Corneal Microstructure Research Articles

Comprehensive assessment of corneal microstructural changes following V4c implantable collamer lens surgery using in vivo confocal microscopy

BackgroundImplantable Collamer Lense (ICL) presents a viable alternative to conventional refractive surgeries, but their impact on corneal microstructure remains unclear. By employing in vivo confocal microscopy (IVCM), we examined changes in stromal and endothelial cells following the insertion of V4c ICLs, with the goal of enhancing post-surgical care and outcomes.MethodsIn this longitudinal investigation, we conducted detailed preoperative assessments on 103 eyes from 53 participants. Follow-up evaluations were carried out after surgery at set intervals: one day, one week, one month, three months, six months, and twelve months. We used IVCM to analyze changes in stromal and endothelial cells. To assess differences between pre- and post-surgery variables and to investigate correlations with age, axial length (AL), and spherical equivalent refraction (SER), we applied a repeated measures mixed-effects model, with statistical significance set at P < 0.05.ResultsNo vision-threatening complications were reported post-surgery. Significant reductions in stromal cell density (SCD) were observed postoperatively, with anterior and mid- SCD reaching their lowest values at 3 months and posterior SCD at 1 month, remaining below baseline at 12 months. endothelial cell density (ECD) and percentage of hexagonal cells (PHC) decreased initially, recovering by 12 months. Conversely, endothelial cellular area (ECA) and coefficient of variation of cell size (CoV) increased postoperatively, with the most significant change at 1 week. Endothelial deposits were detected in 49 of 101 eyes on postoperative day 1, half of them were absorbed within 3 months post-surgery. Changes in posterior SCD were negatively related to AL, while AL, SER, lens thickness showed associated with endothelium changes.ConclusionOur findings elucidate the corneal microstructural changes following V4c ICL implantation, particularly the significant early reductions in stromal and endothelial cell densities. We recommend careful management of viscoelastics during surgery to minimize endothelial deposits that may harm the endothelium. Enhanced early postoperative monitoring and these surgical adjustments can lead to improved surgical and post-surgical care, ultimately supporting better patient recovery.

Corneal Epithelial Cell Density Is Reduced in Young Adults With Conjunctival Ultraviolet Autofluorescence.

The aim of this study was to investigate the effects of chronic ultraviolet (UV) radiation exposure on the corneal microstructure using conjunctival UV autofluorescence (CUVAF) as a marker of exposure. Young healthy adults aged 18 to 35 years were recruited. Participant's demographics and sun exposure behavior were recorded using questionnaires. Images of the bulbar conjunctiva from the right eye were captured and analyzed for the CUVAF area. Corneal microstructure was assessed by in vivo confocal microscopy and anterior segment optical coherence tomography. The presence of palisades of Vogt in all 4 limbal quadrants was recorded. CUVAF was observed in 31 of 52 eyes (60%), with a mean (±SD) nasal, temporal, and total CUVAF area of 5.39 ± 4.16 mm 2 , 4.29 ± 4.27 mm 2 , and 9.67 ± 7.01 mm 2 , respectively. Individuals with CUVAF were significantly more likely to report undertaking moderate-intensity to high-intensity outdoor exercise ( P = 0.021). Individuals with CUVAF were less likely to have visible nasal and temporal palisades of Vogt ( P = 0.011). Corneal basal cell densities anterior to the nerve whorl ( P < 0.001) and nasally ( P = 0.005) were lower in individuals with CUVAF. Wing cell density anterior to the nerve whorl was lower in individuals with CUVAF ( P = 0.011). No significant changes in the central and limbal corneal epithelial thickness were observed. Significant reductions in corneal epithelial cell density were observed in individuals with CUVAF, a biomarker of chronic UV exposure. This observation suggests that chronic UV exposure is damaging to the corneal microstructure.

