Morphological and cytokine profiles as key parameters to distinguish between Gram-negative and Gram-positive bacterial keratitis.

Bacterial keratitis (BK) is an ocular disorder associated with poor visual prognosis. Quantification of the associated inflammatory response may provide insight into the pathogenesis of BK and guide treatment options. In this exploratory study, we evaluated 45 BK patients and 20 healthy controls by optical coherence tomography and pro-inflammatory tear cytokine analysis. The aim was to quantify the differential morphological and cytokine inflammatory response between Gram-negative and Gram-positive BK and to determine the diagnostic value of corneal thickness (CT) and infiltrate thickness (IT) in distinguishing Gram-ve BK in a clinical cohort. Greater CT and IT, at clinical presentation, were indicative of Gram-ve infection with values detected of ≥ 950 µm and ≥ 450 µm, respectively. Combination of these CT and IT values had a 100% sensitivity and 83.3% specificity as a diagnostic indicator of Gram-ve infection. Similarly, there were higher levels of IL-1ß, IL-6 and IL-8 cytokines were quantified in keratitis caused by Gram-negative bacteria. Among the different tear cytokines analysed, a significant reduction after three days of treatment was detected for pro-inflammatory cytokines IL-1ß, IL-2, IL-6, IL-8 and TNF-α, prior to starting with the administration of steroid drops. Overall, this study shows the potential value of serial OCT and tear cytokine measurements in the management of BK.

Corneal Stability of LASIK and SMILE When Combined With Collagen Cross-Linking.

PURPOSE: We investigate whether small incision lenticule extraction (SMILE) is associated with less ectasia than laser-assisted in-situ keratomileusis (LASIK) and whether concomitant collagen cross-linking (CXL) is protective in SMILE Xtra and LASIK Xtra. METHODS: Using an established LASIK rabbit ectasia model, we performed -5 diopter (D) LASIK on six eyes and -5 D SMILE on six eyes; five eyes had -5 D LASIK Xtra, five eyes -5 D SMILE Xtra. Anterior segment optical coherence tomography and corneal topography were performed preoperatively and 2, 4, and 6 weeks postoperatively. Mean (standard deviation [SD]) values of postoperative keratometry (K), maximum posterior elevation (MPE) and minimum corneal thickness (CT) were compared to preoperatively and among the surgical groups (paired t-test, analysis of variance). RESULTS: Mean (SD) K values decreased significantly following SMILE, SMILE Xtra, LASIK, and LASIK Xtra. The MPE increased significantly (P < 0.05) following LASIK, SMILE, and SMILE Xtra, but not following LASIK Xtra (P = 0.12). The MPE was less following SMILE than LASIK, but not statistically significant (week 2, 17.73 [5.77] vs. 22.75 [5.05] µm; P = 0.13); post-LASIK Xtra MPE was less than that following LASIK (week 2. 13.39 [3.05] vs. 22.75 [5.05] µm; P < 0.001). CT decreased significantly in all surgical groups; no differences were detected among the groups. CONCLUSIONS: SMILE may have less potential than LASIK to induce ectasia. LASIK Xtra and SMILE Xtra showed the smallest increase in MPE. TRANSLATIONAL RELEVANCE: Concomitant CXL may be protective following keratorefractive surgery and may reduce further the risk of ectasia.

Early wound healing and refractive response of different pocket configurations following presbyopic inlay implantation.

