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Prof. Leonardo Mastropasqua is Director at the National High-Tech Eye Center, Ophthalmology Clinic, Department of Medicine and Aging Science, University G. D’Annunzio of Chieti-Pescara, Chieti, Italy. In the following editorial, talk about the prospects for corneal lenticule implantation. Mario Nubile is Associate Professor of the same faculty. Both are specialized in corneal, cataract and refractive surgery and focus on imaging technologies of the anterior eye segment. In the following editorial, they talk about the prospects for corneal lenticule implantation and tissue-addition techniques for reshaping the diseased cornea.

Femtosecond lasers have revived the possibility of stromal keratophakia or tissue additive keratoplasty, a technique originally introduced by Prof. Jose Ignacio Barraquer in the 1960s. The surgical technique offers a unique solution to treat keratoconus. In the current study, we reviewed and performed a meta-analysis of the clinical outcomes of the femtosecond laser-assisted stromal keratophakia in the treatment of keratoconus. This is a systematic review and meta-analysis of the estimated outcome difference between pre- and post-lenticule implantations. A total of related 10 studies were found in the literature. No studies reported adverse events, such as persistent haze or graft rejection, at last patients’ visits. We further narrowed down the article selection in accordance to our inclusion criteria to report the composite outcomes (9 studies) and meta-analysis (4 studies). In the composite analysis, we demonstrated that lenticule implantation in keratoconus and post-LASIK ectasia patients appeared to expand the stromal volume of the thin corneas, flattened the cones, and significantly improved uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA) and spherical equivalent (SE). The meta-analysis showed that the random estimated UCVA, BCVA, SE and mean keratometry (Km) differences following the lenticule implantation was −0.214 (95% CI: −0.367 to 0.060; p = 0.006), −0.169 (−0.246 to 0.091; p

The purpose of this study was to evaluate corneal epithelium and stromal remodelling with anterior segment optical coherence tomography in patients who have undergone stromal lenticule addition keratoplasty (SLAK) for advanced keratoconus. This was a prospective non-comparative observational study. Fifteen eyes of 15 patients with advanced keratoconus underwent implantation with a cadaveric, donor negative meniscus-shaped intrastromal lenticule, produced with a femtosecond laser, into a stromal pocket dissected in the recipient cornea at a depth of 120 μm. Simulated keratometry, central corneal thickness (CTT), corneal thinnest point (CTP), central epithelial thickness (CET), central and peripheral lenticule thickness, anterior and posterior stromal thickness were measured. Regional central corneal epithelial thickness (CET) and variations in the inner annular area (IAT) and outer annular area (OAT) were also analysed. All parameters were measured preoperatively and 1, 3, and 6 months postoperatively. The average anterior Sim-k decreased from 59.63 ± 7.58 preoperatively to 57.19 ± 6.33 D 6 months postoperatively. CCT, CTP, CET, and OAT increased and IAT decreased significantly after 1 month. All parameters appeared unchanged at 6-months except that of OAT that further increased. Lenticule thickness was stable. In conclusion we observed that SLAK reshapes the cornea by central flattening with stromal thickening and epithelial thickness restoration.

To evaluate the renewal of corneal nerve structure and function in patients with neurotrophic keratopathy (NK) treated with recombinant human nerve growth factor (rhNGF) eye drops.Prospective, interventional, before-and-after case series.This study included 18 patients with NK with a persistent epithelial defect or corneal ulcer, treated with topical rhNGF, and age-matched healthy controls. Patients underwent clinical examination with corneal fluorescein staining, Schirmer 1 tear test, assessment of corneal sensitivity with the Cochet-Bonnet esthesiometer, and morphologic examination of the nerves by in vivo confocal microscopy (IVCM) at baseline and at 4 and 8 weeks of treatment. IVCM analysis was used to assess corneal sub-basal nerve density, number of nerve branches, and the diameter of nerve fibers.A complete resolution of the epithelial defect was observed in all patients within 8 weeks. Schirmer 1 test showed a significant improvement of tear film secretion. Change from baseline in corneal sensation was significant (P.001) but did not approach that of healthy controls. After 8 weeks of treatment, there was a significant increase in the mean nerve density in affected eyes as compared to baseline (P = .007) as well as in the number of nerve branches (P = .008) and nerve fiber diameter (P = .007).Topical treatment with rhNGF was effective in promoting complete corneal healing of persistent epithelial defects and corneal ulcers in patients with NK. This was associated with an improvement of corneal sensitivity and an increase of sub-basal nerve density, diameter, and number of nerve branches, indicating improvement in structure and function of corneal nerves.

PURPOSE: To investigate the in vivo corneal microscopic changes after femtosecond laser–assisted stromal lenticule addition keratoplasty in keratoconus by means of in vivo confocal microscopy. METHODS: Patients affected by advanced keratoconus were included in the study. Negative meniscus-shaped stromal lenticules, produced with a femtosecond laser (VisuMax; Carl Zeiss Meditec) from eye bank corneas were transplanted into a stromal pocket dissected in the recipient cornea at a depth of 120 µm. In vivo confocal microscopy was performed during the 12-month follow-up to investigate changes of the corneal and lenticule structure. RESULTS: Ten patients were enrolled in the study. No changes of the dendritic cell population were documented during the follow-up period. Mild edema and stromal keratocyte activation gradually decreased during the first month. Subbasal nerve density returned to preoperative values after 6 months. Donor-recipient interfaces appeared hyperreflective but gradually improved over time with significantly reduced reflectivity after 3 months. No evidence of stromal inflammatory cell migration or matrix opacification was observed. Endothelial and keratocyte density remained stable over time. A variable degree of stromal radially distributed folds, not visible on biomicroscopy, was observed in the lenticule and in the posterior recipient stroma. CONCLUSIONS: Stromal lenticule addition keratoplasty produces transitory nerve plexus density reduction and minor inflammatory reaction that rapidly decreases during the first month. Donor-recipient interface reflectivity is comparable to a femtosecond laser refractive procedure with no sign of stromal opacification or stromal rejection in 1 year of follow-up. [ J Refract Surg . 2020;36(8):544–550.]

