Nanoscale helium ion microscopic analysis of collagen fibrillar changes following femtosecond laser dissection of human cornea.

Over the last decade, femtosecond lasers have emerged as an important tool to perform accurate and fine dissections with minimal collateral damage in biological tissue. The most common surgical procedure in medicine utilizing femtosecond laser is LASIK. During the femtosecond laser dissection process, the corneal collagen fibers inevitably undergo biomechanical and thermal changes on a sub-micro- or even a nanoscale level, which can potentially lead to post-surgical complications. In this study, we utilized helium ion microscopy, complemented with transmission electron microscopy to examine the femtosecond laser-induced collagen fibrillar damage in ex vivo human corneas. We found that the biomechanical damage induced by laser etching, generation of tissue bridges, and expansion of cavitation bubble and its subsequent collapse, created distortion to the surrounding collagen lamellae. Femtosecond laser-induced thermal damage was characterized by collapsed collagen lamellae, loss of collagen banding, collagen coiling, and presence of spherical debris. Our findings have shown the ability of helium ion microscopy to provide high resolution images with unprecedented detail of nanoscale fibrillar morphological changes in order to assess a tissue damage, which could not be resolved by conventional scanning electron microscopy previously. This imaging technology has also given us a better understanding of the tissue-laser interactions in a nano-structural manner and their possible effects on post-operative wound recovery.

Hevin plays a pivotal role in corneal wound healing.

BACKGROUND: Hevin is a matricellular protein involved in tissue repair and remodeling via interaction with the surrounding extracellular matrix (ECM) proteins. In this study, we examined the functional role of hevin using a corneal stromal wound healing model achieved by an excimer laser-induced irregular phototherapeutic keratectomy (IrrPTK) in hevin-null (hevin(-/-)) mice. We also investigated the effects of exogenous supplementation of recombinant human hevin (rhHevin) to rescue the stromal cellular components damaged by the excimer laser. METHODOLOGY/PRINCIPAL FINDINGS: Wild type (WT) and hevin (-/-) mice were divided into three groups at 4 time points- 1, 2, 3 and 4 weeks. Group I served as naïve without any treatment. Group II received epithelial debridement and underwent IrrPTK using excimer laser. Group III received topical application of rhHevin after IrrPTK surgery for 3 days. Eyes were analyzed for corneal haze and matrix remodeling components using slit lamp biomicroscopy, in vivo confocal microscopy, light microscopy (LM), transmission electron microscopy (TEM), immunohistochemistry (IHC) and western blotting (WB). IHC showed upregulation of hevin in IrrPTK-injured WT mice. Hevin (-/-) mice developed corneal haze as early as 1-2 weeks post IrrPTK-treatment compared to the WT group, which peaked at 3-4 weeks. They also exhibited accumulation of inflammatory cells, fibrotic components of ECM proteins and vascularized corneas as seen by IHC and WB. LM and TEM showed activated keratocytes (myofibroblasts), inflammatory debris and vascular tissues in the stroma. Exogenous application of rhHevin for 3 days reinstated inflammatory index of the corneal stroma similar to WT mice. CONCLUSIONS/SIGNIFICANCE: Hevin is transiently expressed in the IrrPTK-injured corneas and loss of hevin predisposes them to aberrant wound healing. Hevin (-/-) mice develop early corneal haze characterized by severe chronic inflammation and stromal fibrosis that can be rescued with exogenous administration of rhHevin. Thus, hevin plays a pivotal role in the corneal wound healing.

Mice with a targeted disruption of Slc4a11 model the progressive corneal changes of congenital hereditary endothelial dystrophy.

PURPOSE: To establish an animal model of congenital hereditary endothelial dystrophy (CHED) using Slc4a11 knockout (KO) mice and evaluate the abnormalities in the cornea and kidney. METHODS: The Slc4a11 KO mouse model was generated by gene deletion. Corneal abnormalities were evaluated using slit-lamp photography, anterior segment optical coherence tomography (AS-OCT), immunohistochemistry, RT-PCR, corneal endothelial cell staining, and electron microscopy. The temporal corneal changes were also monitored. Histological and functional changes of the kidney were also evaluated. RESULTS: Successful knockout of the Slc4a11 gene was confirmed by immunohistochemistry and RT-PCR. Slit-lamp photography and AS-OCT showed progressive corneal edema. Increased corneal endothelial cell size with decreased corneal endothelial cell density was observed with increased age. Scanning electron microscopy also revealed progressive cell swelling and distortion of the hexagonal cell morphology with time. Transmission electron microscopy showed characteristic ultrastructural findings of CHED, including endothelial vacuolization, thickening of the Descemet membrane, disorganization of collagen fibril, deposition of amorphous material, and progression of these changes with age. Decreased urine osmolarity and electrolyte concentrations suggesting abnormality in water resorption were also detected. CONCLUSIONS: Our Slc4a11 KO mouse model successfully represents clinical manifestations of human CHED. We were able to show chronological corneal progression for the first time in a knockout mouse model as well as renal abnormalities.

