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Femtosecond laser-assisted keratoplasty has been proposed as a treatment option for corneal transplantation. In this study, we investigated and compared the outcomes of Ziemer Z8 femtosecond laser (FSL)-assisted penetrating keratoplasty (PK) using a liquid interface versus flat interface. Thirty fresh porcine eyes underwent FSL-assisted PK with the Z8 using different levels of energies (30%, 90% or 150%) and different interfaces (liquid or flat). The real-time intraocular pressure (IOP) changes, incision geometry, corneal endothelial damage, as well as the accuracy of laser cutting and tissue reaction, were performed and compared. We found that the overall average IOP at all laser trephination stages was significantly higher with the flat interface, regardless of the energy used (68.9 ± 15.0 mmHg versus 46.1 ± 16.6 mmHg; P 2 versus 139.0 ± 10.4 mm2P = 0.02). The FSL cutting did not cause obvious tissue reaction alongside the laser cut on histological evaluation. The results indicated a liquid interface is the preferable choice in FSL-assisted corneal transplantation.

Significance: Corneal diseases is a major cause of reversible blindness in the world. Monitoring the progression of human corneal edema or corneal scarring to prevent the disease entering into the end stage is crucial. Aim: We present a method for sensing human corneal composition at different depths, namely focused on the epithelium and stromal layer, using high-sensitivity terahertz (THz) broadband spectroscopy. Approach: From the proposed methodology, the THz temporal and absorption spectra of human corneas at different edema stages have been studied. THz wave signals were collected from the direct reflection and four other collection points along the THz wave propagation direction as reviewed from the simulation THz electrical field. Result: Our results show that the epithelium layer acts as a good barrier to maintain hydration level of the stroma, and the quality of the epithelium can be used to predict the level of corneal swelling in corneal edema. At the detection points near to the incident point, the THz frequency spectra demonstrated interference oscillation behavior. At the final edema observing time, results showed that the epithelium lose its barrier properties. The intactness of the epithelium can be used to predict the edema severity in the final stage. When the detection points are further away from the incident point, the THz spectra are believed to contain information from stromal layer. Stromal absorption spectra demonstrated correlation with optical coherence tomography thickness results. Conclusion: The hydration concentration from stromal layer was further quantitatively calculated. At the end of the experiment, all the corneal hydration levels reach to the same value which shows that the edema hydration has reached maximum saturation. The information of individual sublayers of the cornea is obtained by characterizing noninvasively with the use of THz spectroscopy. To our knowledge, this is the first report of using THz for noninvasive characterization of sublayers of the cornea.

Terahertz (THz) technology has emerged recently as a potential novel imaging modality in biomedical fields, including ophthalmology. However, the ocular biological responses after THz electromagnetic exposure have not been investigated. We conducted a rabbit study to evaluate the safety profiles of THz scanning on eyes, at a tissue, cellular, structural and functional level. Eight animals (16 eyes) were analysed after excessive THz exposure (control, 1 h, 4 h, and 1 week after continuous 4-h exposure; THz frequency = 0.3 THz with continuous pulse generated at 40 µW). We found that at all the time points, the corneas and lens remained clear with no corneal haze or lens opacity formation clinically and histopathologically. No thermal effect, assessed by thermographer, was observed. The rod and cone cell-mediated electroretinography responses were not significantly altered, and the corneal keratocytes activity as well as endothelial viability, assessed by in-vivo confocal microscopy, was not affected. Post-exposed corneas, lens and retinas exhibited no significant changes in the mRNA expression of heat shock protein (HSP)90AB1), DNA damage inducible transcript 3 (DDIT3), and early growth response (EGR)1. These tissues were also negative for the inflammatory (CD11b), fibrotic (fibronectin and α-smooth muscle actin), stress (HSP-47) and apoptotic (TUNEL assay) responses on the immunohistochemical analyses. The optical transmittance of corneas did not change significantly, and the inter-fibrillar distances of the corneal stroma evaluated with transmission electron microscopy were not significantly altered after THz exposure. These results provide the basis for future research work on the development of THz imaging system for its application in ophthalmology. National Medical Research Council (NMRC) Published version This research was supported by the Transition Award, National Medical Research Council (R1482/65/2017), Singapore.