Pre-Descemet endothelial keratoplasty (PDEK) is an alternative technique to Descemet membrane endothelial keratoplasty (DMEK). The preparation of PDEK tissue by pneumatic dissection is simple and reproducible. The PDEK clamp helps to consistently obtain a type 1 big bubble. The mean size of type 1 big bubble is 7.255 ± 0.535 × 6.745 ± 0.668 mm. The volume of air required to obtain type 1 big bubble is 0.14 to 0.37 mL. Dissection of PDEK tissue can be achieved by trephination or manual excision. Insertion of tissue into the recipient eye can be by injection or pulling. Unfolding techniques used for PDEK are similar to those used in DMEK. Unlike DMEK, PDEK tissue is easier to handle and unscroll and allows use of younger donors. It could help surgeons converting to endothelial keratoplasty, with significant advantages in preparation, handling, and unscrolling in the eye.

AimsTo describe clinical and in vivo confocal microscopy (IVCM) features of neuropathic corneal pain (NCP) without clinically visible signs.MethodsProspective, observational study of 27 eyes of 14 patients who had continuous severe ocular pain for one or more years, with minimal or no ocular surface signs and were non-responsive to topical lubricants, steroids and/or ciclosporin. All patients were evaluated using Ocular Surface Disease Index, Oxford grading scale, Schirmer test 1, Cochet Bonnet esthesiometry and response to topical anaesthesia. Central and paracentral regions of the cornea of patients and seven healthy controls were studied by IVCM. Corneal epithelial thickness and sub-basal nerve density were measured in patients and controls.ResultsFour patients responded to topical anaesthesia (responsive group (RG)), indicating peripheral NCP while 10 patients did not show any improvement (non-responsive group (NRG)), indicating central NCP. Schirmer-1 test was within normal limits in the RG but significantly greater in the NRG (pConclusionNCP without visible clinical signs does not represent typical dry eye disease. Distinct signs demonstrated on IVCM suggest that peripheral NCP, which responds to topical anaesthesia, and central NCP, which does not, are separate entities.

Background/Purpose Stem cells of the corneal epithelium reside in the limbal palisades and associated limbal epithelial crypts. The limbus constitutes a barrier between the conjunctival and corneal phenotype of cells. When the limbal barrier is breached, stem cell deficiency (LSCD) results with conjunctivalisation of the cornea. The purpose of this study was to document an early manifestation of LSCD which is consistent and distinct across several causes of LSCD. # Methods Seventeen eyes of 11 consecutive patients with diverse known causes of LSCD were included. Patients were examined clinically with the slit lamp and fluorescein staining, and by in vivo confocal microscopy. All corneas were photographed with a slit lamp camera. Seven patients underwent in vivo confocal microscopy examination. # Results There were 6 males and 5 females. Six cases had bilateral involvement and the other 5 were unilateral. All patients showed predominantly superior loss of limbal anatomy with effacement of the palisades of Vogt, superficial vascularization encroaching on the peripheral cornea and alternating columns, 6–20 in number, of late fluorescein staining conjunctivalised and normal epithelium extending for a variable distance towards the corneal centre. These were related to prolonged contact lens wear, atopic keratoconjunctivitis, superficial ocular surface burns, ocular cicatricial pemphigoid, rosacea keratoconjunctivitis, old trachoma and neurotrophic keratopathy. In vivo confocal microscopy confirmed features of LSCD. # Conclusion Alternating columns of conjunctivalized and normal epithelia encroaching on the cornea from the superior limbus is a distinct manifestation of early LSCD across a diverse range of causes. The reason for this manifestation is unclear but could be related to the random dysfunction or loss of the palisade architecture.

Purpose The purpose of this study was to describe the face mask (FM)-related ocular surface changes using clinical tests, in vivo confocal microscopy (IVCM) and impression cytology (IC), and to investigate the Dry Eye-related Quality of life Score (DEQS). Methods Sixty-six patients with dry eye disease (DED) and 62 healthy subjects (group 2) using FM were enrolled. Groups were divided into: groups 1A and 2A: < 3 hours of FM wear; groups 1B and 2B: 3 to 6 hours; and groups 1C and 2C: > 6 hours. Patients underwent DEQS questionnaire, break-up time (BUT), Schirmer test I (STI), fluorescein and lissamine staining (FS and LS), IVCM to determine corneal dendritic cell density (DCD) and goblet cell density (GCD), and IC to measure HLA-DR, at baseline and after 3 months. Results FM use duration before enrollment was 27 ± 2.3 and 30 ± 4.1 (days ± SD) for groups 1 and 2 (P > 0.05). After 3 months, DEQS worsened in groups 1B and 1C, STI in groups 1A to 1C, FS and LS in group 1C (P < 0.05); in controls, BUT and FS worsened only in group 2C (P < 0.05). DCD significantly increased in groups 1A to 1C and HLA-DR in groups 1B and 1C (P < 0.05), whereas GCD did not significantly change. DCD and HLA-DR increased only in group 2C (P < 0.05). DEQS significantly correlated with DCD (P = 0.05, r = 0.698; P < 0.001, r = 0.832) and HLA-DR (P = 0.043, r = −0.687; P < 0.001, r = 0.861) at baseline and 3 months. Conclusions Use of FM increases ocular surface inflammation and negatively impacts the quality of life in patients with DED. Translational Relevance The study of the prolonged use of FM effects may be relevant to managing DED.

