the eye. Hence, it is imperative to diagnose appropriately, suture effectively, taking necessary preventive measures in salvaging these corneas. This manuscript aims at providing tips for handling brittle corneal perforations. It will also discuss the problems encountered during surgery, highlight the suturing techniques that can be customized, and finally give an insight into postoperative care.

Safety and efficacy of combination of suberoylamilide hydroxyamic acid and mitomycin C in reducing pro-fibrotic changes in human corneal epithelial cells.

Corneal haze post refractive surgery is prevented by mitomycin c (MMC) treatment though it can lead to corneal endothelial damage, persistent epithelial defects and necrosis of cells. Suberanilohydroxamic acid (SAHA) however has been proposed to prevent corneal haze without any adverse effects. For clinical application we have investigated the short and long term outcome of cells exposed to SAHA. Human donor cornea, cultured limbal epithelial cells, corneal rims and lenticules were incubated with SAHA and MMC. The cells/tissue was then analyzed by RT-qPCR, immunofluorescence and western blot for markers of apoptosis and fibrosis. The results reveal that short term exposure of SAHA and SAHA + MMC reduced apoptosis levels and increased αSMA expression compared to those treated with MMC. Epithelial cells derived from cultured corneal rim that were incubated with the MMC, SAHA or MMC + SAHA revealed enhanced apoptosis, reduced levels of CK3/CK12, ∆NP63 and COL4A compared to other treatments. In SAHA treated lenticules TGFß induced fibrosis was reduced. The results imply that MMC treatment for corneal haze has both short term and long term adverse effects on cells and the cellular properties. However, a combinatorial treatment of SAHA + MMC prevents expression of corneal fibrotic markers without causing any adverse effect on cellular properties.

Stem cell therapy in coronavirus disease 2019: current evidence and future potential.

The end of 2019 saw the beginning of the coronavirus disease 2019 (COVID-19) pandemic that soared in 2020, affecting 215 countries worldwide, with no signs of abating. In an effort to contain the spread of the disease and treat the infected, researchers are racing against several odds to find an effective solution. The unavailability of timely and affordable or definitive treatment has caused significant morbidity and mortality. Acute respiratory distress syndrome (ARDS) caused by an unregulated host inflammatory response toward the viral infection, followed by multi-organ dysfunction or failure, is one of the primary causes of death in severe cases of COVID-19 infection. Currently, empirical management of respiratory and hematological manifestations along with anti-viral agents is being used to treat the infection. The quest is on for both a vaccine and a more definitive management protocol to curtail the spread. Researchers and clinicians are also exploring the possibility of using cell therapy for severe cases of COVID-19 with ARDS. Mesenchymal stromal cells are known to have immunomodulatory properties and have previously been used to treat viral infections. This review explores the potential of mesenchymal stromal cells as cell therapy for ARDS.

Long-term outcome of custom toric intraocular lens for treating high astigmatism in case of cataract associated with pellucid marginal corneal degeneration.

Pellucid marginal corneal degeneration (PMCD) is a progressive corneal ectasia that ultimately results in high regular astigmatism and correction of this astigmatism is always challenging. However, when a PMCD patient develops a cataract, it provides a golden opportunity to treat this coexisting astigmatism with toric intraocular lens (IOL) implantation. Regular toric IOLs would correct astigmatism only up to 6 diopters in the IOL plane but higher astigmatism would require customization of such IOLs. Our case report describes the long term outcomes of customized toric IOL to tackle this high astigmatism during cataract surgery in PMCD cases.

Resveratrol Rescues Human Corneal Epithelial Cells Cultured in Hyperosmolar Conditions: Potential for Dry Eye Disease Treatment.

