In vivo microscopic and optical coherence tomography classification of neurotrophic keratopathy.

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.

In vivo and ex vivo evaluation of cell-cell interactions, adhesion and migration in ocular surface of patients undergone excimer laser refractive surgery after topical therapy with different lubricant eyedrops.

PURPOSE: To investigate, in vivo by means of in vivo confocal microscopy (IVCM) and ex vivo by impression cytology, epithelial cellular damage after excimer laser refractive surgery in patients under different topical lubricant therapies. METHODS: Two hundred eyes of 100 patients, undergone bilateral excimer laser refractive surgery for medium myopic error correction [spherical equivalent refraction from -1.75 to -3.50 dioptres (D) with refractive astigmatism under -0.75 D], have been recruited. All patients received, in addition to standard therapy for refractive surgery, high weight hyaluronic acid 0.2% eyedrops in one randomly selected eye and carboxymethylcellulose 1% eyedrop in the comparator eye (control eye) 4 times daily for 90 days. Follow-up included a baseline visit and further examination 7-, 30- and 90-day intervals [clinical evaluation with Schirmer test and tear break-up time (TBUT), IVCM and impression cytology]. RESULTS: No significant difference in Schirmer test and TBUT was observed during the follow-up period in eyes under different therapies. IVCM showed an improvement of conjunctival and corneal epithelial cells quality in eye in treatment with high weight hyaluronic acid 0.2% when compared to carboxymethylcellulose. Conjunctival impression cytology demonstrated an evident positivity for CD44 in eyes treated with both treatments in all follow-up controls. ICAM1 expression showed an increasing positivity starting at 30 days that became statistically significant after 90 days of high weight hyaluronic acid 0.2% therapy (p = 0.0167). CONCLUSIONS: In vivo and in vitro results showed the effectiveness of high weight hyaluronic acid 0.2% in facilitating cell-cell interaction, migration, cell proliferation, stabilizing epithelial barrier of the ocular surface. Moreover, use of high weight hyaluronic acid in treatment of corneal tissue damage after refractive surgery, in concordance with standard topical corticosteroids and antibiotics therapy, could be effective in promoting corneal epithelial wound healing with consequent good results in clinical outcome and patients' satisfaction.

Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography Tandem Mass Spectrometry.

Dry eye disease (DED) is a multifactorial disorder of the ocular surface unit resulting in eye discomfort, visual disturbance, and ocular surface damage; the risk of DED increases with age in both sexes, while its incidence is higher among females caused by an overall hormonal imbalance. The role of androgens has recently investigated and these hormones were considered to have a protective function on the ocular surface. In order to correlate DED to tear steroid levels, a robust, specific, and selective method for the simultaneous quantification of cortisol (CORT), corticosterone (CCONE), 11-deoxycortisol (11-DECOL), 4-androstene-3,17-dione (ADIONE), testosterone (TESTO), 17α-hydroxyprogesterone (17-OHP), and progesterone (PROG) was developed and applied for the analysis of tear samples. The method involves a simple extraction procedure of steroids from tears collected on Schirmer strips, followed by a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis. In total, tear samples from 14 DED female patients and 13 healthy female controls were analysed and, CORT, ADIONE, and 17-OHP response levels resulted significantly decreased in dry eye patients respect to controls. The receiver operating characteristic (ROC) curve obtained by the combination of these three steroids (AUC = 0.964) demonstrated the good diagnostic power of the differential tear steroids in identifying DED. In conclusion, the present method made it possible, for the first time, to study steroid profiling directly in tear fluid.

Opaque bubble layer incidence in Femtosecond laser-assisted LASIK: comparison among different flap design parameters.

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.

Confocal microscopy evaluation of stromal fluorescence intensity after standard and accelerated iontophoresis-assisted corneal cross-linking.

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.

In vivo confocal microscopy of the sclerocorneal limbus after limbal stem cell transplantation: Looking for limbal architecture modifications and cytological phenotype correlations.