Alcohol (ethanol) has been used in medicine since time immemorial. In ophthalmic practice, besides as an antiseptic, it was given as retrobulbar injections to relieve severe ocular pain. Alcohol can be applied topically to the surface of neoplastic or suspicious lesions to kill cells that might desquamate and seed during surgical excision, to treat epithelial ingrowth that can occur following corneal surgeries, particularly laser in situ keratomileusis (LASIK), and to treat superficial infectious keratitis. In view of its ability to achieve a smooth cleavage plane between the epithelium and the Bowman's layer, alcohol-assisted delamination (ALD) of the corneal epithelium has been used widely and effectively for a variety of diagnostic and therapeutic indications, at times delivering both outcomes. Diagnostically, ALD yields an intact epithelial sheet which can be fixed flat to provide excellent orientation for histopathological evaluation. Therapeutically, it is most commonly used to treat recurrent corneal erosion syndrome, where its efficacy is comparable to that of phototherapeutic keratectomy but with several advantages. It has also been used to treat various forms of epithelial/anterior stromal dystrophies, which can obviate or delay the need for corneal transplantation for several years. In addition, ALD is performed in corneal collagen cross-linking and corneal refractive surgery for relatively atraumatic removal of the epithelium. In this review, we aimed to provide a comprehensive overview of the diagnostic and therapeutic use of topical alcohol in ophthalmology, to describe the surgical and fixation techniques of ALD, and to highlight our experience in ALD over the past decade.

Stromal lenticule addition keratoplasty (SLAK) is a novel, minimally invasive technique for the treatment of advanced keratoconus in patients with poor vision or contact lens intolerance. A femtosecond laser-shaped lenticule of donor stroma, sculpted by means of femtosecond laser, is implanted into an intrastromal pocket in the recipient cornea. Implanted tissue produces a significant flattening of the cone combined with an increase of the corneal thickness consistent with the thickness profile of the lenticule. These results proved to be stable after 2 years of follow-up, with no evidence of rejection. High-resolution anterior segment-optical coherence tomography (AS-OCT) is an imaging technique necessary in all SLAK surgical phases. In preoperative time it allows to evaluate patient eligibility assessing corneal clarity, stromal and epithelial thickness, and cone apex eccentricity. Intraoperative AS-OCT is useful to detect proper centration and distention of the lenticule in real time. After surgery it allows one to evaluate surgical results and follow them up over time, providing important information about corneal morphological changes. In summary, AS-OCT imaging is essential to approach this novel intrastromal additive corneal surgery.

Femtosecond lasers have revived the possibility of stromal keratophakia or tissue additive keratoplasty, a technique originally introduced by Prof. Jose Ignacio Barraquer in the 1960s. The surgical technique offers a unique solution to treat keratoconus. In the current study, we reviewed and performed a meta-analysis of the clinical outcomes of the femtosecond laser-assisted stromal keratophakia in the treatment of keratoconus.This is a systematic review and meta-analysis of the estimated outcome difference between pre- and post-lenticule implantations.A total of related 10 studies were found in the literature. No studies reported adverse events, such as persistent haze or graft rejection, at last patients' visits. We further narrowed down the article selection in accordance to our inclusion criteria to report the composite outcomes (9 studies) and meta-analysis (4 studies). In the composite analysis, we demonstrated that lenticule implantation in keratoconus and post-LASIK ectasia patients appeared to expand the stromal volume of the thin corneas, flattened the cones, and significantly improved uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA) and spherical equivalent (SE). The meta-analysis showed that the random estimated UCVA, BCVA, SE and mean keratometry (KFemtosecond laser-assisted stromal keratophakia is a feasible technique to correct the refractive aberrations, expand corneal volume and regularize corneal curvature in patients with keratoconus. However, there is a need to standardize the technique (e.g., whether to crosslink or not or to use convex or concave lenticules) and to formulate a mathematical model that accounts for the long-term epithelial thickness changes and stromal remodeling to determine the shape or profile of the lenticules, in order to improve the efficacy of the keratophakia further.

Following refractive surgery, the cornea is denervated and re-innervated, hence a reproducible tool to objectively quantify this change is warranted. This study aimed to determine the repeatability and reproducibility of corneal nerve quantification between automated (ACCMetrics) and manual software (CCMetrics) following refractive surgery. A total of 1007 in vivo confocal microscopy images from 20 post-small incision lenticule extraction (SMILE) or post-laser-assisted in situ keratomileusis (LASIK) patients were evaluated by two independent observers using CCMetrics for corneal nerve fibre density (CNFD), corneal nerve branch density (CNBD), and corneal nerve fibre length (CNFL). Intra-observer and inter-observer reproducibility and repeatability, as well as agreement and correlation between the measurements obtained by ACCMetrics and CCMetrics, were assessed. We found that CNFL demonstrated the best intra- and inter-observer agreement followed by CNFD (intra-class correlation coefficient (ICC) = 0.799 and 0.740, respectively for CNFL, 0.757 and 0.728 for CNFD). CNBD demonstrated poorest intra- and inter-observer ICC. There was an underestimation in ACCMetrics measurements compared to CCMetrics measurements, although the differences were not significant. Our data suggested that both automated and manual methods can be used as reliable tools for the evaluation of corneal nerve status following refractive surgery. However, the measurements obtained with different methods are not interchangeable.

Neurotrophic keratopathy (NK) is a rare degenerative corneal disorder characterized by instability of epithelial integrity with consequent epithelial defects that can worsen up to persistent epithelial defects with stromal melting and ulceration. The pathogenesis of NK springs from a variable degree of damage to the trigeminal nerve plexus, leading to a reduction or total loss of corneal sensitivity. Mackie classification (1995) distinguishes three stages of NK, based on the severity of clinical presentation. The technological innovations in corneal diagnostic imaging allow clinicians to accurately study the morphometry and morphology of corneal structure with microscopic resolution. In this study, 45 patients affected by NK at different stages underwent in vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT) with particular attention to analyze subbasal nerve plexus fibers and the stromal structure. At the light of IVCM and AS-OCT observations, we propose a different staging of NK with respect to the Mackie's classification that takes into account the severity of subbasal nerve fibers damage and the extension in depth of stromal ulceration; this classification better defines, at the time of diagnosis, the cellular and structural alterations in the affected corneas, with possible prognostic and therapeutic values in the management of NK.

