The ultrastructure of rabbit sclera after scleral crosslinking with riboflavin and blue light of different intensities.

PURPOSE: We aimed to determine the ultrastructural changes of collagen fibrils and cells in the rabbit sclera after scleral crosslinking using riboflavin and blue light of different intensities. Scleral crosslinking is known to increase scleral stiffness and may inhibit the axial elongation of progressive myopic eyes. METHODS: The equatorial parts of the sclera of one eye of six adult albino rabbits were treated with topical riboflavin solution (0.5 %) followed by irradiation with blue light (200, 400, 650 mW/cm(2)) for 20 min. After 3 weeks, the ultrastructure of scleral cells and the abundance of small- (10-100 nm) and large-diameter (>100 nm) collagen fibrils in fibril bundles of different scleral layers were examined with electron microscopy. RESULTS: In the scleral stroma of control eyes, the thickness of collagen fibrils showed a bimodal distribution. The abundance of small-diameter collagen fibrils decreased from the inner towards the outer sclera, while the amount of large-diameter fibrils and the scleral collagen content did not differ between different stroma layers. Treatment with riboflavin and blue light at 200 mW/cm(2) did not induce ultrastructural changes of cells and collagen fibrils in the scleral stroma. Treatment with blue light of higher intensities induced scleral cell activation in a scleral layer-dependent manner. In addition, outer scleral layers contained phagocytes that engulfed collagen fibrils and erythrocytes. Blue light of the highest intensity induced a reduction of the scleral collagen content, a decreased abundance of large-diameter collagen fibrils, and an increased amount of small-diameter fibrils in the whole scleral stroma. CONCLUSIONS: The data indicate that in rabbits, scleral crosslinking with riboflavin and blue light of 200 mW/cm(2) for 20 min is relatively safe and does not induce ultrastructural alterations of scleral cells and of the collagen composition of the scleral stroma. Irradiation with blue light of intensities between 200 and 400 mW/cm(2) induces scleral cell activation, which may contribute to scleral scarring and stiffening. Higher intensities cause scleritis.

Scleral cross-linking by riboflavin and blue light application in young rabbits: damage threshold and eye growth inhibition.

BACKGROUND: Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition. METHODS: The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging. RESULTS: Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks. CONCLUSIONS: SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.

Damage threshold in adult rabbit eyes after scleral cross-linking by riboflavin/blue light application.

Several scleral cross-linking (SXL) methods were suggested to increase the biomechanical stiffness of scleral tissue and therefore, to inhibit axial eye elongation in progressive myopia. In addition to scleral cross-linking and biomechanical effects caused by riboflavin and light irradiation such a treatment might induce tissue damage, dependent on the light intensity used. Therefore, we characterized the damage threshold and mechanical stiffening effect in rabbit eyes after application of riboflavin combined with various blue light intensities. Adult pigmented and albino rabbits were treated with riboflavin (0.5 %) and varying blue light (450 ± 50 nm) dosages from 18 to 780 J/cm(2) (15 to 650 mW/cm(2) for 20 min). Scleral, choroidal and retinal tissue alterations were detected by means of light microscopy, electron microscopy and immunohistochemistry. Biomechanical changes were measured by shear rheology. Blue light dosages of 480 J/cm(2) (400 mW/cm(2)) and beyond induced pathological changes in ocular tissues; the damage threshold was defined by the light intensities which induced cellular degeneration and/or massive collagen structure changes. At such high dosages, we observed alterations of the collagen structure in scleral tissue, as well as pigment aggregation, internal hemorrhages, and collapsed blood vessels. Additionally, photoreceptor degenerations associated with microglia activation and macroglia cell reactivity in the retina were detected. These pathological alterations were locally restricted to the treated areas. Pigmentation of rabbit eyes did not change the damage threshold after a treatment with riboflavin and blue light but seems to influence the vulnerability for blue light irradiations. Increased biomechanical stiffness of scleral tissue could be achieved with blue light intensities below the characterized damage threshold. We conclude that riboflavin and blue light application increased the biomechanical stiffness of scleral tissue at blue light energy levels below the damage threshold. Therefore, applied blue light intensities below the characterized damage threshold might define a therapeutic blue light tolerance range.