PURPOSE: Dry eye disease (DED) is a common ocular surface condition across age groups. Recently, vitamin D deficiency has gained importance as a causative factor, and its supplementation alleviates symptoms of DED. Resveratrol (RES) regulates vitamin D receptors (VDRs) and Notch signaling. We investigated the role of RES on vitamin D levels and Notch signaling under hyperosmolar conditions. METHODS: Human corneal epithelial (HCE-T) cells were treated with RES in hyperosmolar and normal conditions. Quantitative real-time polymerase chain reaction (PCR), immunofluorescence, enzyme-linked immunosorbent assay, and western blot analysis were performed for estimating reactive oxygen species, VDR, secreted 25-hydroxyvitamin D3, and Notch signaling pathway molecules in treated and control cells. RESULTS: HCE-T cells in hyperosmolar conditions had increased reactive oxygen species levels and decreased vitamin D levels that got restored in the presence of RES. Hyperosmolarity also reduced VDR expression and Notch activity that normalized to original levels with RES. In the presence of Notch blocker LY-411575, RES could not restore VDR expression or secreted vitamin D levels in HCE-T cells exposed to hyperosmolar conditions, whereas recombinant Jagged1 restored vitamin D and VDR levels. CONCLUSIONS: RES restores vitamin D levels in hyperosmolar conditions most likely through activation of Notch signaling. Hence, RES can be a potential adjuvant in DED for patients considered for vitamin D treatment.

Repeatability of OCT Anterior Surface and Bowman's Layer Curvature and Aberrations in Normal and Keratoconic Eyes.

PURPOSE: To study the repeatability of anterior surface and Bowman's layer curvature in normal and keratoconic eyes using optical coherence tomography (OCT). METHODS: In this study, 96 normal and 96 keratoconic eyes underwent corneal imaging using Pentacam (Oculus Optikgeräte, Wetzlar, Germany) and OCT (Triton, Topcon Corporation, Tokyo, Japan). The elevation data from segmented air-epithelium (A-E) and epithelium-Bowman's layer (E-B) interfaces in OCT scans were used to quantify curvature and aberrations. The wavefront aberrations were evaluated with the ray tracing method and 6th order Zernike polynomials. The intraclass correlation coefficient (ICC), within-subject standard deviation (Sw), and coefficient of variation (CoV) were used to assess repeatability. RESULTS: For curvatures, the Sw was less than 0.25 diopters (D) for the normal and keratoconic eyes. The Sw was highest for root mean square of lower order aberrations (0.14 µm) in keratoconic eyes. The CoV for curvatures was well below 0.5% for both groups. For some aberrations irrespective of groups, the CoV was greater because some individual aberrations (mean of three successive measurements) tended to be smaller in magnitude and even a small Sw resulted in a high CoV. For all variables, the ICC ranged between 0.80 and 0.99 for both the OCT and Pentacam measurements. Most variables were similar between the A-E and E-B interfaces (P > .05) for both groups. However, both differed significantly from all Pentacam variables (P < .05) in normal and keratoconic eyes. CONCLUSIONS: The repeatability of OCT curvatures and aberrations compared well with the Pentacam indices for normal and keratoconic eyes. [J Refract Surg. 2020;36(4):247-252.].

Corneal Viscous Properties Cannot Be Determined From Air-Puff Applanation.

PURPOSE: To assess whether corneal viscous properties are measureable with air-puff applanation in patients. METHODS: The study had 312 normal eyes, 107 fellow eyes of patients with keratoconus, and 289 keratoconic eyes. The Corvis ST (Oculus Optikgeräte GmbH, Wetzlar, Germany) deformation data for all eyes were analyzed using two models. First, a standard linear solid model (SLM) assumed the cornea was an elastic material only. Second, a two-compartment Kelvin-Voigt model (KVM) assumed the cornea was a visco-elastic material. Corneal stiffness and viscosity were calculated. Further, the deflection amplitude was phase shifted virtually relative to the air-puff applanation force to assess whether the KVM was capable of detecting corneal viscous properties from air-puff applanation. This was similar in concept to measured viscoelastic deformations in other soft tissues. The hysteresis area was also calculated with deformation (cornea and whole globe) and deflection (cornea only) amplitude. The greater the magnitude of the hysteresis area, the greater was the magnitude of corneal viscosity (µc). RESULTS: Both the SLM and KVM reported similar magnitudes of corneal stiffnesses (correlation coefficient > 0.99). However, for a given model, corneal stiffness was significantly different between normal, fellow, and keratoconic eyes (P = .001). From the KVM, the corneal viscosity was different between groups (P = .001) but was small in magnitude (order of 10-9). The deflection hysteresis area was also small in magnitude (order of 10-6). In contrast, the KVM detected significant corneal viscosity only when the deflection amplitude was virtually phase shifted with respect to the air-puff applanation force. CONCLUSIONS: No significant corneal viscous response was detected in patients who had air-puff applanation. [J Refract Surg. 2019;35(11):730-736.].