A pilot study on Langerhans cells in keratoconus patients by in vivo confocal microscopy before and after corneal cross-linking and correlation with eye rubbing

PurposeThe aim of this study was to investigate the corneal microstructure and Langerhans cells using in vivo confocal microscopy in keratoconus patients before and after cross-linking, and to correlate the morphologic findings with clinical and patient-reported outcomes, including eye rubbing (ER) behavior. MethodsPatients with progressive keratoconus undergoing iontophoresis-assisted epithelium-on cross-linking (I-CXL) were consecutively enrolled. In vivo confocal microscopy was performed before and 6 months after treatment. Patients were asked to quantify their ER behavior on a Visual Analogue Scale (VAS) and completed the Keratoconus Outcomes Research Questionnaire and the Ocular Surface Disease Index questionnaires at the same time points. Visual acuity, tear osmolarity, topography, aberrometry, and pachymetry of both eyes were assessed. ResultsThirteen patients were included in this pilot study. Preoperatively, the mean Langerhans cells density was 35,615 cells per mm2, and the median morphology was 3. The mean ER VAS before treatment was 7,077 out of 10. The ER VAS showed significant positive correlations with both Langerhans cells density and morphology of the study eye. After treatment, a statistically significant reduction in ER VAS and in Langerhans cells variables was observed. The mean sub-basal plexus nerve density was comparable to pre-operative values 6 months after I-CXL. ConclusionsBased on this preliminary evidence, the presence of high density of mature Langerhans cells in the central cornea of keratoconus patients and its correlation with eye rubbing support the role of inflammation in keratoconus. The reduction in these markers after treatment may suggest a potential of CXL in moderating immune-related inflammation and eye rubbing in the medium term.

Effect of upper eyelid blepharoplasty on the ocular surface, tear film, and corneal microstructure.

This study aimed to investigate the effect of upper eyelid blepharoplasty with the removal of the skin and a strip of orbicularis oculi muscle on the ocular surface, tear film, and dry eye-related symptoms. Twenty-two eyes of 22 consecutive patients operated by a single surgeon (21 females; mean age, 61 years; age range, 41-75 years) were included. All subjects completed the Ocular Surface Disease Index questionnaire, underwent in vivo confocal microscopy, tear film breakup time measurements, the Schirmer test with anesthesia, and fluorescein and lissamine green staining measurements before, 1 month, and 6 months after upper blepharoplasty alone with preseptal orbicularis excision. A significant increase in Ocular Surface Disease Index, and corneal fluorescein and lissamine green staining and a significant decrease in tear film breakup time were observed after 1 month (p=0.003, p=0.004, p=0.029, and p=0.024 respectively) and 6 months (p=0.001 for all findings). No significant difference in the Schirmer test score was observed during the follow-up. None of the in vivo confocal microscopy parameters showed significant changes during the study. An increase in dry eye symptoms and a decrease in tear film stability along with ocular surface staining were observed in patients undergoing upper eyelid blepharoplasty.

Measuring intraocular pressure with OCT: the first approach.

The variability of corneal OCT speckle statistics is indirectly related to changes in corneal microstructure, which may be induced by intraocular pressure (IOP). A new approach is considered, which attempts to estimate IOP based on corneal speckle statistics in OCT images. An area (A) under trajectories of contrast ratio with respect to stromal depth was calculated. The proposed method was evaluated on OCT images from the ex-vivo study on porcine eyeballs and in-vivo study on human corneas. A statistically significant multivariate linear regression model was obtained from the ex-vivo study: IOP = 0.70 · A - 6.11, in which IOP was precisely controlled in the anterior chamber. The ex-vivo study showed good correlation between A and IOP (R = 0.628, at the least) whereas the in-vivo study showed poor correlation between A and clinical air-puff tonometry based estimates of IOP (R = 0.351, at the most), indicating substantial differences between the two studies. The results of the ex-vivo study show the potential for OCT speckle statistics to be utilized for measuring IOP using static corneal imaging that does not require corneal deformation. Nevertheless, further work is needed to validate this approach in living human corneas.

