Femtosecond Laser Cutting of Human Crystalline Lens Capsule and Decellularization for Corneal Endothelial Bioengineering.

The bioengineering of corneal endothelial grafts consists of seeding in vitro cultured corneal endothelial cells onto a thin, transparent, biocompatible, and sufficiently robust carrier which can withstand surgical manipulations. This is one of the most realistic alternatives to donor corneas, which are in chronic global shortage. The anterior capsule of the crystalline lens has already been identified as one of the best possible carriers, but its challenging manual preparation has limited its use. In this study, we describe a femtosecond laser cutting process of the anterior capsule of whole lenses in order to obtain capsule discs of 8 mm diameter, similar to conventional endothelial grafts. Circular marks made on the periphery of the disc indicate its orientation. Immersion in water for 3 days is sufficient to completely remove the lens epithelial cells and to enable the seeding of corneal endothelial cells, which remain viable after 27 days of culture. Therefore, this method provides a transparent, decellularized disc ready to form viable tissue engineered endothelial grafts.

Design and validation of a custom-made system to measure transepithelial electrical impedance in human corneas preserved in active storage machine.

Corneal epithelial barrier represents one of the major limitations to ocular drug delivery and can be explored non-invasively through the evaluation of its electrical properties. Human corneas stored in active storage machine (ASM) could represent an interesting physiological model to explore transcorneal drug penetration. We designed a new system adapted to human corneas preserved in ASM to explore corneal epithelial barrier function ex-vivo. A bipolar set-up including Ag/AgCl electrodes adaptors to fit the corneal ASM and a dedicated software was designed and tested on freshly excised porcine corneas (n = 59) and human corneas stored 14 days in ASM (n = 6). Porcine corneas presented significant and proportional decrease in corneal impedance in response to increasing-size epithelial ulcerations and acute exposure to benzalkonium chloride (BAC) 0.01 and 0.05%. Human corneas stored 14 days in ASM presented a significant increase in corneal impedance associated with the restoration of a multi-layer epithelium and an enhanced expression of tight junctions markers zonula occludens 1, claudin 1 and occludin. These results support the relevance of the developed approach to pursue the exploration and development of human corneas stored in ASM as a physiological pharmacological model.

Nanotopography by chromatic confocal microscopy of the endothelium in Fuchs endothelial corneal dystrophy, pseudophakic bullous keratopathy and healthy corneas.

AIM: To investigate the interest of chromatic confocal microscopy (CCM) to characterise guttae in Fuchs endothelial corneal dystrophy (FECD). METHODS: Descemet's membranes (DM) were obtained during endothelial keratoplasty in patients with FECD and pseudophakic bullous keratopathy (PBK). They were compared with healthy samples obtained from body donation to science. Samples were fixed in 0.5% paraformaldehyde and flat mounted. Surface roughness of DMs was quantified using CCM and the AltiMap software that provided the maximum peak (Sp) and valley (Sv) heights, the mean square roughness (Rq) and the asymmetry coefficient (Ssk). RESULTS: The physiological roughness of healthy samples was characterised by an Rq of 0.12±0.05 µm, which was two times rougher than in PBK (Rq=0.06±0.03 µm), but both were still flat with a symmetrical distribution between peaks and valleys (Ssk close to 0, npeaks=nvalleys), smaller than 1 µm. In FECD, the maximum peak height was 5.10±2.40 µm, up to 5.8 and 8.3 times higher than the control and PBK, respectively. The maximum valley depth was half than the peak (2.28±0.89 µm). The surface with guttae was very rough (Rq=0.45±0.14 µm) and the Ssk=1.84± 0.43 µm, greater than 0, confirms an asymmetric surface with high peaks and low valleys (npeaks>nvalleys). Moreover, the CCM provided quantitative parameters allowing to distinguish different types of guttae from different patients. CONCLUSIONS: CCM is an innovative approach to describe and quantify different morphologies of guttae. It could be useful to analyse the different stages of FECD and define subgroups of patients.

P02-A121†Precut DSAEK stored in an active storage machine: feasibility study.

PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: to measure endothelial viability of precut DSAEK after 3 or 10 days of storage in our ASM in a preclinical study. METHODS: Human pairs of corneas were included. The endothelial cell density (ECD in cells/mm2), and central corneal thickness (CCT in µm) were measured to ensure their initial intra pair comparability. After deswelling (CorneaJet, Eurobio) grafts preparation was performed by cutting the anterior stroma with a Moria linear microkeratome and keeping the anterior lamellae attached during storage. After randomization, one cornea was kept in the corneajet bottle (CJ) and the other was inserted into the ASM allowing a renewal or storage medium (CorneaMax, Eurobio) at 2.6 µL/min with 21 mmHg of pressure in the endothelial chamber. Both group of corneas were stored for 3 or 10 days at 31°C. The final viable ECD (vECD) was determined using the triple staining with Hoechst-Ethidium-Calcein-AM by an independent experimenter in a masked fashion. RESULTS: Initial ECDs were comparable: 2595±878 in ASM versus 2654±954 cells/mm2 in CJ for the 3-days period (n=5 pairs) and 2416±712 in ASM versus 2492±764 cells/mm2 in CJ for the 10-period (n=5 pairs). CCTs were also comparable. The anterior lamellae stayed attached in either the ASM or CJ. vECD was significantly higher in ASM than in CJ with respectively 2062±695 cells/mm2 versus 1632±633 cells/mm2 after 3 days either a cell loss of 20.5% and 38.5% respectively (p=0.0062) and 1082±649 versus 935±691 cells/mm2 for the 10-day period either a cell loss of 132% and 164% respectively (p=0.005). Grafts thickness did not differ after 3 days 219±25 µm in ASM versus 182±39 µm (p=0.063) or 10 days respectively 221±58 µm versus 189±48 µm (p=0.06). CONCLUSION: The storage of precut DSAEKs into the ASM allows a better preservation of grafts without use on deswelling storage medium. Nevertheless, the cell loss remains high after 10 days, suggesting a significant cell stress.

P01-A120†Pre-dissected dmek stored in an active storage machine: feasibility study.

PURPOSE: The number of endothelial grafts precut by eye banks increases. Their shelf life is limited to a few days. We previously demonstrated the superiority of an active storage machine (ASM) over organ culture (passive) for whole corneas. AIMS: To measure the endothelial viability of pre-dissected DMEK after 3 and 10 days of storage in our ASM in a preclinical study. METHODS: Pairs of human corneas were included. The endothelial cell density (ECD in cells/mm2), thickness and transparency of corneas were measured before graft preparation. Descemet's membrane (DM) was peeled using the no-touch technique leaving the graft attached to the center of the cornea (on approx. 1mm2). After randomization, one cornea was kept in organ culture (OC) and the other in the ASM (21 mmHg, 2.6 µL/min) in the same medium (CorneaMax, Eurobio). The final viable ECD was determined using the triple staining with Hoechst-Ethidium-Calcein-AM. In addition, the expression of CD166 and NCAM (lateral membranes), ZO-1 (apical junctions), Na+/K+ ATPase (endothelial pump function) and COX-IV (mitochondrial content) was studied by immunostaining to characterize endothelial cells after the storage. RESULTS: Initial ECDs were comparable: 2185±232 cells/mm2 in the ASM versus 2276±328 in OC for the 3-day period and 2680±416 cells/mm2 in the ASM versus 2644±420 in OC for the 10-day period. The DMs did not fold back in either BR or OC. The viable ECD did not differ significantly between the ASM and OC for either storage period: 2378±501 (ASM) versus 2342±503 (OC) for the 3-day period (n=8 pairs and p=0.624) and 2482±288 (ASM) versus 2579±315 (OC) for the 10-day period (n=5 pairs and p=0.176). Corneas were more transparent and thinner in the ASM than in OC after 3 days (916±86 versus 1193±136µm, p=0.0001) and 10 days (957±128 versus 1220±105µm, p=0.0625). The functional and structural markers studied were expressed in both groups after 3 and 10 days, some better preserved in the ASM. CONCLUSION: The storage of precut DMEKs is possible in ASM and OC for at least 10 days. Interestingly, a pre-dissected endothelium continues to partially exert its pump function into the ASM. In practice, this could allow the stroma to be used for DALK without further deswelling. In addition to improving the storage of whole grafts, the ASM allows the storage of precut DMEKs for up to 10 days with excellent endothelial survival.

P33-A110†Ex vivo models of corneal epithelial regeneration in bioreactor: respective roles of limbal, conjunctival and corneal epithelia.