BACKGROUND: Presbyopic inlays have mostly been implanted under a corneal flap. Implantation in a pocket has advantages including less postoperative dry eye and neurotrophic effect, and better biomechanical corneal stability. This study investigated the effect of different pocket and flocket dimensions on corneal stability and refractive power after Raindrop™ implantation, and the associated wound healing response. METHODOLOGY: Ten New Zealand White rabbits had bilateral pocket Raindrop™ implantation. Eyes were allocated to 4 groups: pockets with 4mm, 6mm, and 8mm diameters, and 8mm flocket. They were examined pre-operatively, at day 1, weeks 1, 2, 3 and 4 post-surgery with anterior segment optical coherence tomography, corneal topography and in-vivo confocal microscopy. After euthanasia (week 4), CD11b, heat shock protein (HSP) 47 and fibronectin corneal immunohistochemistry was performed. RESULTS: Corneal thickness (mean±SD) increased from 360.0±16.2µm pre-operatively to 383.9±32.5, 409.4±79.3, 393.6±35.2, 396.4±50.7 and 405±20.3µm on day 1, weeks 1,2,3 and 4 respectively (p<0.008, all time-points). Corneal refractive power increased by 11.1±5.5, 7.5±2.5, 7.5±3.1, 7.0±3.6 and 6.3±2.9D (p<0.001). Corneal astigmatism increased from 1.1±0.3D to 2.3±1.6, 1.7±0.7, 1.8±1.0, 1.6±0.9 and 1.6±0.9D respectively (p = 0.033). CT, refractive power change and astigmatism were not different between groups. The 8mm pocket and 8mm flocket groups had the least stromal keratocyte reflectivity. CD11b, fibronectin or HSP47 weren't detected. CONCLUSIONS: Anatomical and refractive stability was achieved by 1 week; the outcomes were not affected by pocket or flocket configuration. No scarring or inflammation was identified. The 8mm pocket and flocket showed the least keratocyte activation, suggesting they might be the preferred configuration.

Evaluation of a Micro-Optical Coherence Tomography for the Corneal Endothelium in an Animal Model.

Recent developments in optical coherence tomography (OCT) systems for the cornea have limited resolution or acquisition speed. In this study we aim to evaluate the use of a 'micro-OCT' (µOCT ~1 µm axial resolution) compared to existing imaging modalities using animal models of corneal endothelial disease. We used established cryoinjury and bullous keratopathy models in Sprague Dawley rats comparing ex vivo µOCT imaging in normal and diseased eyes to (1) histology; (2) in vivo confocal microscopy (IVCM); and (3) scanning electron microscopy (SEM). Qualitative and quantitative comparisons amongst imaging modalities were performed using mean endothelial cell circularity [(4π × Area)/Perimeter(2)] with coefficient of variation (COV). We found that µOCT imaging was able to delineate endothelial cells (with nuclei), detect inflammatory cells, and corneal layers with histology-like resolution, comparable to existing imaging modalities. The mean endothelial cell circularity score was 0.88 ± 0.03, 0.87 ± 0.04 and 0.88 ± 0.05 (P = 0.216) for the SEM, IVCM and µOCT respectively, with SEM producing homogenous endothelial cell images (COV = 0.028) compared to the IVCM (0.051) and µOCT (0.062). In summary, our preliminary study suggests that the µOCT may be useful for achieving non-contact, histology-like images of the cornea for endothelial cell evaluation, which requires further development for in vivo imaging.

Application of Graphene as Candidate Biomaterial for Synthetic Keratoprosthesis Skirt.

PURPOSE: Synthetic keratoprostheses are required for visual rehabilitation in patients with end-stage corneal blindness. This study aimed to assess the biocompatibility of graphene material and its potential as a novel synthetic keratoprosthesis skirt material for corneal tissue engineering. METHODS: Human corneal stromal fibroblasts were cultured on material surfaces including pristine graphene film, graphene foam, pristine titanium (Ti) discs, and tissue culture plastic surface (TCPS). Cell attachment was assayed by immunostaining of paxillin and vinculin. Cell viability and proliferation were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Click iT 5-ethynyl-2'-deoxyuridine (EdU) assays. The growth of fibroblasts on three-dimensional graphene foam was examined by scanning electron microscopy, and cytokine release was analyzed by enzyme-linked immunosorbent assay. Graphene films were implanted into rabbit corneal stromal pockets and examined by slit-lamp biomicroscopy, anterior segment optical coherence tomography, in vivo confocal microscopy, and histology. RESULTS: Pristine graphene demonstrated good biocompatibility with human stromal fibroblasts in terms of cell adhesion, viability, and proliferation. Cells on graphene films showed higher number than on TCPS control. Cells grown on graphene had 10% more proliferation than on Ti. The expression levels of IL-6 and IL-8 were reduced when cells were seeded on graphene foam as compared to Ti and graphene film. Implantation of graphene film into rabbit stroma (n = 6) did not show any signs of infection, neovascularization, or inflammation. CONCLUSIONS: Graphene displayed excellent short-term biocompatibility with corneal cells and tissue. This demonstrates that graphene can be developed as a tissue engineering material for use in cornea.