Ovalbumin-Induced Allergic Inflammation Diminishes Cross-Linked Collagen Structures in an Experimental Rabbit Model of Corneal Cross-Linking.

BackgroundAllergic conjunctivitis (AC) is one of the reported potential risk factors of progression in keratoconus patients after corneal cross-linking surgery; however, the causal relationship is still inconclusive. Recent studies have indicated that various inflammatory cytokines play a vital role in the development of primary keratoconus. It is still unclear whether these inflammatory mediators also trigger CXL failures. This study aimed to investigate the impact of AC on the rabbit corneas after trans-epithelial corneal cross-linking (TCXL).MethodsA total of six rabbits were kept untreated as the normal control (NC) group. A total of 18 rabbits were treated by TCXL and divided into three groups (six in each group), namely, no treatment (TCXL group); induction of AC (TCXL + AC group); and induction of AC plus topical prednisolone acetate (TCXL + AC + PA group), according to additional treatment. AC was induced by topical application of ovalbumin after intraperitoneal pre-sensitization with ovalbumin. Rabbits were evaluated by slit lamp, in vivo laser scanning confocal microscopy, anterior segment optical coherence tomography, and measurement of corneal biomechanics. The cornea specimens were collected for the transmission electron microscope, the collagenase I digestion test, and PCR assay for TNF-α, IL-6, IL-1β, matrix metalloproteinase 9 (MMP-9), lysyl oxidase (LOX), and tissue inhibitor of metalloproteinases 1 (TIMP-1) on the day (D) 28.ResultsOn D28, the TNF-α, IL-6, IL-1β, MMP-9, and LOX levels were significantly increased while the TIMP-1 was decreased in the TCXL + AC group when compared with the TCXL and TCXL + AC + PA groups. In vivo confocal microscopy revealed that at a depth of 150–210 μm, a trabecular patterned hyperdense structure surrounded by elongated needle-like processes could be observed in the TCXL and TCXL + AC + PA groups, but hardly seen in the TCXL + AC group. The demarcation lines were indistinct and blurred in the TCXL + AC group. An electron microscope demonstrated less interlacing fibril lamellae and higher interfibrillar spacing in the TCXL + AC group. The stability of corneal biomechanics and resistance to collagenase were decreased in the TCXL + AC group.ConclusionThe corneal microstructures induced by TCXL and biomechanical stability were diminished in rabbits with AC but could be maintained by topical anti-inflammatory treatment. Our results supported the causal relationship between altered cytokine profiles and corneal microstructure after primary corneal cross-linking.

Examination of corneal microstructure in the quiescent phase of vernal keratoconjunctivitis using in vivo confocal microscopy.

This study investigated the morphological characteristics of corneal microstructure in the quiescent phase of vernal keratoconjunctivitis (VKC). Twenty patients with quiescent VKC and 25 healthy subjects were included. In vivo confocal microscopy (IVCM) of the central cornea was performed. Cellular density of each layer and the morphology of subbasal nerve plexus (SBNP) was analysed. Langerhans cell density (LCD), morphology (LCM), and field area (LCF) were also examined. No differences were found either in cell densities nor in SBNP morphology (p > 0.05). LCD, LCM and LCF were significantly higher in the VKC group (p = 0.005, p < 0.001 and p < 0.001, respectively). The severity of papillary hypertrophy had a significant impact on LCD, LCM and LCF (β-coefficient: 19.541, p < 0.001; β-coefficient: 0.283, p < 0.001 and β-coefficient: 595.255, p < 0.001, respectively). In quiescent VKC, LCD, LCM, and LCF were increased, and they were associated with the severity of papillary hypertrophy. Alterations of Langerhans cells indicate a subclinical inflammatory process without ocular symptoms.

In Vivo Confocal Microscopy Evaluation in Patients with Keratoconus.