PURPOSE: Human corneas preserved in bioreactor (BR) are characterized by not only a better endothelial viability, but also a more differentiated and stratified epithelium compared to corneas preserved in organoculture. By using corneal preservation in BR, we aimed to analyze the respective contribution of corneal (C), limbal (L), and conjunctival (Conj) epithelia in corneal epithelial regeneration. METHODS: Five pairs of corneas from body donation to Science were used with a death-to-collection time <20 hours. A 3- to 5-mm-wide conjunctival flange was kept intact. Five patterns were set up by complete mechanical removal of 1, 2, or 3 epithelia (-): C-L+Conj+, C-L-Conj+, C-L+Conj-, C+L-Conj-, C-L-Conj- (control) (n=2 for each pattern). The L epithelia was destroyed by scraping and thermocoagulation. Corneas were then kept in BR (21mmHg, 2.5µl/min of Corneamax Eurobio, 31°C) for 3 weeks to allow epithelial regeneration. The epithelium was then analyzed using immunofluorescence (IF) on flat mounted cornea by targeting CK12 (corneal epithelium) and CK15 (limbal epithelium). Cell nuclei were counterstained with DAPI. Corneal transparency was quantified using a transparometer. RESULTS: No epithelium was reconstituted in the C-L-Conj- control group. In the other 4 models including the C-L-Conj+ group, the cornea was transparent and covered by a pluristratified corneal epithelium, characterized by CK12 expression. CONCLUSION: In this BR model, conjunctival epithelial cells alone allowed the regeneration of a typical corneal epithelium whereas corneal epithelium was able to migrate to the limbus and conjunctiva. We hypothesize that all 3 ocular surface epithelia contain stem cells or progenitors able to migrate throughout the cornea and restore the corneal epithelium independently of each other. The main difference between our ex vivo model and in vivo situation is the absence of neovascularization. This suggests that the main cause of limbic insufficiency is due to the loss of the anti-angiogenic barrier rather than the loss of limbic stem cells.

P03-A138†Comparison of specular and light transmission microscopy for endothelial control of corneas stored in the active storage machine.

PURPOSE: The Active Storage Machine (ASM), designed by Sincler (a company of group Laboratoires Théa) for eyebanks, will be used for long term donor corneas preservation at 31°C before transplantation. In this device, the endothelial cell density (ECD) counting is expected to be performed non-invasively throughout the storage, thus without changing the storage medium nor handling the cornea. To meet these constraints, specular microscopy (SM), also used for cold storage was selected, instead of the standard light transmission microscopy (LTM-NaCl) used in eye banks storing corneas in organ culture at 31-34°C. The purpose of this study is to compare both imaging methods for ECD measurement of corneas preserved in ASM. METHODS: Five human corneas from body donation to Science were preserved in a prototype ASM with 35mmHg in the endothelial chamber, 2.5µl/min of Corneamax® (Eurobio, France) at 31°C for 5 days. The endothelium of the cornea was imaged through the ASM window using the CellChek® D+ SM (Konan Medical, California, United-States) equipped with an add on device at customized stage. The cornea was then immediately removed from the ASM and prepared for standard endothelial assessment (dilation of the intercellular spaces using 0.9% NaCl and light transmission imaging). Finally, the endothelium was stained with alizarine red and trypan blue and observed again with the same microscope, to determine ECD using the referenced method up to now. For each cornea and each observation method, 5 images were acquired: 1 central and 4 paracentral. The SM images were counted with the Konan software. The LTM-NaCl and 'Alizarin Red' counts were performed with a dedicated plugin of ImageJ after microscope calibration. RESULTS: The means ± SD of the ECD calculated for SM, LTM-NaCl and 'Alizarine' images were respectively of 2314 ± 537, 2243 ± 506 and 2354 ± 543 cells/mm2. There was no significant difference between the 3 methods (ANOVA one-way, p-value = 0.1066). The percentage error was -1.7% +/- 3.3% for SM and -4.7 +/- 4.0% for light transmission microscopy. CONCLUSION: Quality control of the endothelium of corneas stored in ASM can be performed non-invasively with a standard eye bank SM. The ECD measured by SM does not differ from that measured by the conventional microscopy technique used until now in organoculture.

P38-A108†Optimization of the triple HEC staining for laboratory assessment of DMEK graft quality.