PURPOSE: To correlate a biomicroscopic evaluation, an in vivo confocal microscopy examination, and impression cytologic findings of the corneal center and sclerocorneal limbus after cultured limbal stem cell transplantation and to test the effectiveness of in vivo confocal microscopy as a diagnostic procedure in ocular surface cell therapy reconstructive surgery. METHODS: Six eyes of six patients affected by limbal stem cell deficiency after chemical burns underwent ex vivo expanded limbal stem cell transplantation (two eyes) and ex vivo expanded limbal stem cell transplantation with subsequent penetrating keratoplasty (four eyes) to restore corneal transparency. One year after surgery, all patients underwent a biomicroscopic evaluation, central cornea impression cytology to detect cytokeratin 12 (CK12) positivity, and in vivo confocal microscopy of the central cornea and the sclerocorneal limbus to investigate the epithelial cellular morphology, limbal architecture, and corneal inflammation level. RESULTS: Impression cytology analysis showed CK12 positivity in five of six cases, in concordance with the biomicroscopic evaluation. Confocal microscopy pointed out irregular limbal architecture with the absence of the palisades of Vogt in all cases; the central epithelial morphology presented clear corneal characteristics in three cases and irregular morphology in the remaining three. CONCLUSIONS: After successful ex vivo expanded limbal stem cell transplantation, in the presence of a complete anatomic architecture subversion, documented by support of in vivo confocal microscopy, the sclerocorneal limbus seemed to maintain its primary function. In vivo confocal microscopy confirmed the procedure was a non-invasive, efficacious diagnostic ocular surface procedure in the case of cell therapy reconstructive surgery.

The expression of LGR5 in healthy human stem cell niches and its modulation in inflamed conditions.

PURPOSE: The aims of this study are to investigate the expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) protein in the normal human cornea and limbus and to analyze modifications of this expression under inflammatory conditions. METHODS: The expression of LGR5 was evaluated in seven limbal epithelial crypts (LECs), collected from healthy cadaver donors, and five inflamed LECs obtained from enucleated eyes. Central corneal buttons were used as controls. LGR5 protein distribution was determined by immunohistochemistry staining analysis. RESULTS: The cytoplasmic expression of LGR5 protein was observed in 100% of healthy LECs. Three out of five inflamed tissues analyzed were completely negative, while in the two remaining cases, we observed a moderate positivity in the basal cells of LECs. No relation was found between the expression of LGR5 and the grade of inflammatory cells. CONCLUSIONS: These findings demonstrate the presence of LGR5-positive cells in human LECs and their decrease in inflamed conditions, which suggests a critical role of this protein during inflammation and its possible use as a marker in normal crypts.

Induced inflammation and apoptosis in femtosecond laser-assisted capsulotomies and manual capsulorhexes: an immunohistochemical study.

PURPOSE: To evaluate cellular inflammation and apoptosis induced in the central portion of capsulorhexes/capsulotomies during cataract surgery, comparing a conventional manual technique and a femtosecond laser-assisted procedure at different energy settings using two laser systems. METHODS: Fifty-six capsulorhexes/capsulotomies were divided into four groups: the manual group (14 capsulorhexes) performed with the manual technique; the 7.0-µJ group (14 capsulotomies) (LensAR laser system; Lensar, Inc., Orlando, FL); the 10-µJ group (14 capsulotomies) (LenSx laser system; Alcon Laboratories, Inc., Fort Worth, TX); and the 13.0-µJ group (14 capsulotomies) (LenSx laser system). All samples were stained for cellular apoptosis analysis (TUNEL assay) and cellular induced inflammation (NF-κB). RESULTS: One-way analysis of variance indicated a statistically significant difference in the percentage of NF-κB and TUNEL positive cells between the four groups, (F [3.52] = 14.717, P < .001) and (F [3.52] = 139.561, P < .001), respectively. Post-hoc analysis indicated a statistically significant difference in the percentage of NF-κB positive cells between the 13.0-µJ group and the manual, 7.0-µJ, and 10-µJ groups (P < .001, = .037, and < .001, respectively). Post-hoc analysis of differences in TUNEL positive cells indicated a significant difference between the 7.0-µJ and 10-µJ groups (P <.017) and between the 13.0-µJ group and the manual, 7.0-µJ, and 10-µJ groups (P < .001, < .001, and < .001, respectively). CONCLUSION: The results show a higher percentage of NF-κB and TUNEL positive cells in the 13.0-µJ group compared to the 7.0-µJ, 10-µJ, and manual groups. Therefore, inflammatory response and cell death increased at increasing energies. An effective capsulotomy in femtosecond laser-assisted cataract surgery with minimal detrimental apoptotic and inflammatory effects is possible if the laser system is set to use the minimum energy level.

In Vivo and Impression Cytology Study on the Effect of Compatible Solutes Eye Drops on the Ocular Surface Epithelial Cell Quality in Dry Eye Patients.

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.

S100 A and B expression in normal and inflamed human limbus.