Dose-dependent collagen cross-linking of rabbit scleral tissue by blue light and riboflavin treatment probed by dynamic shear rheology.

PURPOSE: To determine the visco-elastic properties of isolated rabbit scleral tissue and dose-dependent biomechanical and morphological changes after collagen cross-linking by riboflavin/blue light treatment. MATERIAL: Scleral patches from 87 adult albino rabbit eyes were examined by dynamic shear rheology. Scleral patches were treated by riboflavin and different intensities of blue light (450 nm), and the impact on the visco-elastic properties was determined by various rheological test regimes. The relative elastic modulus was calculated from non-treated and corresponding treated scleral patches, and treatments with different blue light intensities were compared. RESULTS: Shear rheology enables us to study the material properties of scleral tissue within physiological relevant parameters. Cross-linking treatment increased the viscous as well as the elastic modulus and changed the ratio of the elastic versus viscous proportion in scleral tissue. Constant riboflavin application combined with different blue light intensities from 12 mW/cm(2) up to 100 mW/cm(2) increased the relative elastic modulus of scleral tissue by factors up to 1.8. Further enhancement of the applied light intensity caused a decline of the relative elastic modulus. This might be due to destructive changes of the collagen bundle structure at larger light intensities, as observed by histological examination. CONCLUSION: Collagen cross-linking by riboflavin/blue light application increases the biomechanical stiffness of the sclera in a dose-dependent manner up to certain light intensities. Therefore, this treatment might be a suitable therapeutic approach to stabilize the biomechanical properties of scleral tissue in cases of pathological eye expansion.

Laboratory measurement of the absorption coefficient of riboflavin for ultraviolet light (365 nm).

PURPOSE: Corneal cross-linking (CXL) is an increasingly used treatment technique for stabilizing the cornea in keratoconus. Cross-linking (polymerization) between collagen fibrils is induced by riboflavin (vitamin B2) and ultraviolet light (365 nm). Although reported to reach a constant value at higher riboflavin concentrations, the Lambert-Beer law predicts a linear increase in the absorption coefficient. This work was carried out to determine absorption behavior at different riboflavin concentrations and to further investigate the purported plateau absorption coefficient value of riboflavin and to identify possible bleaching effects. METHODS: The Lambert-Beer law was used to calculate the absorption coefficient at various riboflavin concentrations. The following investigated concentrations of riboflavin solutions were prepared using a mixture of 0.5% riboflavin and 20% Dextran T500 dissolved in 0.9% sodium chloride solution: 0%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%, and were investigated with and without aperture plate implementation. An additional test series measured the transmitted power at selected riboflavin concentrations over time. RESULTS: In diluted solutions, a linear correlation exists between the absorption coefficient and riboflavin concentration. The absorption coefficient reaches a plateau, but this occurs at a higher riboflavin concentration (0.1%) than previously reported (just above 0.04%). Transmitted light power increases over time, indicating a bleaching effect of riboflavin. CONCLUSIONS: The riboflavin concentration can be effectively varied as a treatment parameter in a considerably broader range than previously thought.

Scheimpflug imaging of corneas after collagen cross-linking.

PURPOSE: To compare geometrical shape factors of keratoconus corneas after cross-linking (CXL) by means of Scheimpflug imaging with those of untreated fellow eyes. SETTING: Institut für Refraktive und Ophthalmo-Chirurgie, Zürich, Switzerland. METHODS: Scheimpflug imaging of the anterior segments was performed with the Pentacam (Oculus, Wetzlar, Germany) in 21 patients with progressive keratectasia before and after CXL. Only 1 eye per patient was treated with corneal cross-linking using the riboflavin/UV-A approach, the fellow eye serving as control. The following corneal parameters and their postoperative evolution during 1 year after treatment have been evaluated: minimal curvature radius and its location, thickness at the thinnest point, location of the thinnest point, anterior and posterior elevation, conoid asphericity constants of the anterior and posterior surface, and 7 keratoconus indices. Statistical comparison was performed by means of the Wilcoxon test. RESULTS: None of the treated eyes showed topographic progression in contrast to the untreated group where 8 eyes experienced significant progression. Minimal curvature radius increased significantly after 1 year compared with preoperative (6.14-6.21 mm), whereas in the untreated fellow eye, it significantly decreased (6.94-6.86 mm). Minimal corneal thickness was significantly reduced after treatment (P < 0.002 at 12 months). The cornea showed an evolution toward a more regular shape as indicated by a significant reduction in 4 of 7 keratoconus indices. No complications of CXL occurred in this small study group. CONCLUSIONS: After cross-linking, the corneal shape undergoes a process of regularization. This process is active during the first year after treatment and may continue. Longer follow-up is warranted to estimate the full amount of regression of the keratectasia after CXL.