Prophylactic Vancomycin Drops Reduce the Severity of Early Bacterial Keratitis in Keratoprosthesis.

BACKGROUND: Artificial cornea transplantation, keratoprosthesis, improves vision for patients at high risk of failure with human cadaveric cornea. However, post-operative infection can cause visual loss and implant extrusion in 3.2-17% of eyes. Long-term vancomycin drops are recommended following keratoprosthesis to prevent bacterial keratitis. Evidence, though, in support of this practice is poor. We investigated whether prophylactic vancomycin drops prevented bacterial keratitis in an animal keratoprosthesis model. METHODOLOGY: Twenty-three rabbits were assigned either to a prophylactic group (n = 13) that received vancomycin 1.4% drops 5 times/day from keratoprosthesis implantation to sacrifice, or a non-prophylactic group (n = 10) that received no drops. All rabbits had Staphylococcus aureus inoculation into the cornea at 7-12 days post-implantation and were sacrificed at predetermined time-points. Prophylactic and non-prophylactic groups were compared with slit-lamp photography (SLP), anterior segment optical coherence tomography (AS-OCT), and histology, immunohistochemistry and bacterial quantification of excised corneas. Corneal vancomycin pharmacokinetics were studied in 8 additional rabbits. RESULTS: On day 1 post-inoculation, the median SLP score and mean±SEM AS-OCT corneal thickness (CT) were greater in the non-prophylactic than the prophylactic group (11 vs. 1, p = 0.049 and 486.9±61.2 vs. 327.4±37.1 µm, p = 0.029 respectively). On days 2 and 4, SLP scores and CT were not significantly different. Immunohistochemistry showed a greater CD11b+ve/non-CD11b+ve cell ratio in the non-prophylactic group (1.45 vs. 0.71) on day 2. Bacterial counts were not significantly different between the two groups. Corneal vancomycin concentration (2.835±0.383 µg/ml) exceeded minimum inhibitory concentration (MIC) for Staphylococcus aureus only after 16 days of vancomycin drops. Two of 3 rabbits still developed infection despite bacterial inoculation after 16 days of prophylactic drops. CONCLUSIONS: Prophylactic vancomycin drops provided short-term benefit, but did not prevent infection. Achieving MIC in the cornea was not sufficient to prevent Staphylococcus aureus keratitis. Patients should continue to be counselled regarding the risk of infection following keratoprosthesis.

Repeatability and Reproducibility of Corneal Biometric Measurements Using the Visante Omni and a Rabbit Experimental Model of Post-Surgical Corneal Ectasia.