Keratoconus is the most common primary corneal ectasia characterized by progressive focal thinning. Patients experience increased irregular astigmatism, decreased visual acuity and corneal sensitivity. Corneal collagen crosslinking (CXL), a minimally invasive procedure, is effective in halting disease progression. Historically, keratoconus research was confined to ex vivo settings. In vivo confocal microscopy (IVCM) has been used to examine the corneal microstructure clinically. In this review, we discuss keratoconus cellular changes evaluated by IVCM before and after CXL. Cellular changes before CXL include decreased keratocyte and nerve densities, disorganized subbasal nerves with thickening, increased nerve tortuosity and shortened nerve fibre length. Repopulation of keratocytes occurs up to 1 year post procedure. IVCM also correlates corneal nerve status to functional corneal sensitivity. Immediately after CXL, there is reduced nerve density and keratocyte absence due to mechanical removal of the epithelium and CXL effect. Nerve regeneration begins after 1 month, with nerve fibre densities recovering to pre-operative levels between 6 months to 1 year and remains stable up to 5 years. Nerves remain tortuous and nerve densities are reduced. Corneal sensitivity is reduced immediately postoperatively but recovers with nerve regeneration. Our article provides comprehensive review on the use of IVCM imaging in keratoconus patients.

Effect of travoprost, latanoprost and bimatoprost PGF2α treatments on the biomechanical properties of in-vivo rabbit cornea

Prostaglandin F2α analogues (PGF2α), one of the most commonly prescribed classes of hypotensive agents, could decrease collagen fibril density and remodel the extracellular matrix in cornea. We hypothesized that PGF2α′s would induce corneal softening, reduce the accuracy of intraocular pressure (IOP) measurement and lead to uncertainty in the effectiveness of the therapy. We determined the stress-strain behavior of rabbit cornea after PGF2α usage and evaluated the effect of biomechanical changes associated with PGF2α treatment on IOP measurements by Goldmann Applanation Tonometry (GAT). The tangent modulus decreased after PGF2α treatment, while the stromal interfibrillar spacing increased. PGF2α was shown to also affect the lateral eye with lower effect, which did not undergo direct eyedrop treatment. Significant decreases in the numerical predictions of GAT-IOP were predicted in all treated groups relative to control groups. Different PGF2α′s (travoprost, latanoprost and bimatoprost) were associated with different extents of reduction in tissue stiffness and changes in corneal microstructure. PGF2α-induced changes in corneal mechanical properties could reduce the accuracy of IOP measurement and may cause an overestimation of the effect of PGF2α in lowering IOP, possibly leading to uncertainties in glaucoma management.

An in vivo confocal microscopy study of corneal changes in patients with systemic sclerosis

To investigate corneal microstructure of systemic sclerosis (SSc) patients using in vivo confocal microscopy (IVCM). 33 patients with SSc and 30 age-matched healthy subjects were recruited. All participants underwent comprehensive ophthalmic examination including IVCM (Heidelberg Retina Tomograph III, Heidelberg Engineering GmbH, Heidelberg, Germany) and ocular surface evaluation. Subbasal nerve plexus morphology was investigated using automated software analysis (ACCMetrics V3; University of Manchester, Manchester, UK). Keratocyte cell densities in the anterior stroma were significantly lower in patients with SSc compared to controls (P < 0.0001). In 7 SSc patients no keratocyte nuclei were identified in the anterior stroma and in most patients scattered hyperreflective punctate material were observed in the anterior stroma. Significantly lower subbasal nerve fiber parameters were found in patients with SSc compared to healthy subjects (P < 0.05). There were no significant correlations between the duration of SSc and any of the corneal cell density values. Tear break-up time values (4.82 ± 3.15 s) and Ocular Surface Disease Index scores (33.27 ± 30.11) were abnormal, Schirmer values (6.78 ± 5.82 mm) were borderline in SSc patients. In SSc, corneal morphological changes and accumulation of punctate material in the stroma was detected with confocal microscopy. Severe ocular surface disease was observed in SSc patients with significant impairment in subbasal nerve plexus morphology resembling peripheral neuropathy.