Collagen crosslinking with ultraviolet-A and hypoosmolar riboflavin solution in thin corneas.

Corneal collagen crosslinking (CXL) with riboflavin and ultraviolet-A light is a method for treating progressive keratectasia. The currently accepted treatment parameters induce collagen crosslinking in the anterior 250 to 350 microm of corneal stroma. To protect the endothelium, CXL inclusion criteria require a minimum corneal thickness of 400 microm after removal of the epithelium. In advanced keratoconus, however, progressive corneal thinning often leads to a remaining stromal thickness of less than 400 microm. We have therefore modified the current treatment protocol by preoperatively swelling thin corneas to a stromal thickness of at least 400 microm using hypoosmolar riboflavin solution. This treatment protocol was performed in a case series of 20 patients, and no complications were observed. Preoperative swelling of the cornea safely broadens the spectrum of CXL indications to thin corneas that would otherwise not be eligible for treatment.

Efficacy and safety of blue-light scleral cross-linking.

PURPOSE: To evaluate the efficacy of blue-light scleral cross-linking as well as its safety in preventing retinal damage beneath the treated sclera. METHODS: Six rabbits were unilaterally treated with topical riboflavin (0.5%) and blue light (465 nm) on the equatorial sclera using a light emitting diode source with an exposure area of 9 mm in diameter. Four weeks after the treatment, the animals were euthanized and the exposed sclera and contralateral eye sclera excised for comparative testing of biomechanical rigidity and histologic retinal cellular damage. Extensiometry was performed to evaluate the stress-strain curve of treated versus untreated sclera, and light microscopy of the treated sclera and underlying retina were also comparatively evaluated. RESULTS: Blue-light scleral cross-linking showed a three-fold increased stiffening in all tested animals in the stress-strain curve. Histological investigation revealed no retinal damage in any of the treated eyes. CONCLUSIONS: Scleral cross-linking with riboflavin and blue light (465 nm) has a stiffening effect on the sclera without histological tissue damage to the retina.

Ultraviolet A/riboflavin corneal cross-linking for infectious keratitis associated with corneal melts.

PURPOSE: To evaluate the efficacy of ultraviolet-corneal cross-linking (CXL) for treating infectious melting keratitis. METHODS: Five patients with infectious keratitis associated with corneal melting were treated with CXL at the outpatient departments of the Institut für Refraktive und Ophthalmo-Chirurgie and the eye hospital at the University of Zurich. CXL was performed when the infection did not respond to systemic and topical antibiotic therapy. Follow-up after cross-linking ranged from 1 to 9 months. RESULTS: In all cases, the progression of corneal melting was halted after CXL treatment. Emergency keratoplasty was not necessary in any of the 5 cases presented. CONCLUSIONS: CXL is a promising option for treating patients with therapy-refractory infectious keratitis to avoid emergency keratoplasty.

Pregnancy-related exacerbation of iatrogenic keratectasia despite corneal collagen crosslinking.

Iatrogenic keratectasia after laser in situ keratomileusis (LASIK) represents a serious complication of refractive laser surgery. We describe a woman who developed bilateral iatrogenic keratectasia during her first pregnancy 26 months after LASIK. Corneal collagen crosslinking (CCL) with riboflavin and ultraviolet-A was performed in March 2005 (right eye) and April 2005 (left eye). This treatment stopped the progression and even caused the keratometric steepness to regress over a postoperative follow-up of 22 months, as demonstrated by preoperative and postoperative corneal topographies and maximum K-readings. During the patient's second pregnancy, the keratectasia exacerbated. To our knowledge, this is the first case showing exacerbation of keratectasia despite CCL and, as the exacerbation occurred only during pregnancy, suggesting that hormonal changes might affect corneal biomechanical stability.