PURPOSE: To investigate the repeatability and reproducibility of the Visante Omni topography in obtaining topography measurements of rabbit corneas and to develop a post-surgical model of corneal ectasia. METHODS: Eight rabbits were used to study the repeatability and reproducibility by assessing the intra- and interobserver bias and limits of agreement. Another nine rabbits underwent different diopters (D) of laser in situ keratosmileusis (LASIK) were used for the development of ectasia model. All eyes were examined with the Visante Omni, and corneal ultrastructure were evaluated with transmission electron microscopy (TEM). RESULTS: There was no significant intra- or interobserver difference for mean steep and flat keratometry (K) values of simulated K, anterior, and posterior elevation measurements. Eyes underwent -5 D LASIK had a significant increase in mean amplitude of astigmatism and posterior surface elevation with time (P for trend < 0.05). At 2 and 3 months, the -5 D LASIK group had significant greater mean amplitude of astigmatism (P = 0.036; P = 0.027) and posterior surface elevation (both P < 0.005) compared with control group. On TEM, the mean collagen fibril diameter and interfibril distance in the -5 D LASIK eyes were significantly greater than in controls at 3 months (P = 0.018; P < 0.001). CONCLUSIONS: The Visante Omni provided imaging of the rabbit cornea with good repeatability and reproducibility. Application of -5 D LASIK treatment produced a rabbit model of corneal ectasia that was gradual in development and simulated the human condition. TRANSLATIONAL RELEVANCE: The results provide the foundations for the future evaluation of novel treatment modalities for post-surgical ectasia and keratoconus.

Surgical compensation of presbyopia with corneal inlays.

Presbyopia, the physiological change in near vision that develops with ageing, gradually affects individuals older than 40 years and is a growing cause of visual disability due to ageing demographics of the global population. The routine use of computers and 'smartphones', combined with the affluence of the 'baby boomers' generation has set high standards for near vision correction. Corneal inlays are a relatively new treatment modality that is effective at compensating for presbyopia. The dimensions of these devices vary from 2 to 3.8 mm in diameter and 5 to 32 µm in thickness. They are implanted in the anterior corneal stroma of the non-dominant eye, most commonly, in a femtosecond laser created corneal pocket. They improve near vision by increasing the depth of focus, creating a hyper-prolate region of increased central cornea power or providing a refractive add power. This article reviews the literature on the efficacy and safety of corneal inlays.

Conventional versus accelerated collagen cross-linking for keratoconus.

: Collagen cross-linking (CXL) is a procedure that primarily aims to increase corneal stiffness. Although used for a variety of conditions, it is most commonly applied to the treatment of keratoconus. Collagen cross-linking involves irradiation of the cornea with ultraviolet A (UVA) irradiation after it has been soaked with riboflavin (vitamin B), a photosensitizer. In conventional treatment, based on the Dresden protocol, a minimum corneal thickness threshold of 400 µm is recommended and UVA (370 nm) irradiation of 3 mW/cm irradiance is applied for 30 min, resulting in a cumulative dose of 5.4 J/cm. Evidence presented in this review shows that conventional CXL stabilizes the vision and corneal topographic parameters in the majority of treated patients, with only a small failure rate. It has a good safety profile with no endothelial cell loss and a small risk of corneal infiltration and infection. To reduce the treatment duration, accelerated protocols of similar efficacy have been sought. In accelerated protocols, UVA irradiation of higher irradiance, typically 9 mW/cm, is applied for a shorter time, typically 10 min. The evidence, limited to small studies with short follow-up, shows that they may also stabilize the vision and the ectasia, with no additional safety concerns highlighted. Randomized controlled studies are, however, required to confirm the encouraging results and noninferiority to conventional treatment.

Deep sclerectomy versus trabeculectomy: a morphological study with anterior segment optical coherence tomography.

PURPOSE: To investigate the intraocular pressure (IOP) lowering mechanisms of deep sclerectomy (DS) with anterior segment optical coherence tomography (AS-OCT). METHODS: In a prospective cross-sectional study, AS-OCT parameters were compared between DS, trabeculectomy and control cases. Association with IOP and success (IOP≤16 mm Hg without medication) was investigated. RESULTS: 18 DS (15 patients), 17 trabeculectomy (16 patients) and 15 controls (15 patients) were examined. Successful had a taller intrascleral lake (IL) and thicker conjunctival/Tenon's layer (CTL) than non-successful cases (513.3 vs 361.1 µm, p=0.027 and 586.7 vs 251.1 µm, p<0.001, respectively). CTL thickness correlated with IOP (r=-0.6407, p=0.004). CTL thickness was significantly different between controls, DS and trabeculectomy (mean (SD): 203.3 (62.6) vs 418.9 (261.9) vs 604.1 (220.7) µm, p<0.0001). Successful trabeculectomy cases had a taller bleb cavity (BC) than non-successful cases (607.5 vs 176.7 µm, p=0.041). CTL microcysts were detected in 50% of DS and 52.9% of trabeculectomy cases (p=1). CONCLUSIONS: Trans-conjunctival aqueous percolation was identified as a novel DS drainage route. DS had a fluid reservoir below the scleral flap, the IL, in analogy to the trabeculectomy BC. A postoperative tall IL and a thick CTL were associated with good outcome.

