Andri K. Riau

Preservation, sterilization and de-epithelialization of human amniotic membrane for use in ocular surface reconstruction.

In the past 20 years, human amniotic membrane (AM) has become widely used as an ophthalmic surgical patch as well as a substrate for stem cell tissue equivalents for ocular surface reconstruction. AM reduces ocular surface scarring and inflammation, and enhances epithelialization. In addition, it shows limited immunogenicity and some anti-microbial properties. Before being applied clinically, the donor of AM is required to undergo a thorough health screening and the membrane has to undergo an accepted processing routine, which includes preservation, sterilization and de-epithelialization. There have been various articles describing methods in preserving, sterilizing and de-epithelializing AM. Each preparation technique has been reported to have differential effects on the physical and biological properties of the AM. Therefore, it is difficult to establish a standardized procedure. In this review, we discuss the present techniques and several novel, new approaches in the preparation of AM for use in ocular surface reconstruction, and their impact on AM structure and biological activity.

Early Corneal Wound Healing and Inflammatory Responses after Refractive Lenticule Extraction (ReLEx)

PURPOSE To compare the early corneal wound repair and inflammatory responses after refractive lenticule extraction (ReLEx) and LASIK. METHODS Eighteen rabbits underwent ReLEx and another 18 underwent LASIK. Each group was divided into three subgroups of six rabbits each and these were subjected to refractive corrections of -3.00 diopters (D), -6.00 D, and -9.00 D. Slit lamp photography, anterior segment optical coherence tomography (AS-OCT), corneal topography, and in vivo confocal microscopy were performed 1 day after surgery. After euthanatization, the corneas were subjected to immunofluorescent staining for fibronectin, CD11b, Ki-67, and TUNEL assay. RESULTS On slit lamp microscopy, all corneas appeared clear pre- and postoperatively in both ReLEx and LASIK eyes. Corneal topography showed a more significant corneal flattening after LASIK than after ReLEx as the degree of correction was increased (P = 0.916 after -3.00 D correction to P = 0.097 after -9.00 D correction). In vivo confocal microscopy showed less light-scattering particles at the flap interface after ReLEx compared with LASIK. Immunostaining of fibronectin showed a less abundant expression in corneas that underwent ReLEx than LASIK. The differences became more marked as the power of correction was increased. Similar trend was seen in the number of CD11b-positive cells (P = 0.476 after -3.00 D correction to P < 0.001 after -9.00D correction). There was no marked disparity observed in cell death and proliferation between post-ReLEx and -LASIK eyes. CONCLUSIONS This study has shown that the ReLEx procedure may result in less topographic changes, inflammation, and early extracellular matrix deposition than LASIK, especially at high refractive correction.

Femtosecond Lenticule Extraction (FLEx): Clinical Results, Interface Evaluation, and Intraocular Pressure Variation

PURPOSE To characterize the clinical profile of femtosecond lenticule extraction (FLEx) correlated with ultrastructural analysis of the corneal interface and in vivo real-time intraocular pressure (IOP). METHODS Prospective clinical case series with experimental studies; consecutive patients underwent FLEx at a single tertiary center over 10 months with postsurgical follow-up of 3 months. The patients were divided into three groups according to spherical equivalence (SE) (A, < -5.0 diopters [D]; B, ≥ -5.00 D and < -9.00 D; and C, ≥ -9.0 D). Twelve human cadaveric eyes analyzed using scanning electron microscopy after receiving FLEx; 40 rabbit eyes received FLEx with in vivo IOP measurements. The main outcome measures were refractive outcomes from study subjects; with corneal interface and IOP in experimental studies. RESULTS Thirty-three subjects (22 females, 66.7%) underwent FLEx in both eyes (66 eyes). Mean age was 32 years (range, 21 to 46 years). Preoperative mean SE was -5.77 ± 2.04 D with astigmatism of -1.03 ± 0.72 D. There was a slight hyperopic shift (mean SE 0.14 ± 0.53 D); 94% achieved uncorrected visual acuity ≥20/25 3 months postoperatively. Refractive stability was achieved within 1 month (P < 0.001). Ultrastructurally, the smoothness of the corneal interface was independent of ablation depth (mean irregularity scores A, B, C: 8.8 ± 0.6, 10.3 ± 0.4, 8.7 ± 0.6, respectively; P = 0.88). The increase in IOP during FLEx was similar to that in femtosecond (FS)-LASIK, albeit a twofold duration of raised IOP in FLEx (P < 0.001). CONCLUSIONS These results suggest that FLEx is predictable and effective in treating myopia and myopic astigmatism. Experimental studies support the early clinical results and safety of this procedure.

