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Purpose: To evaluate the safety and feasibility of intrastromal injection of human corneal stromal keratocytes (CSKs) and its therapeutic effect on a rodent early corneal opacity model., Methods: Twelve research-grade donor corneas were used in primary culture to generate quiescent CSKs and activated stromal fibroblasts (SFs). Single and repeated intrastromal injections of 2 to 4 × 104 cells to rat normal corneas (n = 52) or corneas with early opacities induced by irregular phototherapeutic keratectomy (n = 16) were performed, followed by weekly examination of corneal response under slit-lamp biomicroscopy and in vivo confocal microscopy with evaluation of haze level and stromal reflectivity, and corneal thickness using anterior segment optical coherence tomography (AS-OCT). Time-lapse tracing of Molday ION-labelled cells was conducted using Spectralis OCT and label intensity was measured. Corneas were collected at time intervals for marker expression by immunofluorescence, cell viability, and apoptosis assays., Results: Injected CSKs showed proper marker expression with negligible SF-related features and inflammation, hence maintaining corneal clarity and stability. The time-dependent loss of injected cells was recovered by repeated injection, achieving an extended expression of human proteoglycans inside rat stroma. In the early corneal opacity model, intrastromal CSK injection reduced stromal reflectivity and thickness, resulting in recovery of corneal clarity, whereas noninjected corneas were thicker and had haze progression., Conclusions: We demonstrated the safety, feasibility, and therapeutic efficacy of intrastromal CSK injection. The cultivated CSKs can be a reliable cell source for potential cell-based therapy for corneal opacities.

With any refractive correction, including Small Incision Lenticule Extraction (SMILE), there may be a residual refractive error that requires a retreatment. Here, we investigated the tissue responses following various retreatment procedures in a rabbit model of SMILE. All rabbits underwent a -6.00D correction with SMILE. Two weeks later, they underwent -1.00D enhancement by: (i) VisuMax Circle, followed by excimer ablation (S+C); (ii) secondary SMILE anterior to the primary procedure (S+SE); or (iii) surface ablation (S+P), and were examined for 28 days. S+P induced corneal edema and haze, and more CD11b- (23±6 cells) and TUNEL-positive (36±4 cells) cells in the central stromal superficial layers early post-operatively (p<0.001 compared to other procedures). The corneas appeared normal on day 28 after S+P, but had a lower number of keratocytes near the laser ablated plane compared to other procedures. S+SE and S+C did not induce corneal haze and resulted similar level of fibronectin. However, S+C resulted in more inflammatory (10±2 cells; p = 0.001) and apoptotic cells (25±2 cells; p<0.001) compared to S+SE (7±1 inflammatory cells and 21±3 apoptotic cells) early post-operatively. In conclusion, each SMILE retreatment method resulted in unique tissue responses. S+SE offers advantages, such as minimal inflammation and cell death, as well as maintaining a 'flap-less' surgery, over other procedures. However, depending on the degree of enhancement, the lenticule may become too thin to be extracted and the procedure becomes more difficult to perform than S+C and S+P. S+P can maintain corneal integrity by avoiding flap creation and is technically more simple to perform than the others, but the surgery needs to be supplemented with mitomycin-C in order to reduce inflammation and modulate better wound healing.

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

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.]., (Copyright 2016, SLACK Incorporated.)

This study aimed at evaluating the effect of intraoperative corneal pocket irrigation in small incision lenticule extraction (SMILE) and compares it to that in femtosecond laser-assisted in situ keratomileusis (FS-LASIK). Sixteen rabbit eyes underwent a SMILE procedure, with 8 eyes having corneal pocket irrigation, while the other 8 eyes were without irrigation. Another 16 eyes underwent a FS-LASIK procedure for comparison, with 8 eyes having flap irrigation, while the other 8 eyes were without irrigation. The results showed that the changes in the total corneal thickness, anterior and posterior lamellar thickness, measured by the anterior segment optical coherence tomography, were comparable between the SMILE with and without irrigation groups, suggesting that the irrigation did not lead to significant changes in the corneal thickness. However, at postoperative 8 hours, in vivo confocal microscopy showed that the interface reflectivity in the SMILE with irrigation group was significantly higher than that in other three groups. The presence of interface fluid was further confirmed by the identification of fluid pockets with undulated collagen shown on histological section in the post-SMILE with irrigation eyes. Our findings might contribute to the occurrence of post-SMILE delayed immediate visual quality recovery and further clinical study is required.

Purpose: To compare flap adhesion strength, stromal bed quality, and tissue responses after flap preparation using nJ- and μJ-energy level femtosecond lasers., Methods: All corneal flaps were created by either VisuMax laser (μJ-energy level) or femto-LDV (nJ-energy level). Flap adhesion strength in the rabbits was measured with a tension meter 1 and 2 months postoperatively. To investigate tissue responses to laser delivery, immunofluorescence staining and TUNEL assay were performed 4 and 24 hours postoperatively. To assess flap bed smoothness, human donor corneas were used. Surface irregularities were graded based on scanning electron microscopy results., Results: The flap adhesion strength in the VisuMax group at month 1 and 2 was 16.95 ± 1.45 kPa and 18.33 ± 1.81 kPa, respectively; and 12.31 ± 4.15 kPa and 13.85 ± 4.78 kPa in the LDV group, respectively. No significant difference was found between the groups. Fibronectin and apoptotic cells were largely absent at the central incision site in the LDV group, but were present in the VisuMax group. The smoothness of flap beds appeared similar for both groups. An observer scored the VisuMax group 8.00 ± 1.00 and the LDV group 7.33 ± 0.58 (P = 0.387)., Conclusions: The flap adhesion strength increased over time after treatment with both lasers. The nJ-energy pulses produced minimal wound healing reaction and apoptotic cells along the incision plane. The application of an nJ-energy laser, which can incise the cornea without inducing significant damage to cells and wound healing reaction, offers great potential at reducing scarring following incisional laser stromal surgery., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To compare four different Circle patterns for flap creation after small incision lenticule extraction (SMILE)., Methods: SMILE was performed on six rabbits. Twenty-eight days after the initial procedure, corneal flaps were created using Circle patterns. Rabbits were divided into four groups (patterns A, B, C, and D). Pattern A creates a circular side cut to meet the cap cut within the clearance zone (outside of the optical zone). Patterns B, C, and D create a lamellar ring posterior, anterior, and at the same depth, respectively, to the cap to meet the cap cut in the clearance zone with the help of a junction cut. Difficulty of flap lift was graded from 1 (easiest) to 5 (most difficult). The bed quality was assessed by scanning electron microscopy., Results: Flaps created by patterns A and D were the easiest to lift (grade 2). The resulting flap bed was smooth and undisrupted. However, pattern A resulted in a reduced re-treatment area. Flaps created by pattern B were the most difficult to lift (grade 4). The stromal dissection was difficult in an attempt to ascertain the original optical zone from the lamellar ring, placed posterior. Flaps produced by pattern C were easy to lift, with minor intrastromal resistance experienced during the lifting process (grade 3). The transition between the lamellar ring and cap cut was hardly discernible in pattern C-treated corneas., Conclusions: Pattern D, a lamellar ring adjacent to the cap cut, was the most optimal to be used for flap creation in cases of SMILE re-treatment., (Copyright 2013, SLACK Incorporated.)