Corneal staining during cataract surgery: Natural course, ASOCT features, and preventive measures.

We report the natural course of the accidental injection of trypan blue into the corneal stroma while performing a routine cataract surgery by a resident during a training session. The corneal staining resolved with conservative medical treatment over 7 weeks. This case describes the anterior segment optical coherence tomography (ASOCT) features of corneal staining. It emphasizes on the relatively benign nature of this dye and the follow-up course. Causes that may be responsible for this untoward complication are highlighted with the necessary preventive measures that need to be taken care are also discussed.

Endoilluminator-assisted pediatric cataract surgery with hazy cornea.

The purpose of this study was to demonstrate the usefulness of endoilluminator in pediatric cataract with hazy corneas. We describe a series of three cases of pediatric cataract where visualization of intraocular structures was inadequate under the operating microscope. The endoilluminator was held at the limbus with light directed obliquely to visualize the details of intraocular structures against the hazy cornea using oblique illumination. It allowed structures behind the hazy cornea to be seen with ease. A simple modification in surgical procedure of pediatric cataract using an endoilluminator helps in better visualization of intraocular structures in difficult situations.

Small lens for a big eye: Successful management of anterior megalophthalmos.

We report a case of anterior megalophthalmos and complicated cataract, with apparently smaller lens in both eyes. The right eye had spontaneous retinal detachment. The child underwent cataract surgery in both the eyes with retinal detachment surgery in the right eye. Due to small size of the lenticular bag, a downsized customized intraocular lens (IOL) was implanted. Postoperatively, the IOL was well centered with ambulatory vision till 3 years of follow-up. This case describes this rare disorder and its association with apparently small-sized lens and discusses the course of its management, highlighting the visual rehabilitation with customization of IOLs.

Surgical Outcome of Congenital Cataract in Eyes With Microcornea.

PURPOSE: To report the intraoperative and postoperative outcomes of congenital cataract surgery in eyes with microcornea. METHODS: In this retrospective, interventional, comparative case series, the authors reviewed 47 eyes of 26 children with microcornea and congenital cataract (21 bilateral and 5 unilateral) who underwent lens aspiration with primary posterior capsulectomy and anterior vitrectomy between 2008 and 2014 with a minimum follow-up period of 6 months. Demographic profiles and systemic and ocular features were documented. Intraoperative and postoperative complications were studied separately for bilateral and unilateral cases. Patients were also divided into two groups on the basis of their ages at surgery (early surgery group: 3 months or younger; late surgery group: older than 3 months) and postoperative complications were compared. Visual outcome was analyzed in those with a follow-up period of more than 1 year. RESULTS: Early surgery was performed in 24 eyes of 13 patients (11 bilateral and 2 unilateral) and late surgery in 23 eyes of 13 patients (10 bilateral and 3 unilateral). Intraoperatively, all eyes had poor pupillary dilatation and 6 (12.8%) eyes needed iris hooks. Postoperatively, the most common early complication was transient corneal edema observed in 22 (46.8%) eyes (13 and 8 eyes in the early and late surgery groups, respectively). Late complications included visual axis opacification in 6 (12.76%) eyes (3 in each group), and secondary glaucoma in 5 (10.64%) eyes (2 and 3 eyes in the early and late surgery groups, respectively). Vision was normal for age in 18 (60%) of the bilateral cases with a follow-up period of more than 1 year. CONCLUSIONS: Early surgical intervention for congenital cataract in eyes with microcornea can result in favorable outcomes with an acceptable rate of postoperative complications. [J Pediatr Ophthalmol Strabismus. 2018;55(1):30-36.].