Implantation of biological corneal inlays, derived from small incision lenticule extraction, may be a feasible method for surgical management of refractive and corneal diseases. However, the refractive outcome is dependent on stromal remodelling of both the inlay and recipient stroma. This study aimed to investigate the refractive changes and tissue responses following implantation of 2.5-mm biological inlays with or without corneal collagen crosslinking (CXL) in a rabbit model. Prior to implantation, rotational rheometry demonstrated an almost two-fold increase in corneal stiffness after CXL. After implantation, haze gradually subsided in the CXL-treated inlays (p = 0.001), whereas the untreated inlays preserved their clarity (p = 0.75). In-vivo confocal microscopy revealed reduced keratocyte cell count at the interface of the CXL inlays at week 8. Following initial steepening, regression was observed in anterior mean curvature from week 1 to 12, being most prominent for the non-CXL subgroups (non-CXL: −12.3 ± 2.6D vs CXL: −2.3 ± 4.4D at 90 μm depth, p = 0.03; non-CXL: −12.4 ± 8.0D vs CXL: −5.0 ± 4.0D at 120 μm depth, p = 0.22). Immunohistochemical analysis revealed comparable tissue responses in CXL and untreated subgroups. Our findings suggest that CXL of biological inlays may reduce the time before refractive stabilization, but longer postoperative steroid treatment is necessary in order to reduce postoperative haze. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

Purpose To investigate the postoperative inflammatory and wound-healing responses after laser scleral microporation for presbyopia. Methods Thirty porcine eyes were used for the optimization of laser intensities first. Six monkeys (12 eyes) received scleral microporation with an erbium yttrium aluminum garnet (Er:YAG) laser, and half of the eyes received concurrent subconjunctival collagen gel to modulate wound-healing response. The intraocular pressure (IOP) and the laser ablation depth were evaluated. The animals were euthanized at 1, 6, and 9 months postoperatively. The limbal areas and scleras were harvested for histologic analysis and immunofluorescence of markers for inflammation (CD11b and CD45), wound healing (CD90, tenascin-C, fibronectin, and HSP47), wound contraction (α-smooth muscle actin [α-SMA]), vascular response (CD31), nerve injury (GAP43), and limbal stem cells (P63 and telomerase). Results In the nonhuman primate study, there was a significant reduction in IOP after the procedure. Overall, the ablation depth was 76.6% to 81.2% at 1 month and slightly decreased to 71.5% to 72.7% at 9 months. Coagulative necrosis around the micropores, as well as expression of CD11b, CD45, tenascin, fibronectin, HSP47, and GAP43, was distinct at 1 month but subsided with time. Collagen gel treatment significantly suppressed the upregulation of CD11b, CD45, fibronectin, and tenascin-C. The expression of CD90, α-SMA, and CD31 was minimal in all eyes. Conclusions The study demonstrated the course of inflammatory and wound-healing responses following laser scleral microporation. The tissue responses were small and self-limited, resolved with time, and were suppressed by concurrent collagen treatment. It provides a useful understanding of this new procedure. Translational relevance The results would be helpful in the laser parameter modification to improve the long-term treatment stability.

Corneal inlays are a relatively new treatment option for presbyopia. Using biological inlays, derived from lenticules extracted from small incision lenticule extraction, may offer advantages over commercialized synthetic inlays in the aspect of biocompatibility. We conducted a non-human primate study to evaluate the safety, predictability, efficacy and tissue response after autogeneic, decellularized xenogeneic and xenogeneic lenticule implantation. The lenticule implantation effectively resulted in central corneal steepening (simulated keratometric values increased by 1.8–2.3 diopters), central hyper-prolate changes (asphericity Q values changed by −0.26 to −0.36), corneal anterior surface elevation (7.7–9.3 μm) and reasonable effective zone (1.5–1.8 times of the lenticule physical diameter), with no differences among the three groups. Slit lamp microscopy, transmission electron microscopy, confocal microscopy, histology and immunohistochemistry analyses confirmed the biocompatibility of the autogeneic and decellularized lenticules, whereas one eye in the xenogeneic group developed corneal stromal rejection during the study period. Our results showed that lenticule implantation has the potential for the management of presbyopia, and provide the basis for future clinical studies. The decellularization process may increase the potential utilization of lenticules without changing the efficacy. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