INTRODUCTION: The quality of the endothelial graft is critical to the success of DMEK and to the survival time of the graft. The peeling technique, preservation method, and skill level of graft preparers need to be evaluated and validated. The most reliable method of evaluation is the viability test based on a triple staining of Hoechst- Ethidium-Calcein AM (H-E-C) which allows the determination of the total number of viable cells on the graft. However, this test has some shortcomings for DMEK grafts: 1) The undesirable fluorescence of the Calcein AM stain prevents accurate viability analysis, especially in cases where the graft is attached to the cornea for preservation; 2) Incompatibility with immunofluorescence (IF) that could provide additional information. The objective of this study is to develop technical tricks to overcome these drawbacks. METHODS: Two strategies were employed to improve Calcein AM staining: 1. Increase the specific fluorescence intensity by changing the diluent and the concentration of Calcein AM; 2. Decrease undesired fluorescence from keratocytes by adding Trypan Blue (BT). In order to combine the IF after the HEC test, an extension wash in PBS was performed. RESULTS: Calcein AM at 4µM diluted in OptiMEM increased fluorescence intensity 3-fold (p=0.0017, n=5) compared with conventional staining at 2µM in PBS. BT decreased the undesired fluorescence of Calcein and thus optimized count variability between different operators by 42% (p=0.0027, n=10) and saved 40% (p=0.0002, n=10) of count time. To perform IF after HEC, prolonged washing in PBS is an effective method to remove residual Calcein fluorescence and allows release of the FITC/Alexa 488 filter. CONCLUSION: This study provides effective technical tips for optimizing the endothelial viability assay using Calcein AM and for performing IF after the viability assay.

Investigating the Role of TGF-ß Signaling Pathways in Human Corneal Endothelial Cell Primary Culture.

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial-mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-ßs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-ß signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-ß1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-ß-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-ß1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-ß receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-ß1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.

Shotgun proteomics identification of proteins expressed in the Descemet's membrane of patients with Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a slowly evolving, bilateral disease of the corneal endothelium, characterized by an abnormal accumulation of extracellular matrix (ECM) in the basement membrane (Descemet's membrane, DM). This results in the formation of small round excrescences, called guttae, and a progressive disappearance of endothelial cells. In the intermediate stage, the numerous guttae create significant optical aberrations, and in the late stage, the loss of endothelial function leads to permanent corneal edema. The molecular components of guttae have not been fully elucidated. In the current study, we conducted shotgun proteomics of the DMs, including guttae, obtained from patients with FECD and revealed that 32 proteins were expressed only in the FECD-DMs but not in the DMs of control subjects. Subsequent enrichment analyses identified associations with multiple ECM-related pathways. Immunostaining of flat-mounted DMs confirmed that 4 of the top 5 identified proteins (hemoglobin α, SRPX2, tenascin-C, and hemoglobin γδεß) were expressed in FECD-DMs but not in non-FECD-DMs. Fibrinogen α was strongly expressed in FECD-DMs, but weakly expressed in non-FECD-DMs. We also demonstrated that matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) can display the in situ spatial distribution of biomolecules expressed in the DM, including the guttae.

Keratoconus and the Impact of Treatment on Patients' Quality of Life: A Qualitative Study.

INTRODUCTION: Keratoconus has a significant impact on patients' quality of life (QoL), from diagnosis to the advanced stages of the disease. The aim of this research was to identify domains of QoL affected by this disease and its treatment. METHODS: Phone interviews were conducted using a semi-structured interview guide, with patients with keratoconus stratified according to their current treatment. A board of keratoconus experts helped identify the guide's main themes. RESULTS: Thirty-five patients (rigid contact lenses, n = 9; cross-linking, n = 9; corneal ring implants, n = 8; and corneal transplantation, n = 9) were interviewed by qualitative researchers. Phone interviews revealed several QoL domains affected by the disease and its treatments: "psychological", "social life", "professional life", "financial costs" and "student life". All domains were impacted, independently of the treatment history. Few differences were found between treatment regimens and keratoconus stages. Qualitative analysis enabled the development of a conceptual framework based on Wilson and Cleary's model for patient outcomes common to all patients. This conceptual model describes the relationship between patients' characteristics, their symptoms, their environment, their functional visual impairment and the impact on their QoL. CONCLUSIONS: These qualitative findings supported the generation of a questionnaire to evaluate the impact of keratoconus and its treatment on patients' QoL. Cognitive debriefings confirmed its content validity. The questionnaire is applicable for all stages of keratoconus and treatments and may help tracking change over time in regular clinical settings. Psychometric validation is yet to be performed before its use in research and clinical practices.