Early Corneal Nerve Damage and Recovery Following Small Incision Lenticule Extraction (SMILE) and Laser In Situ Keratomileusis (LASIK)

PURPOSE We compared early corneal nerve changes after small incision lenticule extraction (SMILE) and laser in situ keratomileusis (LASIK). METHODS A total of 12 rabbits underwent LASIK in one eye and SMILE in the fellow eye. Baseline and follow-up evaluations at 1, 2, and 4 weeks postoperatively were performed with in vivo confocal microscopy to evaluate 5 different areas within the treated zone: center, superior, inferior, nasal, and temporal. Cryosections of the corneas and whole mount of the extracted SMILE lenticules were analyzed with immunostaining of βIII-tubulin. RESULTS One week after SMILE and LASIK, a decrease in nerve length and density was observed in all evaluated areas. A trend toward greater subbasal nerve length and density (SLD), more eyes with subbasal nerves (ESN), more eyes with subbasal nerves longer than 200 μm (SNL), and higher mean number of subbasal nerves by frame (NSN) in SMILE than in LASIK groups was observed at subsequent follow-up time points. Only the SMILE group showed a recovery of SLD, ESN, and NSN by week 4 (P > 0.05). A trend toward more eyes with sprouting subbasal nerves and greater mean number of sprouting nerves was observed in LASIK than in SMILE, indicating that more subbasal nerves were disrupted and undergoing regeneration after LASIK. Immunostaining at postoperative week 4 revealed a faster stromal nerve recovery in post-SMILE eyes compared to post-LASIK eyes. CONCLUSIONS Our findings suggest that SMILE results in less nerve damage and faster nerve recovery than LASIK.

Refractive lenticule re-implantation after myopic ReLEx: a feasibility study of stromal restoration after refractive surgery in a rabbit model.

PURPOSE To investigate the potential of refractive lenticule (RL) storage and re-implantation in vivo as a method for reversing RL extraction (ReLEx) and restoring corneal stromal volume. METHODS ReLEx [-6.00 diopter (D) correction] was performed on six New Zealand White rabbits in one eye. Each extracted RL was tagged and orientated before storage at -80°C for 28 days. Each RL was then re-implanted autologously in the correct orientation after flap relifting. All animals were monitored for 28 days before being euthanized for immunohistochemical analysis. Unoperated fellow eyes were used as controls. All animals had regular pre- and postoperative slit lamp photography, in vivo confocal microscopy, anterior segment optical coherence tomography (AS-OCT), keratometry, and topography. RESULTS No intra-operative complications occurred and RL re-implantation was performed without complication. A mild intrastromal haziness was noted on day 3 after re-implantation (corneal haze grade: 2.20 ± 0.45), but corneas were clear on day 28 (0.20 ± 0.27). RL re-implantation restored central corneal thickness, and keratometric and topographic indices to near pre-operative values. Wound healing processes, marked by fibronectin and tenascin, and a few inflammatory cells were present along the re-implanted lenticular interfaces. No myofibroblasts formation, and Ki67- and TUNEL-positive cells were observed in the corneal stroma on postoperative day 28. CONCLUSIONS RL storage and re-implantation is a feasible technique for restoring stromal volume after myopic ReLEx, and may provide a method for restoring tissue in ectatic corneas, or provide an opportunity for further refractive surgery and presbyopic treatment.

Transmembrane water-flux through SLC4A11: a route defective in genetic corneal diseases

Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4a11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. Slc4a11−/− mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.