Corneal opacities are a leading cause of global blindness. They are conventionally treated by the transplantation of donor corneal tissue, which is, restricted by a worldwide donor material shortage and allograft rejection. Autologous adult stem cells with a potential to differentiate into corneal stromal keratocytes (CSKs) could offer a suitable choice of cells for regenerative cell therapy. Postnatal periodontal ligament (PDL) contains a population of adult stem cells, which has a similar embryological origin as CSK, that is cranial neural crest. We harvested PDL cells from young adult teeth extracted because of non‐functional or orthodontic reason and differentiated them towards CSK phenotype using a two‐step protocol with spheroid formation followed by growth factor and cytokine induction in a stromal environment (human amnion stroma and porcine corneal stroma). Our results showed that the PDL‐differentiated CSK‐like cells expressed CSK markers (CD34, ALDH3A1, keratocan, lumican, CHST6, B3GNT7 and Col8A2) and had minimal expression of genes related to fibrosis and other lineages (vasculogenesis, adipogenesis, myogenesis, epitheliogenesis, neurogenesis and hematogenesis). Introduction of PDL spheroids into the stroma of porcine corneas resulted in extensive migration of cells inside the host stroma after 14‐day organ culture. Their quiescent nature and uniform cell distribution resembled to that of mature CSKs inside the native stroma. Our results demonstrated the potential translation of PDL cells for regenerative corneal cell therapy for corneal opacities. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version

Purpose Pterygium is a common ocular surface disorder. Conjunctival autografting (CAG) following pterygium resection is the gold standard treatment. CAGs without Tenon's tissue provide better results but are more technically difficult to achieve. In this study, we evaluated the feasibility and reproducibility of femtosecond laser (FSL)-assisted CAG preparation. Methods Fifteen porcine globes were fixed in a suction holder and CAGs of different diameters were created by 1) an experienced consultant and 2) a less experienced fellow using the Ziemer LDV Z8. The CAG's dimension was measured and thickness analyzed by optical coherence tomography (OCT) and histology (HE). Statistical analysis was performed by Mann-Whitney-U, Wilcoxon and Spearman-test. Results FSL-assisted CAGs prepared at 100 μm (146.4 ± 45.7 μm) showed a significantly higher deviation from desired depth ( p = 0.04) and a higher variability ( p = 0.03) in thickness than those prepared at 60 μm (71.4 ± 12.7 μm). The experienced (68.3 ± 14.3 μm) and inexperienced surgeon (73.9 ± 11.9 μm) produced 60 μm grafts of comparable thickness ( p = 0.6) and variability ( p = 0.7). The CAG area measured after dissection (37.5 ± 12.1 mm 2 ) did not differ significantly from the FSL settings (40.6 ± 12.7 mm 2 , p = 0.3). FSL cutting time at 60 μm took 18.1 ± 2.2 s, at 100 μm 20.7 ± 2.4 s. Graft separation time was not significantly influenced by depth or surgeon. No buttonholes or CAG tags occurred during surgery. Conclusions The FSL allowed the accurate and reliable preparation of very thin CAGs, independent of surgeon experience and may represent a valuable tool in pterygium surgery.

Refractive surgical treatment of hyperopia still remains a challenge for refractive surgeons. A new nomogram of small incision lenticule extraction (SMILE) procedure has recently been developed for the treatment of hyperopia. In the present study, we aimed to evaluate the wound healing and inflammatory responses of this new nomogram (hyperopic-SMILE), and compared them to those of hyperopic-laser-assisted in situ keratomileusis (LASIK), using a rabbit model. A total of 26 rabbits were used, and slit lamp biomicroscopy, autorefractor/keratometer, intraocular pressure measurement, anterior segment optical coherence tomography, corneal topography, and in vivo confocal microscopy examinations were performed during the study period of 4 weeks. The corneas were then harvested and subject to immunofluorescence of markers for inflammation (CD11b), wound healing (fibronectin) and keratocyte response (HSP47). The lenticule ultrastructual changes were also analyzed by transmission electron microscopy. Out results showed that hyperopic-SMILE effectively steepened the cornea. Compared to hyperopic-LASIK, hyperopic-SMILE had less postoperative wound healing response and stromal interface reaction, especially in higher refractive correction. However, compared to myopic-SMILE, hyperopic-SMILE resulted in more central deranged collagen fibrils. These results provide more perspective into this new treatment option for hyperopia, and evidence for future laser nomogram modification.