Waveform analysis of deformation amplitude and deflection amplitude in normal, suspect, and keratoconic eyes.

PURPOSE: To evaluate the performance of waveform-derived variables in distinguishing normal, suspect, and keratoconic eyes. SETTING: Narayana Nethralaya Eye Hospital, Bangalore, India. DESIGN: Retrospective case series. METHODS: Scheimpflug tomography (Pentacam) and dynamic Scheimpflug analysis (Corvis ST) of 253 normal (253 patients) eyes and 205 keratoconic eyes (205 patients) were evaluated. Among the 205 patients, 62 had keratoconus in 1 eye, while the unaffected eye was suspect. From deformation amplitude, deflection amplitude and whole-eye movement were extracted. A biomechanical model was used to derive a linear (kc [constant]) and nonlinear measure (kc [mean]) of corneal stiffness. Multivariate logistic regression was performed to determine sensitivity and specificity. The analysis was validated in another dataset of 59 normal, 45 suspect, and 160 keratoconic eyes. RESULTS: Deformation amplitude maximum, applanation 1 time and deformation amplitude, applanation 2 time, kc (constant), kc (mean), and deflection amplitude maximum were significantly different between normal and keratoconic eyes (P < .001). The deformation characteristics of the suspect eyes were similar to those of the keratoconic eyes, particularly grade 1 (P > .05). The kc (constant) and kc (mean) had the highest area under curve (>0.98), sensitivity, and specificity greater than 90% and 91%, respectively. Logistic regression using kc (constant) and kc (mean) improved the area to 1.0, with a sensitivity and specificity equal to 99.6% and 100%, respectively. In the validation dataset, the same cutoff yielded a sensitivity, specificity, and accuracy of 99.5%, 100%, and 99.6%, respectively. CONCLUSION: Corneal stiffness and waveform analyses could be reliable differentiators of suspect and keratoconic eyes from normal eyes.

Noncontact Quantification of Topography of Anterior Corneal Surface and Bowman's Layer With High-Speed OCT.

PURPOSE: To quantify keratometry and wavefront aberration of the anterior corneal surface and epithelium-Bowman's layer interface using anterior segment optical coherence tomography (OCT). METHODS: Twenty-five normal eyes and 25 eyes with keratoconus were retrospectively analyzed. The anterior corneal edge and epithelium-Bowman's layer interface were segmented from 12 distortion-corrected OCT B-scans. Axial tangential curvatures and wavefront aberration were calculated by ray tracing and 6th order Zernike analyses. All eyes underwent simultaneous imaging with Pentacam (Oculus Optikgeräte GmbH, Wetzlar, Germany). The Pentacam elevation data were used for aberration analyses using the same ray-tracing method. The paired t test was used to compare the variables. RESULTS: In normal eyes, mean steep axis and maximum keratometry of OCT of the anterior corneal surface and epithelium-Bowman's layer interface were significantly greater than the same of the Pentacam anterior corneal surface (P < .05). Mean root mean square of higher order aberrations of the OCT surfaces was greater than the same of the Pentacam surface by a factor of 4. In eyes with keratoconus, mean steep axis and maximum keratometry of the OCT epithelium-Bowman's layer interface was the greatest (P < .05). Mean root mean square of the higher order aberrations and vertical coma of the OCT epithelium-Bowman's layer interface was the greatest (P < .05). In general, the aberrations of the OCT epithelium-Bowman's layer interface were significantly greater than those of the Pentacam anterior corneal surface. CONCLUSIONS: A noncontact method to quantify the topography and aberrations of corneal surfaces with OCT was presented. OCT measurements yielded greater curvature and aberrations than Pentacam in both normal and keratoconic eyes. [J Refract Surg. 2017;33(5):330-336.].