Neurotrophic Keratopathy (NK) refers to a condition where corneal epitheliopathy leading to frank epithelial defect with or without stromal ulceration (melting) is associated with reduced or absent corneal sensations. Sensory nerves serve nociceptor and trophic functions, which can be affected independently or simultaneously. Loss of trophic function and consequent epithelial breakdown exposes the stroma making it susceptible to enzymatic degradation. Nerve pathology can range from attrition to aberrant re-generation with corresponding symptoms from anaesthesia to hyperaesthesia/allodynia. Many systemic and ocular conditions, including surgery and preserved medications can lead to NK. NK can be mild (epithelium and tear film changes), moderate (non-healing epithelial defect) or severe (stromal melting and perforation). Moderate and severe NK can profoundly affect vision and adversely impact on the quality of life. Medical management with lubricating agents from artificial tears to serum/plasma drops, anti-inflammatory agents, antibiotics and anti-proteases all provide non-specific relief, which may be temporary. Contact lenses, punctal plugs, lid closure with botulinum toxin and surgical interventions like tarsorrhaphy, conjunctival flaps and amniotic membrane provide greater success but often at the cost of obscuring sight. Corneal surgery in a dry ocular surface with reduced sensation is at high risk of failure. The recent advent of biologicals such as biopolymers mimicking heparan sulfate; coenzyme Q10 and antisense oligonucleotide that suppress connexin 43 expression, all offer promise. Recombinant nerve growth factor (cenegermin), recently approved for human use targets the nerve pathology and has the potential of addressing the underlying deficit and becoming a specific therapy for NK.

A new non-invasive method, based on the interferometric analysis of diff ractive and polarizing effects related to the birefringent properties of corneal collagen fibrils, has been developed to objectively evaluate the optical properties of the stroma. The new method shows a relevant impact on corneal surgeries specifically for lamellar transplantation where, due to the polarizing properties of the stroma, the alignment between collagen fibrils of donor corneas with patient collagen fibril orientation has shown an improvement of visual acuity postoperatively. Further studies on the regularity of the corneal isogyre pattern are showing this new method has a strong impact in early-stage diagnosis of corneal disease.

AimsTo investigate and define the nature of big bubbles (BB) formed by injection of viscoelastic in deep anterior lamellar keratoplasty.MethodsIntrastromal injections of 0.1 and 0.3 mL of sodium hyaluronate 1.2% and 0.6% were made into sclera-corneal discs (n = 32) at superficial (anterior-third), midstromal (middle-third) and deep (posterior-third) levels to simulate deep anterior lamellar keratoplasty. Postinjection optical coherence tomograms (OCT) were obtained with the needle in situ. The samples were sectioned and examined histologically. Twelve control samples were injected with air.ResultsWith superficial injections (n=8) only intrastromal accumulation of viscoelastic was noted. With midstromal injections (n=10) intrastromal accumulation of viscoelastic (n=6) and intrastromal big bubbles (IBB) (n=4) with substantial and variable stromal tissue in the walls were noted. No type 1, type 2 or mixed BB were noted. With deep injections (n=14), type 1 BB (n=4), IBB (n=4) and mixed BB (n=6) were obtained.There was no difference in the results with the two different concentrations of viscoelastic used. With air injection (n=12), 10 type 1 and 1 type 2 BB and 1 mixed BB were obtained. No IBB was noted.ConclusionsBB obtained by injection of viscoelastic and air can be different. The former tends to occur at the site of injection, especially with midstromal injections, takes the form of tissue separation by stretch and tearing and does not cleave in a consistent plane like air. Surgeons should be aware of IBB created by viscodissection and not confuse it for a type1 BB. Intraoperative OCT should help identify IBB.

To detect corneal inflammation and apoptosis induced after small incision lenticule extraction (SMILE) at different refractive corrections for moderate to high values of myopia.Fifty patients (50 eyes) suffering from medium to high myopia (spherical equivalent refraction from -3.75 to -10.00 diopters (D) with refractive astigmatism under -0.75 D) underwent SMILE in order to correct myopic error. In vivo evaluation was done by corneal confocal microscopy (IVCM) and ex vivo by immunohistochemistry. After surgery, all corneal lenticules were checked for regularity, entirety, and fixed in formalin for immunohistochemistry evaluation of apoptosis (TUNEL) and inflammation (CD11b) levels. Postoperative assessments took place during the first week and the first and third months after surgery. Patients returned for IVCM examination for analysis of the corneal stromal femtosecond laser treatment interfaces reflectivity.No correlation was observed between treated myopic refractive error and number of CD11bSMILE, as an innovative all-femto surgical procedure, results in a reduced tissue inflammation and apoptosis levels with a minimum tissue response, in terms of interface reflectivity, and there are no statistically significant differences among variable treated refractive error range.

Pseudomonas aeruginosa (PA) is the leading cause of corneal blindness worldwide. A constant increase in multi-drug resistant PA strains have heightened the challenge of effectively managing corneal infections with conventional antibiotics. Antimicrobial peptides are promising antibiotic analogs with a unique mode of action. Cathelicidin-derived shorter peptides (FK13 and FK16) have previously been shown to kill a range of pathogens in both in vitro and in vivo systems. Here, our aim was to exploit the potential of FK13 or FK16 to enhance the anti-Pseudomonas activity of vancomycin, which normally has low clinical efficacy against PA. Our results have demonstrated that FK16 is more potent than FK13 against different PA strains including a clinical isolate from a patient’s ocular surface. FK16 was shown to enhance the membrane permeability of PAO1 at sub-inhibitory concentrations. Moreover, FK16 at lower concentrations was shown to increase the antibacterial susceptibility of vancomycin against PA strains up to eightfold. The bactericidal synergism between FK16 and vancomycin was shown to be stable in the presence of physiological tear salt concentration and did not cause toxic effects on the human corneal epithelial cells and human red blood cells. Our results have revealed that sub-inhibitory concentration of FK16 could augment the antimicrobial effects of vancomycin against PA. It is anticipated that the future exploitation of the peptide design approach may enhance the effectiveness of FK16 and its application as an adjuvant to antibiotic therapy for the treatment of multi-drug resistant infections.