Corneal confocal microscopy in Fabry patients with cornea verticillata

To study the changes in corneal microstructure and nerve fibers in patients with cornea verticillata in Fabry disease. The study included patients with a confirmed diagnosis of Fabry disease. All patients underwent standard ophthalmological examination. Corneal confocal microscopy was performed when slit-lamp examination revealed cornea verticillata. Cornea verticillata was found in 64.5% of patients. Corneal microscopy revealed hyperreflective deposits in the basal cell layer of corneal epithelium in all patients with cornea verticillata. Characteristic features were desquamation of epithelial cells with islands of epithelial cells deficiency, as well as hyperreflective intracellular deposits in the basal layer. Changes in keratocytes in the form of perinuclear white microdots were found in the stroma of 25.8% of patients. Endothelial layer was not changed and had normal cell density, reflectivity and morphology in all patients with cornea verticillata. Corneal confocal microscopy is a valuable diagnostic tool in patients with Fabry disease and may be important in evaluation of disease progression and monitoring of treatment efficacy.

Finite element analysis of determining corneal biomechanical properties in vivo based on Corvis ST

The decrease of corneal stiffness is the key factor leading to keratoconus, and the corneal collagen fiber stiffness and fiber dispersion are closely related to the corneal biomechanical properties. In this paper, a finite element model of human cornea based on corneal microstructure, namely collagen fiber, was established before and after laser assisted in situ keratomileusis (LASIK). By simulating the Corvis ST process and comparing with the actual clinical results, the hyperelastic constitutive parameters and corneal collagen fiber stiffness modulus of the corneal material were determined before and after refractive surgery. After LASIK, the corneal collagen fiber stiffness modulus increased significantly, and was highly correlated with central corneal thickness (CCT). The predictive relationship between the corneal collagen fiber stiffness modulus and the corresponding CCT before and after surgery was: k 1 before = exp(9.14 - 0.009CCT before), k 1 after = exp(8.82 - 0.008CCT after). According to the results of this study, the central corneal thickness of the patient can be used to estimate the preoperative and postoperative collagen fiber stiffness modulus, and then a personalized corneal model that is more consistent with the actual situation of the patient can be established, providing a theoretical reference for more accurately predicting the safe surgical cutting amount of the cornea.

Nearly-incompressible transverse isotropy (NITI) of cornea elasticity: model\xa0and experiments with acoustic micro-tapping OCE

The cornea provides the largest refractive power for the human visual system. Its stiffness, along with intraocular pressure (IOP), are linked to several pathologies, including keratoconus and glaucoma. Although mechanical tests can quantify corneal elasticity ex vivo, they cannot be used clinically. Dynamic optical coherence elastography (OCE), which launches and tracks shear waves to estimate stiffness, provides an attractive non-contact probe of corneal elasticity. To date, however, OCE studies report corneal moduli around tens of kPa, orders-of-magnitude less than those (few MPa) obtained by tensile/inflation testing. This large discrepancy impedes OCE’s clinical adoption. Based on corneal microstructure, we introduce and fully characterize a nearly-incompressible transversely isotropic (NITI) model depicting corneal biomechanics. We show that the cornea must be described by at least two shear moduli, contrary to current single-modulus models, decoupling tensile and shear responses. We measure both as a function of IOP in ex vivo porcine cornea, obtaining values consistent with both tensile and shear tests. At pressures above 30 mmHg, the model begins to fail, consistent with non-linear changes in cornea at high IOP.

Keratoconus Detection Based on a Single Scheimpflug Image.