Topography-guided surface ablation for forme fruste keratoconus.

PURPOSE: To evaluate the efficacy of customized surface ablation in cases of forme fruste keratoconus. DESIGN: Prospective noncomparative case series. PARTICIPANTS: Eleven eyes of 8 contact lens-intolerant patients with forme fruste keratoconus treated at the Institute of Refractive and Ophthalmic Surgery and the University Eye Clinic Zurich. INTERVENTION: Topography-guided customized surface ablation by means of a scanning spot excimer laser. MAIN OUTCOME MEASURES: Visual acuity, refraction, quality of vision (ghosting), corneal topography including the Zernike parameter Z3. RESULTS: Statistically significant reduction of manifest refractive error, corneal irregularity, and ghosting. The spherical equivalent was reduced by -2.8+/-0.62 diopters (D) (P = 0.0007), the cylinder by 1.34 +/- 0.18 D (P = 0.015), Z3 was reduced by 41% (P<0.001), and all patients had less ghosting compared to their preoperative status. No eye lost > or =1 lines in best spectacle-corrected visual acuity; however, 7 of 11 eyes gained > or = 1 line. CONCLUSION: Topography-guided surface ablation is a promising option to rehabilitate vision in contact lens-intolerant patients with forme fruste keratoconus.

Corneal and total wavefront aberrations in phakic and pseudophakic eyes after implantation of monofocal foldable intraocular lenses.

PURPOSE: To compare the correlation between corneal and total wavefront aberrations in normal phakic and pseudophakic eyes after implantation of foldable monofocal intraocular lenses (IOLs). SETTING: University Hospital, Eye Clinic, Zurich, Switzerland. METHODS: Wavefront aberrations and corneal topography of 29 eyes that had cataract surgery with implantation of hydrophobic monofocal foldable IOL (AcrySof, Alcon Labs) were measured at least 2 months postoperatively and compared with wavefront measurements performed in 33 normal young phakic eyes. The total wavefront aberrations were measured by means of a Tscherning wavefront sensor at a wavelength of 660 nm (Allegro Wave Analyzer, WaveLight Laser Technology). The corneal aberrations were derived from corneal topography measurements ascertained with a Placido-based topography system (Keratograph 70600, Oculus). The correlations between corneal and total wavefront aberrations were calculated for all Zernike coefficients from 2nd up to 6th order. RESULTS: There was a significant correlation between corneal and total wavefront aberrations in astigmatism C3 and C5 as well as for all 3rd-order Zernike coefficient in both groups (except C8 in the pseudophakic group). The correlation between corneal and total astigmatism (C3 and C5) was higher in the pseudophakic than in the phakic eyes. In contrast, the correlation for the coma-like aberrations was weaker in the pseudophakic eyes (R>0.18) than in the group of phakic eyes (R>0.58). In both groups, there was no significant correlation between spherical aberration C12 of the cornea and the C12 of the total eye. CONCLUSION: After cataract surgery with an IOL implantation, both vertical and horizontal coma, as well as spherical aberration, were of higher value than in normal eyes. The compensation effect for corneal aberrations of the natural lens is absent in the IOL and explains these findings. The corneal aberrations in pseudophakic eyes reflect better the optical quality of the total eye than the phakic eyes. Nevertheless, the missing correlation in some specific aberrations, such as C8 and C10, shows the inability of corneal topography to provide suitable information on the optical quality of the total eye after cataract surgery. Thus, both corneal and total wavefront measurements are relevant for the assessment of outcomes after cataract surgery.

Q-factor customized ablation profile for the correction of myopic astigmatism.