Impact of graft thickness on visual acuity after Descemet's stripping endothelial keratoplasty.

Objective To evaluate the relationship, over time, between central graft thickness and visual acuity following Descemet's stripping endothelial keratoplasty (DSEK). Methods A retrospective analysis of 70 consecutive cases of DSEK. All donor lenticules were dissected manually. Serial postoperative measurements of central graft and total corneal thicknesses were made using anterior segment optical coherence tomography. Visual acuity, refraction and patient demographics were collected from case notes. The correlation between central graft thickness and visual acuity at serial time points was calculated. Results The median age at surgery was 75 years (lower quartile (LQ) 66, upper quartile (UQ) 83, range 36-90 years). Nineteen eyes were excluded from statistical analysis, leaving 51 eyes of 46 patients remaining. Last follow-up occurred a median of 12 months postoperatively (LQ 6, UQ 23, range 4-38 months). The median preoperative visual acuity was 0.71 logarithm of the minimum angle of resolution (logMAR), improving to 0.34 logMAR postoperatively (p<0.001, n=43). Median graft thickness decreased from 209 µm at day 1 to 142 µm at last follow-up (p<0.001). No statistically significant correlation was found between central total corneal thickness and visual acuity at any time point. Except for a single time point, no statistically significant correlation was found between central graft thickness and visual acuity. Conclusion There is no clear association between central graft, or total corneal, thickness and visual acuity following DSEK.

Pseudoexfoliation and opacification of intraocular lenses.

PURPOSE: To report two cases of spoke-like intraocular lens (IOL) opacification that resembles pseudoexfoliation of the crystalline lens. METHODS: Case series presentation. RESULTS: Patient 1 developed a circle of spoke-like opacification on the anterior IOL surface 7 years after phacoemulsification. Patient 2 had paracentral anterior surface IOL opacification 18 months after cataract extraction and clinical pseudoexfoliation in the fellow eye. CONCLUSION: Spoke-like anterior IOL opacification should raise the suspicion of pseudoexfoliation syndrome.

Assessment of the use of anterior segment optical coherence tomography in microbial keratitis.

PURPOSE: To investigate the imaging capabilities of anterior segment optical coherence tomography (AS OCT) in microbial keratitis and to assess whether measurements of the quantitative parameters, infiltrate thickness and corneal thickness, were possible. DESIGN: Prospective, noncomparative, observational case series. METHODS: The study was conducted at a university hospital clinical setting. Seven patients (eyes) with suspected microbial keratitis underwent standard clinical examination and treatment based on slit-lamp clinical findings. AS OCT scanning was performed on presentation and at two follow-up appointments. All scans were carried out with the scanning beam passing through the center of the infiltration and at a specific meridian. Examination was carried out by the same operator. RESULTS: Corneal infiltration was imaged as a hyperreflective area in the corneal stroma on high-resolution AS OCT scans. Retrocorneal pathologic features and anterior chamber inflammatory cells could be imaged. Corneal and infiltrate thickness could be measured with calipers in six cases. In one case, corneal and infiltrate thickness could not be measured because of a thick inflammatory plaque attached to the endothelium. In this case, the width of the plaque was measured on serial scans. CONCLUSIONS: AS OCT imaging provides a range of parameters that can be used to assess microbial keratitis and the treatment response objectively.