PurposeTo introduce a new approach for keratoconus detection based on corneal microstructure observed in vivo derived from a single Scheimpflug image.MethodsScheimpflug single-image snapshots from 25 control subjects and 25 keratoconus eyes were analyzed; from each group, five subjects were randomly selected to provide out-of-sample data. Each corneal image was segmented, after which the stromal pixel intensities were statistically modeled with a Weibull distribution. Distribution estimated parameters α and β, characterizing corneal microstructure, were used in combination with a macrostructure parameter, central corneal thickness (CCT), for the detection of keratoconus. In addition, receiver operating characteristic curves were used to determine the sensitivity and specificity of each parameter for keratoconus detection.ResultsThe combination of CCT (sensitivity = 88%; specificity = 84%) with microscopic parameters extracted from statistical modeling of light intensity distribution, α (sensitivity = 76%; specificity = 76%) and β (sensitivity = 96%; specificity = 88%), from a single Scheimpflug image was found to be a successful tool to differentiate between keratoconus and control eyes with no misclassifications (sensitivity = 100%; specificity = 100%) with coefficients of variation up to 2.5%.ConclusionsThe combination of microscopic and macroscopic corneal parameters extracted from a static Scheimpflug image is a promising, non-invasive tool to differentiate corneal diseases without the need to perform measurements based on induced deformation of the corneal structure.Translational RelevanceThe proposed methodology has the potential to support clinicians in the detection of keratoconus, without compromising patient comfort.

In vivo Slit Scanning Confocal Microscopic Observation in a Patient with Keratoconus Wearing Miniscleral Contact Lens for One Year: A Case Report

The purpose of the present case report was to evaluate corneal microstructure in a patient with keratoconus wearing miniscleral contact lens (MS CL) for 1 year. A 38 years old patient diagnosed with stage III keratoconus in right eye and corneal graft (penetrating keratoplasty) in left eye was referred to optometry department of a public hospital in Malaysia. Based on the MS CLs fitting performance on the patient’s eye, MS CLs were prescribed in both eyes. However, patient opted for MS CL only in the right eye. In vivo slit scanning confocal microscopy was performed at baseline and after 1 year of contact lens wear in right eye. Qualitative analysis was done using a grading scale designed by Hollingsworth et al. (2005a), while quantitative analysis was performed using NAVIS software in a fixed frame of 0.06 mm². After 1 year of MS CL wear, qualitative observation of right cornea showed no change in stromal haze and architecture of nerve fibers. However, morphological alterations like dark bands were observed both in anterior and posterior stroma along with tendency of clustering keratocyte nuclei in posterior stroma. Quantitative analysis showed that anterior and posterior stroma keratocyte density and endothelium cell density were relatively low after 1 year of MS CL wear (817.3, 667.2, and 2577 cells/mm², respectively) caompared to baseline observation (850.7, 705.0, and 2666 cells/mm², respectively). Mean cell area of anterior stroma, posterior stroma and endothelium were also different in post MS CL wear. Polymegathism and pleomorphism calculated by NAVIS software were varied after 1 year (49.6% and 37.5%, respectively) compared to baseline (67.1% and 29.6%, respectively). In the discussed case, noticeable changes were observed in corneal microstructure after 1 year of MS CL wear. Thereby, in conclusion we recommend future studies with more number of keratoconic eyes wearing MS CLs to emphasize the findings on microstructural changes in keratoconic cornea wearing MS CLs.

Assessing Corneal Speckle in Optical Coherence Tomography: A New Look at Glaucomatous Eyes.