PURPOSE: To compare the results of the Q-factor customized aspheric ablation profile with the wavefront-guided customized ablation pattern for the correction of myopic astigmatism. SETTING: Institute for Refractive and Ophthalmic Surgery, Zurich, Switzerland. METHODS: Thirty-five patients were enrolled in a controlled study in which the nondominant eye was treated with the Q-factor customized profile (custom-Q study group) and the dominant eye was treated with wavefront-guided customized ablation (control group). Preoperative and 1-month postoperative high-contrast visual acuity, low-contrast visual acuity, and glare visual acuity, as well as aberrometry and asphericity of the cornea, were compared between the 2 groups. All eyes received laser in situ keratomileusis surgery, and the laser treatment was accomplished with the Wavelight Eye-Q 400 Hz excimer laser. RESULTS: For corrections up to -9 diopters (D) of myopia, there were no statistically significant differences between the 2 groups regarding any visual or optical parameter except coma-like aberrations (3rd Zernike order), where the wavefront-guided group was significantly better 1 month after surgery (P = .002). For corrections up to -5 D (spherical equivalent), the Q-factor optimized treated eyes had a significantly smaller shift toward oblate cornea: DeltaQ15 = 0.25 in Q-factor customized versus DeltaQ15 = 0.38 in wavefront-guided treatment (P = .04). CONCLUSIONS: Regarding safety and refractive efficacy, custom-Q ablation profiles were clinically equivalent to wavefront-guided profiles in corrections of myopia up to -9 D and astigmatism up to 2.5 D. Corneal asphericity was less impaired by the custom-Q treatment up to -5 D of myopia.

The effect of phenylephrine and cyclopentolate on objective wavefront measurements.

PURPOSE: To investigate the impact of phenylephrine and cyclopentolate on wavefront refraction and fourth order spherical aberration C12. METHODS: This cohort study comprised 151 eyes with sphere up to -10.00 diopters (D) and cylinder -3.75 D. Aberrometry was performed using the ALLEGRO WAVE (WaveLight Laser Technologies AG, Erlangen, Germany) after instillation of phenylephrine 5% yielding objective phenylephrine refraction in accommodated steady-state, as well as after cyclopentolate 0.5% providing objective cyclopentolate refraction in non-accommodated state. Accommodation target fogging was turned off. Wavefront aberrations were expressed by Zernike expansion up to the sixth order, and paraxial curvature matching with Taylor series was used to calculate objective wavefront sphere. RESULTS: Objective wavefront sphere was not influenced by pupil size. Eyes showed substantial accommodation after phenylephrine with a myopic shift of -0.66 D comparing objective to subjective manifest sphere (r=0.942, P<.001). Cycloplegic eyes behaved like a model eye, with a difference of -0.08 D between objective and subjective cycloplegic sphere (r=0.976, P<.001). C12 increased ten-fold from 4.0- to 7.0-mm pupil size, keeping the same sign. Comparing cyclopentolate with phenylephrine, the sign of C12 changed in a positive direction by an average +0.124 +/- 0.109 microm (range: -0.052 to +0.632 microm) at 7.0 mm, whereas the total higher order aberrations changed very little. A good correlation was found between C12 and the change in objective wavefront sphere between cyclopentolate and phenylephrine (r=0.75, P<.001). CONCLUSIONS: Fogging of the accommodation target should be used for wavefront measurements. Weaker cycloplegic agents, such as tropicamide, may be used to ensure relaxed but not completely paralyzed accommodation, which would yield "manifest" aberration values close to the natural resting state.

Treatment-induced shifts of ocular reference axes used for measurement centration.

PURPOSE: To determine the shifts of the main corneal reference points in dependence of the chosen centration axis for the treatment. SETTING: Federal Institute of Technology Zurich, Institute of Biomedical Engineering, Zurich, Switzerland. METHODS: Computer simulations were performed on several variants of the Gullstrand-Emsley schematic eye, which was modified by an off-axis fovea. Refractive corrections were simulated by centering Munnerlyn's formula on each of the 4 corneal reference points determined in the preoperative eye: the optical axis, the line of sight, the visual axis, and the first corneal reflex. Subsequently, the postoperative locations of these axes were determined and compared with the preoperative values. RESULTS: The postoperative line of sight was found to depend least on the choice of the preoperative centration axis for both myopic and hyperopic treatments. It undergoes a maximum movement of 0.040 mm when centering a +5 diopter correction on the preoperative line of sight, whereas the corneal reflex, which is used for centering most topography systems, can move by more than 0.10 mm. CONCLUSIONS: Centration of the correction on the preoperative line of sight enabled good comparability between preoperative and postoperative measurements that use the line of sight as a reference axis. Yet, centration of the treatment on the preoperative line of sight does not ensure comparability between preoperative and postoperative measurements that use the corneal reflex as a reference axis such as most corneal topography systems. Axis shifts might lead to misinterpretation of data such as a wrong diagnosis of a decentered ablation or changes in the Zernike representation.