PURPOSE: To study the expression of S100 A and B family proteins in normal human limbus and to analyze modification of the expression in inflammatory conditions. METHODS: The total expression of members of the S100 family and the expression of A4, A8, A9, and B individually were evaluated in nine normal human corneal limbi, collected from cadaver healthy donors, in particular in the limbal epithelial crypts (LECs), and in five inflamed limbi obtained from enucleated eyes. S100 protein distribution was determined with immunohistochemistry staining analysis. RESULTS: Cytoplasmic expression of total S100 proteins was observed in 100% of LECs; in contrast, the inflamed tissues were completely negative, and faint positivity was observed in only one case. Moreover, cytoplasmic expression of S100 A4 and A9 was uniformly found in the entire LECs in all samples analyzed, while S100 A8 positivity was observed in only 44.4% of cases and only in the cells localized in the central area of the LEC. Positivity for S100 B was not observed in all samples analyzed. CONCLUSIONS: As reported in the literature, normal limbal epithelial cells show strong expression of S100 proteins. A novel finding of this study was the expression for the limbal epithelial crypts. In particular, S100 A4 and A9, which are normally involved in regulating a wide range of biologic effects, including cell motility, survival, and differentiation, are the most expressed members in healthy limbal crypts. In inflamed tissues, expression of S100 proteins was dramatically decreased. S100 proteins, and in particular S100 A4 and S100 A9, can be useful as markers of early changes in stem cell niches due to inflammation.

Pathological changes of the anatomical structure and markers of the limbal stem cell niche due to inflammation.

PURPOSE: The corneoscleral limbus is the site of corneal epithelial stem cells (SC). The aim of this study is to evaluate the expression of different SC markers in the normal human limbus and to determine how this is affected by inflammation. METHODS: Corneoscleral specimens from healthy and inflamed donor eyes were examined by immunohistochemistry/immunofluorescence for p63, vimentin, laminin 5, integrin α6, ß1, ß4, ABCG2, desmoglein 3, connexin 43, N-cadherin, and cytokeratins 12 and 15. The distribution and anatomic structure of the limbal crypts and the percentage of SC marker antigens in healthy donors were analyzed. In inflamed tissues, we evaluated the anatomic structure of the limbal epithelial crypt (LEC) and the positivity for SC markers. RESULTS: In normal limbus, the niche structures were distributed differently. The variability of their number correlated with the percentage of p63 positivity. Integrin ß1 staining directly correlated with p63 positivity while the remaining proteins were variably and widely distributed. Double staining for p63 and vimentin did not reveal any co-localization. In inflamed eyes, the basal cells in the crypts were "stretched" and surrounded by inflammatory cells, and only a few SC markers were still present. CONCLUSIONS: Diseases involving the limbus may result in marked changes of expression of SC markers within the LEC and also alter the crypt structure.

Expression of CREB in primary pterygium and correlation with cyclin D1, ki-67, MMP7, p53, p63, Survivin and Vimentin.

AIM: Ultraviolet (UV) B irradiation induces gene expression that leads to skin cancer. Among the transcription factors induced by UVB radiation exposure, the cyclic AMP response element-binding protein (CREB) is significant. Since several factors downstream of CREB signaling are known to be involved in pterygium pathogenesis, we investigated CREB expression in pterygial and human conjunctival tissues to evaluate if a similar expression pattern is present. Moreover, we analyzed the correlation with CREB expression and other known pterygium markers. METHODS: Primary pterygium samples and normal bulbar conjunctivas surgically removed were analyzed. Formalin-fixed, paraffin-embedded tissues were stained by immunohistochemistry with anti-CREB, anti-vimentin, anti-ki-67, anti-survivin, anti-MMP7, anti-p63, anti-cyclin D1, or anti-p53 antibodies. RESULTS: 94.4% of pterygium samples were positive for CREB with a significant difference compared to the control group (p = 0.002). The staining was localized in the epithelium and absent in the stroma. An increased expression was found for cyclin D1 (p = 0.019), ki-67 (p = 0.005), vimentin (p = 0.003), survivin (p < 0.001), p63 (p = 0.003), and MMP7 (p = 0.002). CREB expression showed a significant correlation with cyclin D1 (ρ = 0.49; p = 0.035), ki-67 (ρ = 0.61; p = 0.007), and p53 (ρ = 0.57; p = 0.013) in pterygium. CONCLUSIONS: These results permit to hypothesize that CREB is involved in pterygium pathogenesis. Since various molecules have been discovered to inhibit CREB, these data could be of interest for pterygium treatment.

Epithelial and stromal remodelling following femtosecond laser-assisted stromal lenticule addition keratoplasty (SLAK) for keratoconus.

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.

In Vivo Evaluation of Corneal Nerves and Epithelial Healing After Treatment With Recombinant Nerve Growth Factor for Neurotrophic Keratopathy.

PURPOSE: 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. DESIGN: Prospective, interventional, before-and-after case series. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Femtosecond laser arcuate keratotomy for the correction of high astigmatism after keratoplasty.