Comprehensive mass spectrometry lipidomics of human biofluids and ocular tissues.

Evaluating lipid profiles in human tissues and biofluids is critical in identifying lipid metabolites in dysregulated metabolic pathways. Due to various chemical characteristics, single-run lipid analysis has not yet been documented. Such approach is essential for analyzing pathology-related lipid metabolites. Age-related macular degeneration, the leading cause of vision loss in western countries, is emblematic of this limitation. Several studies have identified alterations in individual lipids but the majority are based on targeted approaches. In this study, we analyzed and identified approximately 500 lipid species in human biofluids (plasma and erythrocytes) and ocular tissues (retina and retinal pigment epithelium) using the complementarity of hydrophilic interaction liquid chromatography (HILIC) and reversed-phase chromatography (RPC), coupled to high-resolution mass spectrometry. For that, lipids were extracted from human eye globes and blood from 10 subjects and lipidomic analysis was carried out through analysis in HILIC and RPC, alternately. Furthermore, we illustrate the advantages and disadvantages of both techniques for lipid characterization. RPC showed greater sensitivity in hydrophobicity-based lipid separation, detecting diglycerides, triglycerides, cholesterol, and cholesteryl esters, whereas no signal of these molecules was obtained in HILIC. However, due to coelution, RPC was less effective in separating polar lipids like phospholipids, which were separated effectively in HILIC in both ionization modes. The complementary nature of these analytical approaches was essential for the detection and identification of lipid classes/subclasses, which can then provide distinct insights into lipid metabolism, a determinant of the pathophysiology of several diseases involving lipids, notably age-related macular degeneration.

Technique of flat-mount immunostaining for mapping the olfactory epithelium and counting the olfactory sensory neurons.

The pathophysiology underlying olfactory dysfunction is still poorly understood, and more efficient biomolecular tools are necessary to explore this aspect. Immunohistochemistry (IHC) on cross sections is one of the major tools to study the olfactory epithelium (OE), but does not allow reliable counting of olfactory sensory neurons (OSNs) or cartography of the OE. In this study, we want to present an easy immunostaining technique to compensate for these defects of IHC. Using the rat model, we first validated and pre-screened the key OSN markers by IHC on cross sections of the OE. Tuj-1, OMP, DCX, PGP9.5, and N-cadherin were selected for immunostaining on flat-mounted OE because of their staining of OSN dendrites. A simple technique for immunostaining on flat-mounted septal OE was developed: fixation of the isolated septum mucosa in 0.5% paraformaldehyde (PFA) preceded by pretreatment of the rat head in 1% PFA for 1 hour. This technique allowed us to correctly reveal the olfactory areas using all the 5 selected markers on septum mucosa. By combining the mature OSN marker (OMP) and an immature OSN marker (Tuj-1), we quantified the mature (OMP+, Tuj-1-), immature (OMP-, Tuj-1+), transitory (OMP+, Tuj-1+) and total OSN density on septal OE. They were respectively 42080 ± 11820, 49384 ± 7134, 14448 ± 5865 and 105912 ± 13899 cells per mm2 (mean ± SD). Finally, the same immunostaining technique described above was performed with Tuj-1 for OE cartography on ethmoid turbinates without flat-mount.

Dropless penetrating keratoplasty using a subconjunctival dexamethasone implant: safety pilot study.

BACKGROUND/AIM: Rejection is the main cause of graft failure after penetrating keratoplasty (PK). Its prevention by repeated instillation of steroid eye-drops has not evolved in decades. Poor adherence and discontinuous nature of eye-drop treatment may explain some PK failures. In a rabbit model, we previously demonstrated that a subconjunctival dexamethasone implant was well tolerated and prevented rejection efficiently in the first 5-6 weeks. This clinical trial investigates its tolerance and safety after PK. METHODS: Single-centre, phase II non-randomised tolerance and safety pilot study (NCT02834260). Designed to analyse the risk of elevated intraocular pressure (IOP), discomfort and resorption time. Fourteen patients with a low rejection risk indication of PK were enrolled between January 2017 and August 2018. The implant was injected in the 12 o'clock position, 5 mm from the limbus, at the end of PK. A steroid eye-drop treatment was planned when implant resorption was complete. Patients were monitored regularly for 12 months: IOP (main outcome measure at 1 month), discomfort and redness scores, implant status, rejection episode and central corneal thickness by optical coherence tomography. An independent data safety monitoring committee verified safety aspects. RESULTS: No increase in IOP or other adverse event related to the implant was observed. Average resorption time was 6 weeks. The switch to steroid eye-drops was uneventful. One patient, included despite preoperative corneal neovascularisation (unintended protocol deviation) experienced a rejection. CONCLUSIONS: This is the first proof of concept that dropless immunosuppression is possible after low rejection risk PK. TRIAL REGISTRATION NUMBER: NCT02834260.