Neurotrophic keratitis (NK) is a rare degenerative disease of the cornea caused by trigeminal nerve damage, which leads to loss of corneal sensitivity, corneal epithelium breakdown, and poor healing. Though extremely uncommon, NK is increasingly recognized for its characteristics as a distinct and well-defined clinical entity rather than a rare complication of various diseases that can disrupt trigeminal innervation. Indeed, the defining feature of NK is loss of corneal sensitivity, and its clinical findings do not correlate with the wide range of systemic or ocular conditions that underlie trigeminal nerve damage. Despite increasing awareness of NK as a distinct condition, its management continues to be challenged by the lack of treatments that target nerve regeneration. This review focuses on the role of corneal nerves in maintaining ocular surface homeostasis, the consequences (such as alterations in neuromediators and corneal cell morphology/function) of impaired innervation, and advances in NK diagnosis and management. Novel therapeutic strategies should aim to improve corneal innervation in order support corneal renewal and healing. J. Cell. Physiol. 232: 717-724, 2017. © 2016 Wiley Periodicals, Inc.

To investigate the feasibility of the procedure and the modifications of the corneal curvature and profile obtained with a novel technique of stromal-lentoid implantation in ex vivo human corneas. Experimental ex vivo study in human corneas. Twelve stromal lentoids were produced by means of hyperopic femtosecond lenticule extraction (FLEx) with the VisuMax femtosecond laser (Carl Zeiss Meditec, Jena, Germany) with a refractive power of +8.00 D and optical zone of 6.0 (six lenticules) and 5.0 mm (six lenticules), respectively. The posterior surface of these stromal lentoids presented a higher curvature with respect to the anterior surface and, therefore, the thinnest point is located at the center of the lenticule, gradually thickening towards the periphery. Another 12 corneas underwent femtosecond laser intrastromal pocket creation at a depth of 115 microns, for lenticule insertion. After intrastromal lenticule implantation the changes of corneal profiles were evaluated by means of corneal topography and anterior segment optical coherence tomography (OCT). The implantations of intrastromal lenticules were successful in all cases with proper distention and centration. A mid-peripheral forward shift of the anterior corneal surface along with a thickening (greater in periphery) was induced. There was a significant flattening of anterior corneal central areas of 7.31 ± 1.52 D (p = 0.002). Diameters of the flattening area were found to be consistently correlated with the optical zone diameter of the implanted lenticules (p = 0.006). Central flattening was similar in both groups. OCT pachymetry maps showed an increase of corneal thickness consistently correlated with implanted lentoid thickness and diameter. The implantation of modified hyperopic-shaped intra-corneal stromal lentoid is a feasible and reproducible technique for achieving central corneal flattening while increasing thickness. Whether this procedure may be beneficial in the treatment of refractive errors or ectatic corneal disorders such as keratoconus merits further study.

Purpose To evaluate the scrolling propensity of pre-Descemet endothelial keratoplasty (PDEK) tissue and to compare it with each component of the PDEK tissue, namely the pre-Descemet layer (Dua's layer [PDL]) and the Descemet membrane (DM). Design Experimental laboratory investigation. Methods Fourteen human donor sclerocorneal discs in which a type 1 big bubble was obtained by stromal injection of air were studied. The wall of the type 1 big bubble was excised and its grade of scrolling noted. The components of the wall (ie, the DM and PDL) were then separated and the scrolling of each was individually graded. Statistical comparison of the grade of scrolling of each layer and correlation with age was carried out; 25-μm slices of anterior and posterior stroma obtained with the femtosecond laser from 4 control samples were used for comparison. The main outcome measure was the grade of scrolling of PDEK tissue in comparison with PDL and DM. Results Mean donor age was 67 years. The mean grade of the scroll formed by PDEK tissue was1.6 compared to 0.64 for PDL alone and 3.6 for DM alone. The difference was statistically significant. No correlation between donor age and degree of scrolling for any of the tissues tested was found. Conclusion PDEK tissue scrolls less than DM. PDL scrolls the least. This demonstrates that PDL tissue splints the DM and reduces its scrolling in PDEK. This feature has relevance to tissue preparation, handling, and unscrolling in the eye during endothelial keratoplasty.

PURPOSE: To investigate the in vivo effect of a novel femtosecond laser–assisted procedure termed stromal lenticule addition keratoplasty for advanced keratoconus. METHODS: Ten patients with stage III and IV stable keratoconus were included. Negative meniscus-shaped stromal lenticules were produced from corneoscleral eye bank buttons with a refractive lenticule extraction procedure with a 500-kHz VisuMax femtosecond laser (Carl Zeiss Meditec, Jena, Germany). Recipient corneas underwent a modified femtosecond laser flap-cut procedure to produce an intrastromal pocket and lenticules were implanted. Patients were followed up for 6 months after surgery with determination of uncorrected (UDVA) and corrected (CDVA) distance visual acuity, subjective refraction and topographic corneal curvature changes, anterior segment optical coherence tomography (AS-OCT), and in vivo confocal microscopy. RESULTS: Comparison of preoperative and 6-month postoperative UDVA and CDVA showed statistically significant improvements ( P = .024 and .007, respectively) from 1.58 ± 0.36 to 1.22 ± 0.37 and from 1.07 ± 0.17 to 0.70 ± 0.23 logMAR. Eight of 10 eyes showed an improvement in UDVA ( P < .001) that ranged between one and three lines, whereas all but one eye presented improved CDVA. Corneal topography documented a decrease between preoperative and 6-month postoperative anterior mean curvature (AVG-K at 3 mm) and anterior Q values ( P = .005). AS-OCT showed a significant increase in thickness of the central and mid-peripheral cornea produced by the lenticule implantation ( P = .005). CONCLUSIONS: The stromal lenticule addition keratoplasty procedure was clinically efficient in improving the corneal shape and vision in patients with keratoconus. Negative meniscus-shaped lenticule addition induced a flattening of the cone while increasing corneal thickness. [ J Refract Surg. 2018;34(1):36–44.]