One for all: A standardized protocol for ex vivo culture of limbal, conjunctival and oral mucosal epithelial cells into corneal lineage.

BACKGROUND AIMS: Autologous transplantation of ex vivo cultured cells the treatment of choice for patients with limbal stem cell deficiency. The most commonly used cell sources for transplantation limbal, conjunctival or oral mucosal tissue. Protocols vary for culturing each tissue type, and there are no comparative studies on transplantation outcomes using these different culture techniques. To overcome this limitation, we devised a simple protocol that can uniformly promote growth and differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. METHODS: Biopsies were cultured as explants on de-epithelialized human amniotic membrane in the presence of recombinant epidermal growth factor and insulin. Cultured cells were characterized using immunohistochemistry and quantitative reverse transcriptase polymerase chain reaction for stem/progenitor markers (ABCG2 and P63α) and differentiation markers (CK3, CK12, CK4, CK13, CK15 and CONNEXIN 43). Fluorescence-activated cell sorter analysis was performed for ABCG2. RESULTS: The results revealed that cells of all three biopsies differentiated into the corneal lineage. Positivity of CK3/12, CK4, CK12 and CONNEXIN 43 immunostaining and the relative mRNA expression of CK3, CK4, CK12, CK13, CK15 and CONNEXIN 43 could be detected in the cultured biopsies. CONCLUSIONS: Unlike tissue-specific protocols, our protocol can unequivocally promote differentiation of cells from a limbal, conjunctival or oral mucosal biopsy into the corneal lineage. This simple standardized protocol can be adapted for ocular surface reconstruction using stem cell transplantation.

Characterization of ex vivo cultured limbal, conjunctival, and oral mucosal cells: A comparative study with implications in transplantation medicine.

PURPOSE: Limbal epithelial stem cell deficiency is caused by exposure of the cornea to thermal, chemical, or radiation burns or by diseases (aniridia and Stevens-Johnson syndrome). Autologous cell transplantation is a widely used therapeutic modality for restoring the corneal surface in such pathological conditions. Ex vivo cultured limbal, conjunctival, and oral biopsies have been widely used to reconstruct the corneal surface with variable outcomes. Culture characterization of the ex vivo cultured cells would provide insight and clues into the underlying signaling mechanisms that would aid in determining the probable transplantation outcome. Comparison of the vital proteins and genes among the three ex vivo cultured tissues has implications in clinical practice. To address this issue, we characterized and compared the proliferative and differentiated properties of ex vivo cultured limbal, conjunctival, and oral biopsies used for cell-based therapy for corneal surface restoration. METHODS: Limbal, conjunctival, and oral biopsies were collected with informed patient consent. Explant cultures were established on the denuded human amniotic membrane with corneal lineage differentiation medium. The day 14 cultures were characterized for epithelial and corneal lineage-specific markers using reverse transcription (RT)-PCR for cytokeratin 3, 4, 12, 13, 15, connexin 43, vimentin, p63α, and ABCG2 markers. mRNA expression was estimated in day 14 cultures with real-time quantitative real time (qRT)-PCR for pluripotency markers (OCT4, SOX2, NANOG), putative corneal stem cell markers (ABCG2 and p63α), proliferation markers (cyclin d1, Ki-67, PCNA, and CDC20), apoptotic markers (BCL2, BAX, caspase 3, and caspase 9), Notch signaling pathway markers (Notch1, Jagged1, Hes1, Hes3, Hes5, and Hey1), and autophagic markers (LC3A, LC3B, ATG7, RAB7, LAMP1, and LAMP2). Fluorescence-activated cell sorter profiling was performed for pluripotent markers and putative corneal stem cell markers ABCG2 and p63α. RESULTS: The protein and mRNA expression levels of the pluripotent markers were lower, whereas those of the putative stem/progenitor markers ABCG2, ΔNp63α, and Notch signaling molecules (Notch1 and Jagged1) were elevated in limbal cultures. The gene expression levels of the autophagy markers (LC3A, LC3B, and LAMP1) were significantly increased in the limbal cultures compared to the oral and conjunctival cultures. CONCLUSIONS: In conclusion, the limbal epithelial cultures showed higher expression of proliferative, limbal stem cell marker, Notch signaling, and autophagy markers suggesting a role in stem cell maintenance and differentiation. This implicates the probable factors that might drive a successful transplantation. Our findings provide the initial steps toward understanding transplantation medicine in an ex vivo model.