Comparison of four different VisuMax circle patterns for flap creation after small incision lenticule extraction.

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.

Aberrant DNA methylation of matrix remodeling and cell adhesion related genes in pterygium.

Background Pterygium is a common ocular surface disease characterized by abnormal epithelial and fibrovascular proliferation, invasion, and matrix remodeling. This lesion, which migrates from the periphery to the center of the cornea, impairs vision and causes considerable irritation. The mechanism of pterygium formation remains ambiguous, and current treatment is solely surgical excision, with a significant risk of recurrence after surgery. Here, we investigate the role of methylation in DNA sequences that regulate matrix remodeling and cell adhesion in pterygium formation. Methodology/Principal Findings Pterygium and uninvolved conjunctiva samples were obtained from the same eye of patients undergoing surgery. The EpiTYPER Sequenom technology, based on differential base cleavage and bisulfite sequencing was used to evaluate the extent of methylation of 29 matrix and adhesion related genes. In pterygium, three CpG sites at −268, −32 and −29 bp upstream of transglutaminase 2 (TGM-2) transcription initiation were significantly hypermethylated (p<0.05), whereas hypomethylation was detected at CpGs +484 and +602 bp downstream of matrix metalloproteinase 2 (MMP-2) transcription start site, and −809, −762, −631 and −629 bp upstream of the CD24 transcription start site. RT-qPCR, western blot and immunofluorescent staining showed that transcript and protein expression were reduced for TGM-2 and increased for MMP-2 and CD24. Inhibition of methylation in cultured conjunctival epithelial cells increased these transcripts. Conclusions/Significance We found regions of aberrant DNA methylation which were consistent with alteration of TGM-2, MMP-2, and CD24 transcript and protein expression, and that inhibition of methylation in cultured cells can increase the expression of these genes. Since these genes were related to cell adhesion and matrix remodeling, dysregulation may lead to fibroblastic and neovascular changes and pterygium formation. These results have implications for the prognostication of pterygium in clinical practice, for example, detection of epigenetic changes may have a role in predicting post surgical recurrence of aggressive lesions.

Comparative study of nJ- and μJ-energy level femtosecond lasers: evaluation of flap adhesion strength, stromal bed quality, and tissue responses.

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.

Reversible Femtosecond Laser-Assisted Myopia Correction: A Non-Human Primate Study of Lenticule Re-Implantation after Refractive Lenticule Extraction

LASIK (laser-assisted in situ keratomileusis) is a common laser refractive procedure for myopia and astigmatism, involving permanent removal of anterior corneal stromal tissue by excimer ablation beneath a hinged flap. Correction of refractive error is achieved by the resulting change in the curvature of the cornea and is limited by central corneal thickness, as a thin residual stromal bed may result in biomechanical instability of the cornea. A recently developed alternative to LASIK called Refractive Lenticule Extraction (ReLEx) utilizes solely a femtosecond laser (FSL) to incise an intrastromal refractive lenticule (RL), which results in reshaping the corneal curvature and correcting the myopia and/or astigmatism. As the RL is extracted intact in the ReLEx, we hypothesized that it could be cryopreserved and re-implanted at a later date to restore corneal stromal volume, in the event of keratectasia, making ReLEx a potentially reversible procedure, unlike LASIK. In this study, we re-implanted cryopreserved RLs in a non-human primate model of ReLEx. Mild intrastromal haze, noted during the first 2 weeks after re-implantation, subsided after 8 weeks. Refractive parameters including corneal thickness, anterior curvature and refractive error indices were restored to near pre-operative values after the re-implantation. Immunohistochemistry revealed no myofibroblast formation or abnormal collagen type I expression after 8 weeks, and a significant attenuation of fibronectin and tenascin expression from week 8 to 16 after re-implantation. In addition, keratocyte re-population could be found along the implanted RL interfaces. Our findings suggest that RL cryopreservation and re-implantation after ReLEx appears feasible, suggesting the possibility of potential reversibility of the procedure, and possible future uses of RLs in treating other corneal disorders and refractive errors.