PURPOSE: Femtosecond laser-assisted small incision lenticule extraction (SMILE) was performed to correct myopic astigmatism in a 39-year-old patient who had previously undergone deep anterior lamellar keratoplasty (DALK) for keratoconus, with clinically significant anisometropia and contact lens intolerance. METHODS: Case report. RESULTS: SMILE was planned in the right eye to reduce the refractive error and to allow spectacle correction. The surgical procedure was centered on the visual axis, a 5.20-mm optical zone was used, and the refractive lenticule was extracted through a 3.25-mm incision located inside the graft edge. The refractive target was achieved 1 week after surgery and remained stable until the 3-month follow-up visit, while the corrected distance visual acuity improved from 20/100 to 20/40. Complications were not observed and the graft remained clear. In vivo confocal microscopy and anterior segment optical coherence tomography highlighted a regular wound healing and refractive interface profile. CONCLUSIONS: This case report highlights the feasibility of using SMILE as an alternative procedure for correcting refractive errors after keratoplasty, but further prospective studies are needed to prove the long-term refractive outcomes and safety of this technique. [ J Refract Surg. 2015:31(9):634–637.]

The aim of this study is to investigate in vivo and ex vivo ocular surface alterations induced by dry eye disease and modification after osmoprotective therapy. Forty-eight eyes of 24 patients suffering from dry eye have been recruited. All patients received Optive (compatible solutes) eye drops in one randomly selected eye and Hylogel (sodium hyaluronate 0,2%) in the other. Follow-up included a baseline visit and further examination 30-, 60-, and 90-day intervals (which comprises clinical evaluation, in vivo confocal microscopy—IVCM—of the ocular surface, and conjunctival impression cytology). No significant difference in Schirmer I Test, TBUT, and vital staining results was observed during the follow-up period in both groups. IVCM showed in all patients an improvement of ocular surface epithelial morphology and signs of inflammation (oedema and keratocyte activation). However, these modifications were more evident in patients treated with Optive therapy. A significant reduction of the expression of MMP9 and IL6 in Optive group was observed during the follow-up period in comparison to Hylogel treatment. Our results show that in dry eye disease therapy based on osmoprotective eye drops determines a reduction of inflammatory activation of ocular surface, with consequent improvement of the quality of corneal and conjunctival epithelium.

To compare the features of capsulotomy obtained during femtosecond laser-assisted cataract surgery with those of continuous curvilinear capsulorhexis (CCC) obtained using a standard manual technique.Ophthalmology Clinic, Department of Medicine and Science of Ageing, University G. D'Annunzio Chieti-Pescara, Chieti, Italy.Prospective randomized clinical study.Candidates for cataract extraction were randomized into 1 of 3 groups as follows: Lensx femtosecond laser-assisted cataract surgery capsulotomy (laser group 1), Lensar femtosecond laser-assisted cataract surgery capsulotomy (laser group 2), and manual CCC (manual group).Each group comprised 30 eyes (30 patients). The capsulotomies in laser group 1 and laser group 2 showed significantly better circularity than the manual CCCs at 7 days (P.001). There was a significant correlation between the intended versus achieved capsulotomy size in the 2 laser groups. Both laser groups had better intraocular lens (IOL) centration than the manual group at all timepoints (P.001). Between-group differences in uncorrected and corrected distance visual acuities were not statistically significant. The residual spherical equivalent and mean absolute error were statistically significantly smaller in the 2 laser groups than in the manual group (P=.038) and increased significantly over time in all the groups (P.001).Femtosecond laser capsulotomies showed better circularity with more predictable size than manual CCCs. In addition, IOL centration was better immediately after surgery and over time with better refractive results in the 2 laser groups.

Ocular surface diseases (OSDs) represent a widely investigated field of research given their growing incidence and the negative impact on quality of life. During OSDs, cytokines generated by damaged epithelia trigger and deregulate the lymphoid cells composing the eye-associated lymphoid tissues, inducing an immune-mediated chronic inflammation that amplifies and propagates the disease during time. The conjunctiva-associated lymphoid tissue (CALT), given its particular position that permits immune cells covering the cornea, might play a crucial role in the development of OSDs. Despite the recognized inflammatory role of mucosa-associated lymphoid tissues in other stations taking contact with the external environment (gut or bronchus), CALT did not gain the deserved consideration. In the last years, the diffusion of the in vivo confocal microscopy (IVCM) stimulated the interest to CALT, especially in dry eye, ocular allergy, and glaucoma. Though the initial stimuli were different, IVCM documented similar changes, represented by increased lymphoid cells within the diffuse layer, follicles and interfollicular spaces. These findings, which need to be validated by immunohistology, support the CALT stimulation during OSDs. However, while an involvement of the CALT in OSDs is hypothesizable, the exact role of this structure in their pathogenesis remains unclear and warrants further investigations.

The aim of this study is to determine modifications in stromal fluorescence intensity after different corneal cross-linking (CXL) procedures and to correlate stromal fluorescence to corneal biomechanical resistance. For confocal microscopy study, 15 human cadaver corneas were examined. Three served as control (group 1), three were just soaked with iontophoresis procedure (group 2), three were treated with standard epi-off technique (group 3), and six underwent iontophoresis imbibition. Three of later six were irradiated for 30 min with 3 mW/cm2 UVA (group 4) and three for 9 min at 10 mW/cm2 UVA (group 5). Confocal microscopy was performed to quantify the fluorescence intensity in the cornea at different stromal depths. For biomechanical study, 30 human cadaver corneas were randomly divided into five groups and treated as previously described. Static stress–strain measurements of the corneas were performed. Iontophoresis imbibition followed by 10mW/cm2 irradiation proved to increase stromal fluorescence into the corneal stroma and significant differences were revealed between group 3 and 5 both at 100 (p = 0.0171) and 250 µm (p = 0.0024), respectively. Biomechanical analysis showed an improvement of corneal resistance in group 5. Iontophoresis imbibition followed by accelerated irradiation increased the stromal fluorescence and is related to an improvement of biomechanical resistance. This approach may represent a new strategy to achieve greater concentrations of riboflavin without removing corneal epithelium and improve clinical results while reducing the side effects of CXL.