PURPOSE: To investigate the early corneal wound healing and inflammatory responses after small incision lenticule extraction (SMILE) with different power of corrections and surgical experiences using a rabbit model. METHODS: Twenty-four rabbits underwent SMILE with −2.00, −4.00, and −8.00 diopters (D) correction. One eye of each rabbit was operated on by surgeon 1 (experienced) and the contralateral eye was operated on by surgeon 2 (novice). Slit-lamp examination, anterior segment optical coherence tomography (AS-OCT), and in vivo confocal microscopy were performed at 1 day and 1 week postoperatively. The corneas were harvested for immunofluorescence of markers for inflammation (CD11b), wound healing (fibronectin), and keratocyte response (HSP47). RESULTS: All corneas appeared clear throughout the follow-up period. In vivo confocal microscopy showed a greater reflectivity after −8.00 D than −2.00 D correction at day 1 at the lenticule anterior, posterior, and extracted lenticule planes (surgeon 1: P = .004, .041, and .038; surgeon 2: P = .012, .045, and .031). Different refractive corrections did not significantly affect the expression of CD11b, fibronectin, and HSP47. In the −2.00 D group, eyes operated on by surgeon 2 had thicker central corneal thickness evaluated by AS-OCT ( P = .049) and exhibited more CD11b- and HSP47-positive cells at day 1 at the small vertical incision ( P = .039 and .042). CONCLUSIONS: The early inflammatory and wound healing responses after SMILE were minimal. In the early postoperative period, less surgical experience resulted in an increased inflammatory response in low myopic corrections. Greater keratocyte response was seen in high myopic corrections irrespective of surgeon experience. [ J Refract Surg. 2016;32(5):346–353.]

Purpose: To compare the postoperative higher-order-aberrations (HOAs) after hyperopic small incision lenticule extraction (SMILE), hyperopic laser-assisted in situ keratomileusis (LASIK), and lenticule implantation for correction of hyperopia. Methods: Eighteen monkeys were divided to six groups: +2.00 D and +4.00 D hyperopic SMILE, +2.00 D and +4.00 D hyperopic LASIK (n = 6 eyes for each), and lenticule implantation with a −2.00 D and −4.00 D lenticule (n = 3 eyes for each). The corneal HOAs were evaluated preoperatively and 3-month postoperatively. Results: At 3-month postoperatively, the spherical aberrations significantly increased toward negative direction in all +4.00 D groups (all P < 0.05). There was a significant change toward more negative values in the third-order vertical coma in the SMILE +4.00 D and LASIK +4.00 D groups (P = 0.026 and P = 0.036, respectively). There were also significant changes in the third-order horizontal trefoil (P = 0.034) and oblique secondary astigmatism (P = 0.012) in the LASIK +4.00 D group. In the eyes that underwent +4.00 D lenticule implantation, the fourth-order horizontal quatrefoil significantly increased (P = 0.029). In low hyperopia correction (+2.00 D), treatment with lenticule implantation tended to have less changes in HOAs, compared to the other two groups. Conclusions: In hyperopic SMILE, hyperopic LASIK or lenticule implantation surgery, significant induction of third- and fourth-order HOAs were seen in moderate hyperopia correction but not in low hyperopia correction. In low hyperopia treatment, lenticule implantation might offer a favorable trend in the aspect of HOAs. Translational Relevance: The results provided the knowledge of surgically induced HOAs and understanding of the effects of surgery in different types of hyperopic correction. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