Treatment of corticosteroid-resistant Graves' orbitopathy with tocilizumab: a single-centre prospective study.

PURPOSE: To assess tocilizumab (TCZ) as an emergent treatment for corticosteroid-resistant active Graves' orbitopathy (GO). METHODS: We conducted a single-centre prospective study. We assessed TCZ in patients with active corticosteroid-resistant GO. Each patient received intravenous TCZ every four weeks until symptom stabilization. Our primary outcome was GO activity evaluated by the clinical activity score (CAS). The secondary outcomes included variation in thyroid-stimulating immunoglobulin (TSI). RESULTS: We included ten patients. Three patients had compressive neuropathy with visual field impairment and vision loss. CAS improved significantly in 100% of the patients included in the analysis, with a decrease in the mean CAS of 4.5 ± 1.2 points (p = .003). There was a significant decrease in the TSI after therapy, from 21.7 ± 22.9 at baseline to 4.0 ± 3.3 (p = .006). A mean of three infusions was necessary to drastically decrease the TSI amount. The baseline mean before TCZ was 4.7 ± 1.2 and the final mean after TCZ IV infusion was 0.2 ± 0.4. CONCLUSIONS: Our study showed the efficiency of TCZ in patients with GO resistant to corticosteroid therapy, as shown in previous studies. Our present work adds two important pieces of information: TCZ might be particularly useful for GO with compressive neuropathy and it is efficient regardless of initial TSI level. Considering the numerous advantages over steroids (high response rate and lower rate of adverse events), further randomized controlled trials should be conducted to assess the possible place of TCZ as a first-line treatment.

Femtosecond laser-cut autologous anterior lens capsule transplantation to treat refractory macular holes.

BACKGROUND: To report the long-term outcomes of round autologous anterior lens capsules (ALCs) cut by a femtosecond laser (FSL) and transplanted onto refractory macular holes (MHs) in a prospective interventional study. METHODS: Three eyes of three patients were included for persistent MH after reattached rhegmatogenous retinal detachment (RRD) (n = 2) or RRD recurrence by persistent MH (n = 1), in a university hospital. A 6 mm diameter ALC disc was carefully extracted during FSL-assisted lens extraction, stained with 0.06% trypan blue, decellularised, transplanted using a catheter and unfolded over the MH. Gas or silicone-oil tamponade was used. At 1 year, the main criterion was anatomic success, defined as complete MH closure. Secondary criteria were changes in best corrected visual acuity (BCVA), ellipsoid zone (EZ) and external limiting membrane (ELM) defects, complications. RESULTS: Baseline data were: minimum and maximum diameters, respectively 887, 1079 and 1180 µm; 1260, 1213 and 1350 µm; central posterior staphyloma in two highly myopic eyes; number of prior surgeries 2 ± 1. At 1 year, the three MHs were closed with stable transplanted ALCs. Distant BCVA improved respectively from 3.0, 0.8, 3.0 to 1.0, 0.2, 0.7 logMAR, i.e. all eyes achieved ≥0.3 logMAR improvement. All patients had decreased EZ and ELM defects, without reaching normal profile. No adverse event occurred. CONCLUSIONS: FSL-cut ALC helps standardise this challenging surgery: it prevents from tears and facilitates manipulation, so that the ALC disc is perfectly transparent and biocompatible, with a large MH overlap. One-year follow-up highlighted that this technique helps safely close refractory MHs with satisfactory visual recovery.

Sodium hyaluronate 0.30% ocular gel versus sodium hyaluronate 0.18% eye drop in the treatment of moderate to severe dry eye disease.