Optical coherence tomography speckle is used here in an unconventional way as the indirect source of information on tissue microstructure. The study reveals that the corneal speckle of glaucoma suspects has a similar relationship between the parameters of scattering exhibited in glaucoma patients. The purpose of this study was to evaluate the potential of optical coherence tomography imaging of the cornea in early diagnosis of glaucoma. Corneas of 64 subjects of three groups, including 18 primary open-angle glaucoma patients, 24 glaucoma suspects with normal levels of IOP and uncompromised visual field, and 22 age-matched controls, were imaged with spectral optical coherence tomography. A central region of interest in each optical coherence tomography image visualizing the stroma was automatically extracted, and the intensity data within that region were fit with the generalized gamma distribution. Its parameters describe the scatterer cross section and scatterer density and indirectly assess corneal microstructure. In addition, subjects underwent measurements of IOP, visual field, Heidelberg Retinal Tomography, retinal nerve fiber layer thickness, and biometry. Group means of all parameters were tested for equality. Within each group of subjects, correlation was evaluated between the statistical parameters of the corneal speckle. Glaucoma suspects had statistically significantly different IOP, visual field parameters, mean retinal fiber layer thickness, and central corneal thickness from those of glaucoma patients but not from those of the control group. The parameters of the corneal speckle were not significantly different between the groups, but they showed a markedly higher and statistically significant coefficient of determination for glaucoma patients and suspects than that for the control group (0.927 and 0.707 vs. 0.336). The results indicate that glaucoma suspects have similar relationship between the corneal scatterer cross section and scatterer density to that exhibited in the glaucoma patients but markedly different from that of healthy controls.

Corneal Properties of Keratoconus Based on Scheimpflug Light Intensity Distribution

We introduce a new approach to assess the properties of corneal microstructure in vivo of healthy control and keratoconus eyes, based on statistical modeling of light intensity distribution from Scheimpflug images. Twenty participants (10 mild keratoconus and 10 control eyes) were included in this study. Corneal biomechanics was assessed with a commercial Scheimpflug camera technology. Sets of 140 images acquired per measurement were exported for further analysis. For each image, after corneal segmentation, the stromal pixel intensities were statistically modeled, leading to parametric time-series that characterizes distributional changes during the measurement. From those time series, a set of 10 newly introduced parameters (microscopic parameters) was derived to discriminate normal from keratoconic corneas and further compared against clinical parameters available from the same measuring device, including central corneal thickness, IOP, and deformation amplitude (macroscopic parameters). Biomechanical microscopic parameters extracted from statistical modeling of light intensity distribution were good discriminators between mild keratoconus and control eyes (Mann-Whitney U test, P < 0.05/N [Bonferroni]). The combination of available macroscopic and novel microscopic parameters was the most successful tool to differentiate between keratoconus and control eyes with no misclassifications. For the first time to our knowledge, a set of parameters related to corneal microstructure, acquired from statistical modeling of light intensity distribution of dynamic Scheimpflug image acquisition was introduced. This novel approach showed the potential of combining macroscopic and microscopic corneal properties derived from a single clinical device to discriminate successfully between mild keratoconus and control eyes.

Tissue effects of intra-tissue refractive index shaping (IRIS): insights from two-photon autofluorescence and second harmonic generation microscopy.

Intra-tissue refractive index shaping (IRIS) is a novel, non-ablative form of vision correction by which femtosecond laser pulses are tightly focused into ocular tissues to induce localized refractive index (RI) change via nonlinear absorption. Here, we examined the effects of Blue-IRIS on corneal microstructure to gain insights into underlying mechanisms. Three-layer grating patterns were inscribed with IRIS ~180 µm below the epithelial surface of ex vivo rabbit globes using a 400 nm femtosecond laser. Keeping laser power constant at 82 mW in the focal volume, multiple patterns were written at different scan speeds. The largest RI change induced in this study was + 0.011 at 20 mm/s. After measuring the phase change profile of each inscribed pattern, two-photon excited autofluorescence (TPEF) and second harmonic generation (SHG) microscopy were used to quantify changes in stromal structure. While TPEF increased significantly with induced RI change, there was a noticeable suppression of SHG signal in IRIS treated regions. We posit that enhancement of TPEF was due to the formation of new fluorophores, while decreases in SHG were most likely due to degradation of collagen triple helices. All in all, the changes observed suggest that IRIS works by inducing a localized, photochemical change in collagen structure.

In vivo confocal microstructural analysis of corneas presenting Kayser-Fleischer rings in patients with Wilson's disease.