Purpose.To quantify the effect of small incision lenticule extraction (SMILE) on the corneal biomechanics using Scheimpflug noncontact tonometer (Corvis ST).Methods.Twenty eyes of twenty patients, evaluated as eligible for surgery, with high myopia and/or moderate myopic astigmatism, underwent small incision lenticule extraction (SMILE). All patients underwent Corvis ST preoperatively and postoperatively after 1 week, and 1 and 3 months to observe alterations of corneal biomechanical properties. The main outcome measures were Deformation Amplitude, 1st-AT, and 2nd-AT. The relationship between the amount of stroma removed and the percentage variation of the measured parameters from baseline was evaluated with generalized linear model from each time point. For completeness also intraocular pressure (IOP), central corneal thickness (CCT), and their variations after surgery were evaluated.Results.The ratio between the amount of removed refractive error and, respectively, changes of Deformation Amplitude, 1st-AT, and 2nd-AT were significantly modified at the 1st week after surgery(P=0.005; P=0.001; P=0.024). At 1 and 3 months these values did not show statistically significant alterations. Intraocular pressure and central corneal thickness showed statistically significant changes during follow-up.Conclusions.No significant modifications in biomechanical properties were observed after SMILE so this procedure could induce only minimal transient alterations of corneal biomechanics.

The purpose of this study was to evaluate the incidence of opaque bubble layer (OBL) in femtosecond laser-assisted in situ keratomileusis (LASIK) flaps created with the support of Visumax Carl Zeiss femtosecond laser, planned with different flap diameters (7.90, 8.0, and 8.20 mm) and the same laser energy and power settings. Incidence of intraoperative OBL in flaps of consecutive 108 patients (216 eyes) subjected to bilateral femtosecond-assisted LASIK was considered. Flap creation was performed with the same laser design parameters (spot distance and energy offset) and different presetting diameters of 7.90 mm (72 eyes, group 1), 8 mm (72 eyes, group 2), and 8.20 mm (72 eyes, group 3). The incidence of OBL was considered and its extension was reported measuring involvement of different four corneal flap quadrants in which was theoretically divided the entire flap area; based on these data, OBL presence was classified as none (no evidence of OBL), minimal (minimal presence in not more that one quadrants corneal flap), mild (OBL presence in almost two or three quadrants without tendency to invade central cornea), and moderate (OBL presence in almost three quadrants with tendency to invade central cornea). In group 1, the incidence of OBL was of 23.6 % (17 eyes) with a mild/moderate presence; in group 2, incidence was 20.8 % (15 eyes) with mild presence. Group 3 presented a reduced OBL incidence (4.1 %, 3 eye) with a minimal presence. No statistically significant difference was found between group 1 and 2 (p = 0.8414).We found statistically significant differences between group 1 and group 3 (p = 0.0012) and between groups 2 and 3 (p = 0.0044). A significant reduction and extension of OBL incidence were evident when LASIK flap settings diameter was increased, and flap edge was closer to the contact glass border; this is probably consequent to a more effective gas dispersion outside of corneal flap.

Objective: To analyze the three-dimensional (3D) features of conjunctival epithelial microcysts (CEM) in eyes affected with primary open angle glaucoma (POAG). Methods: This was a case series study. Nine patients that underwent successful trabeculectomy and four eyes with medically controlled POAG were enrolled. Patients were examined with confocal laser-scanning microscope (Heidelberg Retina Tomograph/Rostock Cornea Module). Sequential images 300 × 300 μm (384 × 384 pixels) derived from automatic scans were acquired throughout the upper bulbar conjunctiva, 2 mm from the limbus. Image acquisition was performed in z-scan automatic volume mode and a series of 40 images to a maximum depth of 40 μm were captured. The 3-D volume tissue reconstruction with a maximal size of 300 × 300 × 40 μm and voxel size of 0.78 × 0.78 × 0.95 μm was performed with the AMIRA volume-rendering software package, to provide a 3-D characterization of conjunctival epithelial microcysts (CEM). Results: In the enface view, CEM appeared as empty, optically clear, round or oval shaped sub-epithelial structures. In eyes that underwent trabeculectomy CEM showed greater density and larger area compared to medically controlled glaucomatous eyes. The 3-D spatial reconstruction showed microcysts as oval-shaped, optically clear, differently sized structures, often surrounded by a well-defined and mildly thick wall. All microcysts were embedded in the extra cellular spaces and located 10 μm below the epithelial surface. Conclusions: Conjunctival epithelial microcysts were proposed as hallmark of trans-scleral aqueous humor outflow in eyes with glaucoma. They can be effectively imaged with a 3-D reconstruction system, which permits to better clarify their microscopic anatomy and patho-physiological significance.

To describe in vivo confocal microscopy (IVCM) findings in patients with limbal stem cell deficiency (LSCD).23 eyes of 17 consecutive patients suffering from LSCD were included in this study. A detailed examination by IVCM was performed in addition to a routine slit-lamp biomicroscopy. Size and density of corneal epithelial and conjunctival epithelial cells on cornea were measured and statistically analysed using SPSS version 8.0 software. Results were compared with histology in select cases.Anatomical and morphological differences were observed between normal corneal cells and conjunctival epithelial cells on cornea. Size and density differences reached statistically significant levels between the normal corneal cells and the conjunctival epithelial cells on cornea (p0.01). Goblet cells were visible throughout the conjunctivalised corneal epithelium in eight eyes. Several IVCM features could be correlated with histology in six of our patients.A number of features were demonstrated by laser IVCM in patients presenting clinically with LSCD. Some of these features were corroborated with features observed on histological examination of tissue samples.

Purpose: To assess the reproducibility and repeatability of peripapillary retinal nerve fibre layer (RNFL) thickness measurements using a spectral-domain optical coherence tomography (SD-OCT) device in healthy subjects. Methods: In this observational study, 68 young Caucasian healthy volunteers (68 eyes) were subjected to Cirrus™ high-definition (HD) OCT (Zeiss) peripapillary RNFL thickness measurements by two experienced examiners in two different sessions. Average, 4-quadrant and 12-clock-hour sector RNFL thicknesses were analysed. For each option, intra-observer, intrasession repeatability and interobserver, intersession reproducibility were tested. To assess the repeatability of measurements, the Bland and Altman plots were used and the coefficient of repeatability was calculated. Interobserver and intersession reproducibilities were analysed by means of concordance correlation coefficients (CCCs). Results: The sample age ranged from 21 to 39 years (mean 29.09, standard deviation ±5.21). The average RNFL thickness ranged from 90.97 to 91.46 and from 91.34 to 91.78 µm, for the first and the second operator, respectively. The highest repeatability and reproducibility were obtained for average RNFL thickness with coefficients of repeatability of 5.30 and 6.05 µm for the first and the second operator, interoperator CCCs of 0.95 and 0.96 for the first and the second session, and intersession CCCs of 0.96 and 0.97 for the first and the second operator, respectively. Conclusions: Cirrus OCT peripapillary average RNFL thickness measurement in young healthy subjects showed high interoperator and intersession reproducibility. Intrasession repeatability as tested by coefficient of repeatability was next to the device resolution, with very similar results between the two operators. When analysing quadrant and clock hour sector RNFL thickness measurements, both repeatability and reproducibility tend to decrease.