Safety profile of accelerated corneal cross-linking versus conventional cross-linking: a comparative study on ex vivo-cultured limbal epithelial cells.

AIM/BACKGROUND: To compare the effects of accelerated corneal collagen cross-linking (ACXL) and corneal collagen cross-linking (CXL) on ex vivo-cultured limbal epithelial cells (LECs). METHODS: Day 14 cultured LECs were either unexposed (control) or exposed to different intensities of ultraviolet-A (UV-A) irradiance for different durations (3 mW for 30 min, 9 mW for 10 min, 18 mW for 5 min and 30 mW for 3 min) in the presence and absence of riboflavin. These cells were further processed for quantitative real-time PCR, vital staining, immunofluorescence staining and fluorescence-activated cell sorting (FACS) staining to evaluate the apoptotic status. Statistical analysis was performed using a Student t test. RESULTS: Vital staining showed a significantly higher (p=0.004) dead cell population with 3 mW for 30 min when compared with 30 mW for 3 min exposure (p=0.225). Quantitative PCR results revealed significantly reduced abcg2 and Δnp63 mRNA levels, while FACS analysis showed an increase in ABCG2-Annexin V positive population in cells exposed to 3 mW for 30 mins. Neither reduction of mRNA expression of abcg2 and Δnp63 nor increase in FACS-stained ABCG2-Annexin V positivity was detected in cells exposed to 30 mW for 3 min. Additionally, enhanced caspase activity was detected with fluorochrome inhibitor of caspases staining and mRNA expression of caspase 3 and 9 was upregulated in cells exposed to 3 mW for 30 min, but not at 30 mW for 3 min. CONCLUSIONS: The 30 mW UV-A irradiation used in ACXL appears to be safe on cultured LECs in comparison with 3 mW used in CXL.

Potential apoptotic effect of ultraviolet-A irradiation during cross-linking: a study on ex vivo cultivated limbal epithelial cells.

AIM/BACKGROUND: To study the effects of ultraviolet-A (UV-A) irradiation, in the presence or absence of riboflavin, on ex vivo cultured limbal epithelial cells (LECs). METHODS: The study was carried out in a super specialty ophthalmic hospital. Ex vivo cultured LECs were grown on denuded amniotic membranes and exposed to similar levels of UV-A radiation used during corneal cross-linking (CXL), in the presence or absence of the photosensitiser, riboflavin. These cells were then used for extraction of RNA, cDNA conversion, and antibody staining. Quantitative PCR and immunofluorescence staining were performed to evaluate the apoptotic state of treated and non-treated LECs. Statistical analyses were evaluated using a Student's t test. RESULTS: We found that bcl-2, an antiapoptotic gene, was downregulated, whereas, bax, a proapoptotic gene, was upregulated. After LECs were exposed to UV-A radiation, a significant upregulation of both caspase 3 and caspase 9 was observed in treated cells when compared with untreated LECs. CONCLUSIONS: These results indicate that exposure of LECs to UV-A dosages similar to those used in the CXL procedure promotes the expression of genes known to promote apoptosis. In the presence of riboflavin, the damage caused by UV-A treatment was marginalised, but not totally blocked.