To evaluate microstructural differences between corneas with and without Kayser-Fleischer rings in age-matched subjects with Wilson's disease with neurological symptoms, using confocal laser scanning microscopy. The study included 12 subjects with Wilson's disease with neurological symptoms. Twelve corneas presented clinically with classic Kayser-Fleischer rings, visible on slit lamp examination; the other 12 served as controls. The subjects underwent a comprehensive clinical examination. Microstructural analysis using confocal laser scanning microscopy evaluated increased corneal thickness, decreased number of cells, increased debris or specific deposits, and unusual microstructures. Clinically, the subjects with Kayser-Fleischer rings had similar corneal findings and normal intraocular pressure; two had typical sunflower cataracts and decreased visual acuity. The control eyes all presented normal visual acuity, intraocular pressure, and corneal appearance. The microstructural analysis demonstrated similar findings in all the affected corneas. Compared with the control corneas, there were fewer keratocytes in the anterior stroma (17.380 vs. 22.380/mm3). Round, "hollow" dark areas were observed between the keratocytes; these were universal and similar in appearance in all affected corneas and all cornea layers. In the peripheral posterior stroma, there were dust-like, bright, granular deposits that tended to increase in number and density toward Descemet's membrane, masking the peripheral endothelium. The control corneas presented a normal microstructure apart from dust-like granular deposits in the periphery. In vivo confocal microscopy is a useful tool for evaluating the corneal microstructure when a Kayser-Fleischer ring is clinically present. The ring consists of granular, bright particles that increase in density toward Descemet's membrane, and is associated with a decreased number of keratocytes and peculiar dark, round areas in all stromal layers, probably a sign of corneal damage. When the ring is not visible in subjects with Wilson's disease, changes to the corneal microstructure are insignificant.

Long term safety and tolerability of Tafluprost 0.0015% vs Timolol 0.1% preservative-free in ocular hypertensive and in primary open-angle glaucoma patients: a cross sectional study

BackgroundThe effects of preservatives of antiglaucoma medications on corneal surface and tear function have been widely shown in literature; it’s not the same as regards the active compounds themselves. The purpose of our study was to compare Ocular Surface Disease (OSD) signs and symptoms of Tafluprost 0.0015% versus preservative free (PF) Timolol 0.1% eyedrops in ocular hypertensive (OH) and in primary open-angle glaucoma (POAG) patients.MethodsA cross-sectional study included patients in monotherapy for at least 36 months with Tafluprost 0.0015% (27) or PF Timolol 0.1% (24) and 20 healthy age and sex-matched volunteers. All subjects underwent clinical tests (Schirmer I and break-up time), in vivo confocal microscopy (IVCM) and were surveyed using Ocular Surface Disease Index (OSDI) and Glaucoma Symptoms Scale (GSS) questionnaires. The groups were compared with ANOVA, Kruskal-Wallis test, t-test, Mann-Whitney test and Bonferroni’s adjustment of p-values.ResultsNo significant differences were found in questionnaires scores, clinical tests, IVCM variables between therapy groups. Tafluprost 0.0015% group showed significantly higher OSDI score, basal epithelial cells density, stromal reflectivity, sub-basal nerves tortuosity (p = 0.0000, 0.037, 0.006, 0.0000) and less GSS score, number of sub-basal nerves (p = 0.0000, 0.037) than controls but similar clinical tests results (p > 0.05). PF Timolol group had significantly higher OSDI score, basal epithelial cells density, stromal reflectivity and sub-basal nerve tortuosity (p = 0.000, 0.014, 0.008, 0.002), less GSS score, BUT and number of sub-basal nerves (p = 0.0000, 0.026, 0.003) than controls.ConclusionsCompared to PF Timolol 0.1%, Tafluprost 0.0015% showed similar safety with regards to tear function and corneal status and a similar tolerability profile. Both therapy groups show some alterations in corneal microstructure but no side effects on tear function except for an increased tear instability in PF Timolol 0.1% group. Ophtalmologists should be aware that even PF formulations may lead to a mild ocular surface impairment.