PURPOSE: To determine the feasibility and initial outcomes of using a femtosecond laser to perform arcuate keratotomies to correct high post-keratoplasty astigmatism. DESIGN: Prospective noncomparative interventional case series. PARTICIPANTS: 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. METHODS: 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. MAIN OUTCOME MEASURES: 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). RESULTS: 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 (P<0.05). 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. CONCLUSIONS: 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. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Orientation teeth in nonmechanical femtosecond laser corneal trephination for penetrating keratoplasty.

PURPOSE: To evaluate femtosecond laser-assisted corneal full-thickness trephination with orientation teeth and notches for penetrating keratoplasty (PKP). DESIGN: Interventional case series. METHODS: Four eyes of four patients aged 34 to 55 years underwent PKP using a femtosecond laser system enabling corneal trephination with orientation teeth and notches. Patients were affected by advanced keratoconus (n = 2) or postinfectious corneal scar (n = 2). Scanning electron microscopy (SEM) was performed to evaluate the cut quality of donor and recipient cornea. RESULTS: All procedures were completed without any complications. In all cases, the donor and the recipient cornea were cut with the same size of 8.0-mm diameter. A complete perforating cut was achieved in all eyes, and a correct intraoperative matching of teeth and notches was obtained. SEM of donor and recipient specimens showed a full-thickness cut with smooth and regular shape of orientation teeth and notches. CONCLUSIONS: Orientation teeth and notches shaped PKP using the femtosecond laser technology is feasible and may offer further promising approaches toward customized trephination techniques.

Interference figures of polarimetric interferometry analysis of the human corneal stroma.

A rotating polarimetric 90°-cross linear-filter interferometry system was used to detect the morphological characteristics and features of interference patterns produced in in-vivo corneal stroma in healthy human corneas of 23 subjects. The characteristic corneal isogyres presenting with an evident cross-shaped pattern, grossly aligned with the fixation axis, were observed in all patients with centers within the pupillary dark area, impeding the exact determination of the center point. During the rotational scan in 78.3% of the eyes the cross-shaped pattern of the isogyre gradually separated to form two distinct hyperbolic arcs in opposite quadrants, reaching their maximal separation at 45 degrees with respect to angle of cross-shaped pattern formation. The corneal cross and hyperbolic-pattern repeated every 90° throughout the 360° rotational scan. While the interpretation of the isogyres presents particular difficulties, two summary parameters can be extracted for each cornea: the presence/orientation of a single or two dark areas in post-processed images and isochromes. However, the development of dedicated software for semi-quantitative analysis of these parameters and enantiomorphism may become available in the near future. The possible application of polarimetric interferometry in the field of both corneal pathologies and corneal surgery may be of great interest for clinical purposes.

In Vivo and Ex Vivo Evaluation of Inflammation and Apoptosis Induced after SMILE Procedures for Different Refractive Error Range.

PURPOSE: To detect corneal inflammation and apoptosis induced after small incision lenticule extraction (SMILE) at different refractive corrections for moderate to high values of myopia. METHODS: 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. RESULTS: No correlation was observed between treated myopic refractive error and number of CD11b+ and TUNEL+ cell in all analyzed extracted lenticules. IVCM at 1 week and 1 month of follow-up showed numerous reflective particles at the laser treatment interface with a moderate light scattering. In semi-quantitative analysis of reflectivity intensity at the laser interfaces, a statistical difference was evident only between 1 week and 1 month (p = 0.0213). CONCLUSIONS: SMILE, 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.

Small Incision Lenticule Extraction After Deep Anterior Lamellar Keratoplasty.

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.

Big bubble deep anterior lamellar keratoplasty: the collagen layer in the wall of the big bubble is unique.

PURPOSE: In big bubble (BB), deep anterior lamellar keratoplasty intracorneal injection of air separates Descemet's membrane (DM) and the pre-Descemet's layer (Dua's layer [DL]) to create a type 1 BB. We tested the hypothesis that air injection after excision or ablation of DL will fail to produce a BB. METHODS: Nine human sclero-corneal discs were used. Three served as controls. In three, a type 1 BB was created, the bubble wall was excised and more air injected in an attempt to create another BB. In three samples, the DM was removed and 22µ of posterior cornea were ablated by phototherapeutic keratectomy (PTK). Air was injected to induce formation of a BB. Tissue from these experiments was subjected to light and electron microscopy. RESULTS: In all three control eyes, a type 1 BB (DL + DM) was obtained. Air injection after excision of the type 1 BB wall in three samples failed to produce another BB. Following PTK of DL, injection of air failed to create a BB in all three samples. Multiple points of air leak from the deep stroma were observed in all six samples. Light and electron microscopy showed a clear distinction between the ablated and non-ablated areas of cornea. CONCLUSIONS: This study supports the hypothesis that a BB cannot be created once the DL is excised or ablated. This adds to evidence that DL is unique. It also demonstrates that DL is not a random separation of deep stroma of the cornea.