Purpose: To investigate the drug release profiles of a tacrolimus-loaded poly(D,L-lactide-co-ε-caprolactone) (PLC) microfilm, and to evaluate its efficacy on the treatment of allergic conjunctivitis using a mouse model. Methods: The in vitro and in vivo drug release profiles were first characterized. Balb/c mice were immunized with short ragweed (SRW) injection followed by re-challenges with topical SRW solution. The mice were divided into six groups (n = 12 in each): negative control (NC); positive control (PC); tacrolimus eye drops (Te); subconjunctival tacrolimus microfilm (Tm); dexamethasone eye drops (De); and tacrolimus + dexamethasone eye drops (Te+De). The mice were evaluated for 28 days by a scoring system for allergic conjunctivitis. Histopathologic and immunohistochemical staining with CD11c, CD4, and IL-4 were performed. Results: The microfilms were biocompatible and delivered clinically sufficient dose in a sustained manner, with a steady rate of 0.212 to 0.243 μg/day in vivo. Compared to the PC groups, the Te, Tm, De, and Te+De groups significantly reduced the allergic clinical scores throughout the study period (all P < 0.01; 0.0 ± 0.0, 5.6 ± 0.9, 3.3 ± 0.9, 3.2 ± 0.9, 1.9 ± 0.4 and 1.7 ± 0.8 for the NC, PC, Tm, Te, De, and Te+De groups, respectively, at 4 weeks after treatment). The suppressed eosinophils, CD11c, CD4, and IL-4 expression were also observed in all treatment groups, with more reduction in the Te+De group. Conclusions: Tacrolimus-loaded microfilms display good biocompatibility and desirable sustained drug release. It was as effective as conventional tacrolimus eye drops on the treatment of allergic conjunctivitis, providing a promising clinically applicable alternative for controlling allergic disease activity, or other immune-mediated ocular diseases. NRF (Natl Research Foundation, S’pore) NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Published version

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

Background: Presbyopic inlays have mostly been implanted under a corneal flap. Implantation in a pocket has advantages including less postoperative dry eye and neurotrophic effect, and better biomechanical corneal stability. This study investigated the effect of different pocket and flocket dimensions on corneal stability and refractive power after Raindrop™ implantation, and the associated wound healing response. Methodology: Ten New Zealand White rabbits had bilateral pocket Raindrop™ implantation. Eyes were allocated to 4 groups: pockets with 4mm, 6mm, and 8mm diameters, and 8mm flocket. They were examined pre-operatively, at day 1, weeks 1, 2, 3 and 4 post-surgery with anterior segment optical coherence tomography, corneal topography and in-vivo confocal microscopy. After euthanasia (week 4), CD11b, heat shock protein (HSP) 47 and fibronectin corneal immunohistochemistry was performed. Results: Corneal thickness (mean±SD) increased from 360.0±16.2μm pre-operatively to 383.9±32.5, 409.4±79.3, 393.6±35.2, 396.4±50.7 and 405±20.3μm on day 1, weeks 1,2,3 and 4 respectively (p

PURPOSE To investigate the quality of the ultrathin corneal grafts prepared by femtosecond laser from the endothelial side for Descemet stripping endothelial keratoplasty. METHODS Thirty human corneoscleral buttons were cut from the endothelial side by laser Doppler velocimetry (LDV) with or without viscoelastic materials coating. Two cutting depths were selected: 70 and 90 μm. The postcut endothelial count was determined by specular microscopy, and the graft thickness was evaluated by anterior segment optical coherence tomography. The endothelial viability was determined using Trypan blue/Alizarin red staining, calcein-AM/EthD-1 live/dead cell assay, and scanning electron microscope (SEM). The graft interface smoothness was evaluated by SEM. Another 18 corneoscleral buttons were used as controls for the comparisons. RESULTS The overall targeted cutting depth and achieved cutting depth were significantly highly correlated (r = 0.84). The central to peripheral corneal thickness ratio was 0.976 and 0.998 for the 70- and 90-μm grafts. The percentage of the damaged endothelial cells assessed by vital staining and SEM showed the 70-μm grafts had noticeably more endothelial damage compared with the 90-μm grafts. But the damage was significantly reduced, to the control corneas level, after coating the endothelium with Viscoat. The 90-μm grafts had a slightly rougher graft interface than the 70-μm grafts, but all the grafts dissected by a Chansue dissector exhibited a generally smooth interface. CONCLUSIONS The corneal endothelial grafts prepared by LDV femtosecond laser with endothelial approach produced consistently ultrathin grafts in uniform shape with high accuracy and good endothelial and stromal interface quality.