Clinical photoablation with a 500-Hz scanning spot excimer laser.

PURPOSE: The aim of this study was to use a 500-Hz scanning spot laser (Concept500, WaveLight Laser Technologie AG, Erlangen, Germany) to investigate potential side effects that might be associated with the use of a high repetition rate laser platform. METHODS: Seven eyes were treated using a 500-Hz scanning spot laser for laser in situ keratomileusis (LASIK). The local frequency of the ablation was kept below 40 Hz to avoid local heating of corneal tissue. With the exception of the high repetition rate (500 Hz), all other laser parameters such as fluence, algorithm, ablation profile, and spot diameter were identical to a standard WaveLight Allegretto laser system. Patients were examined at 1 month and 1 year after initial treatment. Preoperative and postoperative examination included manifest sphere and cylinder, uncorrected and best spectacle-corrected visual acuity (BSCVA). RESULTS: All eyes were treated for myopia or myopic astigmatism. Five eyes received spherocylindrical and two eyes spherical ablation only. No adverse events correlated with the use of a high repetition rate laser system were observed during surgery or at any point during follow-up. All eyes maintained or had improved BSCVA at 12 months after treatment when compared to preoperative values. CONCLUSION: The use of an excimer laser with a maximal repetition rate of 500 Hz and a local repetition rate of less than 40 Hz was free of any specific side effect that might be associated with the use of such a high repetition rate.

Transferring wavefront measurements into corneal ablations: an overview of related topics.

We give an overview of possible side effects that are specific for, or of particular relevance in, customized treatments. Certain processes involved in customized ablations have the potential to alter the quality of the optical correction. Professionals associated with customized treatment should be informed and trained with respect to possible sources of error.

Conservative treatment of vertical diplopia in a patient with silent sinus syndrome.

Silent sinus syndrome is a rare disease of the maxillary sinus characterized by bony absorption processes leading to progressive sinus wall thinning with consecutive enophthalmos and hypoglobus. It represents a benign cause of acquired enophthalmos and is often accompanied by painless vertical diplopia, the latter treated surgically in all cases published to date. We report a 56-year-old patient with silent sinus syndrome in whom vertical diplopia was treated with prisms showing that conservative treatment alone may, in mild cases, be an effective alternative to reconstructive surgery.

Light damage susceptibility and RPE65 in rats.

A sequence variation in the pigment epithelial protein RPE65 has been shown to correlate with RPE65 protein levels, rhodopsin regeneration kinetics and light damage susceptibility in different mouse strains. Here, we tested whether such a correlation can also be found in rats. We examined four rat strains for RPE65 protein levels and the Rpe65 gene sequence. In two strains, we additionally determined Rpe65 mRNA levels, rhodopsin regeneration and light damage susceptibility (LDS).RPE65 protein levels were higher in Lewis and Brown Norway rats compared to Wistar and Long Evans. The albino strains Wistar and Lewis were investigated further. Lewis had higher Rpe65 mRNA levels than Wistar. Sequence analysis of the coding region of the Rpe65 cDNA revealed no relevant sequence variations in the two strains. Content and regeneration of rhodopsin were comparable in both strains. However, Wistar rats were more susceptible to light damage than Lewis. We conclude that lower RPE65 protein levels in Wistar may have been caused by decreased gene expression and not by a sequence variation as suggested for mice. In rats, RPE65 may not be a limiting factor for rhodopsin regeneration. Since LDS in rats did not directly correlate with RPE65 protein levels and rhodopsin regeneration, other yet unidentified (genetic) factors may account for the susceptibility differences observed in rats.