PURPOSE: Compare 0.30% sodium hyaluronate (0.30%HA) ocular gel with 0.18%HA eye drops in terms of improvement of ocular signs and symptoms, in patients with moderate to severe dry eye disease (DED). METHODS: This was a multicentric, randomized, investigator-masked, non-inferiority, comparative study conducted over 84 days. Three visits were scheduled, testing fluorescein corneal and conjunctival staining (Oxford and Van Bijsterveld scores), tear film break-up time (TBUT), Schirmer test, DED symptoms, 5-Item-Dry-Eye-Questionnaire (5-DEQ), patient and investigator satisfaction and frequency of instillation. RESULTS: At Day 35 (D35) and Day 84 (D84), both groups (n = 35 each) had a significant improvement in corneal staining (p < 0.001) with no inter-group difference. Van Bijsterveld score improved earlier (D35) for 0.30%HA suggesting a faster effect on conjunctival epithelium healing. There was no difference between the two concentrations in terms of TBUT or Schirmer improvements; however, the Schirmer test increase was only significant for 0.30%HA at D35 (p = 0.040). At D35 and D84, both groups showed similar improvements of DED symptoms and DEQ-5 score. Furthermore, treatment satisfaction was similar for the 2 formulations suggesting that daily use of 0.30%HA do not cause gel-related blurred vision disturbances. Frequency of instillation was similar for both groups. CONCLUSION: Our study demonstrates the non-inferiority of 0.30%HA gel compared to 0.18%HA solution in patients with moderate to severe DED. Because of its gel formulation and higher HA concentration providing prolonged comfort without causing visual disturbances, 0.30%HA gel might be adapted for bedtime use or during the day in more severe conditions.

Characterization of Femtosecond Laser and Porcine Crystalline Lens Interactions by Optical Microscopy.

The use of ultrafast laser pulses for eye anterior segment surgery has seen a tremendous growth of interest as the technique has revolutionized the field, from the treatment of myopia, hyperopia, and presbyopia in the cornea to laser-assisted cataract surgery of the crystalline lens. For the latter, a comprehensive understanding of the laser-tissue interaction has yet to be achieved, mainly because of the challenge of observing the interaction zone in situ with sufficient spatial and temporal resolution in the complex and multi-layered tissue of the crystalline lens. We report here on the dedicated characterization results of the laser-tissue interaction zone in the ex vivo porcine lens using three different methods: in situ and real-time microscopy, wide-field optical imaging, and phase-contrast microscopy of the histological cross sections. These complementary approaches together revealed new physical and biological consequences of laser irradiation: a low-energy interaction regime (pulse energy below ~1 µJ) with very limited cavitation effects and a stronger photo-disruption regime (pulse energy above 1 µJ) with a long cavitation duration from seconds to minutes, resulting in elongated spots. These advances in the understanding of the ultrafast laser's interactions with the lens are of the utmost importance for the preparation of the next-generation treatments that will be applied to the lens.

Corneal epithelium in keratoconus underexpresses active NRF2 and a subset of oxidative stress-related genes.

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.

Objective measurement of adherence to topical steroid medication after penetrating keratoplasty using an electronic monitoring aid: A pilot study.

Objectives: Corneal transplantation is the most common transplant worldwide and its success critically depends on the management of corneal graft rejection through topical steroid therapy during the first 12 months after surgery. There is currently no published data on adherence after keratoplasty. This pilot study aims to explore the adherence to topical steroid after penetrating keratoplasty using a smart electronic device. Methods: Thirty patients undergoing penetrating keratoplasty were included to evaluate the adherence to topical dexamethasone medication for 12 months after surgery. Patients received the usual post-transplantation treatment (topical dexamethasone) and follow-up after surgery (day 15, months 1, 2, 3, 4, 5, 6, 9, and 12). Adherence to treatment was monitored using the KaliJAR device (Kali Care, Santa Clara, CA, USA), which recorded the number of single-dose units (SDU) discarded. At control visits, data recorded by the device were compared to the manually count of SDU. Adherence ratio and individual adherence curve were explored for all patients. Results: Data from 27 patients showed a high agreement between adherence ratio calculated based on the device data and obtained from manual counting of the discarded SDU (intraclass coefficient correlation of 0.87 [95% CI: 0.738-0.938]). Mean adherence to the treatment over the 12-month study period was 95.2 ± 4%. Conclusions: Adherence to topical dexamethasone for 12 months after corneal transplantation was high. The connected device was able to record accurately the discarded SDU. This approach would be a particular interest in the early identification and personalized follow-up of poorly adherent patients.