Purpose To ascertain in vivo confocal microscopic features of corneal/conjunctival intraepithelial neoplasia (CIN) and correlate these with histology of the same lesions. Design Observational case series with evaluation of diagnostic technology. Methods Four patients with unilateral CIN (3 men and 1 woman) were examined with the Heidelberg Retina Tomograph II (HRT II) Rostock Cornea Module (RCM) confocal microscope before, during, and after treatment. Corneal epithelial samples were taken by alcohol delamination technique in 2 patients, while impression cytology (IC) samples were obtained in the other 2 patients. Morphometric analysis of confocal and histologic images was carried out and the findings correlated. Four controls (all men, 2 with limbal stem cell deficiency, 1 with a limbal lesion, and 1 with diffuse keratoconjunctival proliferation) were similarly examined. Two of these had biopsy for histologic examination. Main outcome measures comprised the degree of correlation between histology and confocal microscopic features of CIN. Results Dysplastic cellular changes were noticed on histopathology and correlated well with confocal microscopy, corresponding to the different corneal epithelial layers. Bright nucleoli within huge nuclei and ill-defined cell borders were a feature of the basal epithelium on histopathology and confocal microscopy. Subbasal corneal nerves were not visualized on confocal microscopy in areas affected by CIN. These features disappeared in response to treatment cycles as the basal epithelium reverted to its normal pattern, as seen by confocal microscopy. Conclusion Confocal microscopy findings highly correlate with histologic features in CIN. Confocal microscopic features of CIN as defined in this study will enable a reliable diagnosis in a noninvasive manner. Confocal microscopy will also allow real-time monitoring of the condition during treatment.

PURPOSE To perform in vivo assessment of corneal alterations in patients with Cogan syndrome (CS) and to correlate these findings with prior histopathologic descriptions. METHODS Four consecutive patients (8 eyes) presenting with typical CS underwent confocal microscopy examination. At the moment of evaluation, ocular inflammation was quiescent in all the patients. The images were studied singularly, and then compared to those obtained from 5 healthy controls. Statistical analysis was performed with Student t test. RESULTS All the cases showed multiple brightly reflective deposits in the stroma with moderate reflectivity of the surrounding stromal tissue. The outer corneal layers and the endothelium were spared. In 3/4 of the patients, the nerve fibers of the subepithelial plexus were thin and poorly reflective, with interruptions and lack of the typical branching pattern. No cells different from keratocytes could be detected in either case or control eyes. Corneal vascularization was present in one case. In CS-affected eyes, corneal thickness was 552+/-62 microm on average, and endothelial cell count was 2466+/-288 cells/mm(2). Statistical significance was not reached compared to the control group. CONCLUSIONS Confocal microscopy was able to provide detailed images of corneal alterations in CS. This confirmed the interstitial nature of the keratitis and showed an excellent accordance with prior histopathologic findings. The absence of inflammatory cell infiltration may be explained by the prolonged control of ocular inflammation in our patients, which may also have limited stimuli for corneal neovascularization.

To determine the feasibility and initial outcomes of using a femtosecond laser to perform arcuate keratotomies to correct high post-keratoplasty astigmatism.Prospective noncomparative interventional case series.Twelve eyes of 12 consecutive patients (mean age 44.9+/-9.5 years) who presented with a high degree of astigmatism, noncorrectable with spectacles or contact lenses (10 post-penetrating keratoplasty, 2 post-deep lamellar keratoplasty), and were candidates for relaxing incisional corneal surgery.The Femtec (20/10 Perfect Vision, GmbH, Heidelberg, Germany) femtosecond laser performed paired 90-degree angled arcuate incisions on the graft button. The incision sites and depths were programmed at 1.00 mm inside the graft edge and at 90% of the corresponding local graft thickness, whereas the angular lengths of the cuts were determined by analyzing the locations and extents of the steepest meridians in the topographic map.Changes in uncorrected visual acuity (UCVA), best spectacle-corrected visual acuity (BSCVA), mean subjective and topographically determined astigmatism; imaging of incisions by anterior segment optical coherence tomography (AS-OCT); and wound healing by in vivo confocal microscopy (IVCM).Postoperative follow-up extended to 6 months. Mean uncorrected logarithm of the minimum angle of resolution (logMAR) BSCVA and UCVA improved from preoperative values of 0.25+/-0.16 and 1.05+/-0.18 to 6-month values of 0.11+/-0.12 (standard deviation) and 0.55+/-0.34, respectively (P0.05). Mean subjective astigmatism was 7.16+/-3.07 diopters (D) preoperatively and 2.23+/-1.55 D at 1 month after surgery (P = 0.002) and remained stable to the end of follow-up. Anterior segment optical coherence tomography image analysis showed that the depth and location of the incisions were consistent with the preoperative surgical plan. In vivo confocal microscopy showed mild edema and keratocyte activation along the incision edges, together with initial epithelial ingrowth inside the wound, followed by subsequent moderate fibrotic scarring.Arcuate keratotomies performed with the femtosecond laser were effective in reducing post-keratoplasty astigmatism. Laser-generated incisions within the graft button presented precise geometry and reliable depth of incision, with a wound healing pattern characterized by epithelial ingrowth and mild fibrosis.The author(s) have no proprietary or commercial interest in any materials discussed in this article.