The effect of amniotic membrane de-epithelialization method on its biological properties and ability to promote limbal epithelial cell culture.

PURPOSE: We characterized the de-epithelialized human amniotic membrane (HAM), and compared cell attachment and proliferation efficiencies. METHODS: HAM was de-epithelialized by 20% ethanol (AHAM), 1.2 U/mL Dispase (DHAM), 0.02% EDTA (EHAM), 0.25% trypsin-EDTA (THAM), and 5 M urea (UHAM), respectively, followed by gentle scraping with a #15 blade. Surface topology, extracellular matrix (ECM), and growth factor content were characterized and compared to intact HAM by electron microscopies (EM), atomic force microscopy (AFM), immunohistochemistry, and Western blotting. Primary human limbal epithelial cells (LEC) attachment and proliferation efficiencies were assayed. Statistical significance was calculated by SPSS and Fisher's least significant difference test. RESULTS: EHAM, THAM, and UHAM had intact basal lamina and smooth basement membrane surface shown under transmission and scanning EM, and AFM. Cell remnants stayed on AHAM. Disrupted basement membrane and stroma was found in DHAM. Immunostaining intensity quantification and hierarchical clustering revealed that ECM composition of EHAM and UHAM resembled intact HAM. In contrast, DHAM and THAM had drastic loss of ECM and growth factor content. LEC attachment efficiency at 24 hours after seeding was the highest in EHAM (51% as on conventional culture surface), followed by UHAM and AHAM. However, cell proliferation indices at day 10 of culture were similar among different HAM substrates, suggesting repair of ECM and basement membrane by growing epithelial cells. CONCLUSIONS: Urea denudation preserved the basement membrane integrity, ECM, and growth factor composition, and had higher cell attachment and proliferation efficiencies. With its short processing time, urea treatment offers a novel alternative for HAM de-epithelialization.

In vivo biocompatibility of two PEG/PAA interpenetrating polymer networks as corneal inlays following deep stromal pocket implantation.

This study compared the effects of implanting two interpenetrating polymer networks (IPNs) into rabbit corneas. The first (Implant 1) was based on PEG-diacrylate, the second (Implant 2) was based on PEG-diacrylamide. There were inserted into deep stromal pockets created using a manual surgical technique for either 3 or 6 months. The implanted corneas were compared with normal and sham-operated corneas through slit lamp observation, anterior segment optical coherence tomography, in vivo confocal scanning and histological examination. Corneas with Implant 1 (based on PEG-diacrylate) developed diffuse haze, ulcers and opacities within 3 months, while corneas with Implant 2 (based on PEG-diacrylamide) remained clear at 6 months. They also exhibited normal numbers of epithelial cell layers, without any immune cell infiltration, inflammation, oedema or neovascularisation at post-operative 6 month. Morphological studies showed transient epithelial layer thinning over the hydrogel inserted area and elevated keratocyte activity at 3 months; however, the epithelium thickness and keratocyte morphology were improved at 6 months. Implant 2 exhibited superior in vivo biocompatibility and higher optical clarity than Implant 1. PEG-diacrylamide-based IPN hydrogel is therefore a potential candidate for corneal inlays to correct refractive error.

Plastic compressed collagen as a novel carrier for expanded human corneal endothelial cells for transplantation.

Current treatments for reversible blindness caused by corneal endothelial cell failure involve replacing the failed endothelium with donor tissue using a one donor-one recipient strategy. Due to the increasing pressure of a worldwide donor cornea shortage there has been considerable interest in developing alternative strategies to treat endothelial disorders using expanded cell replacement therapy. Protocols have been developed which allow successful expansion of endothelial cells in vitro but this approach requires a supporting material that would allow easy transfer of cells to the recipient. We describe the first use of plastic compressed collagen as a highly effective, novel carrier for human corneal endothelial cells. A human corneal endothelial cell line and primary human corneal endothelial cells retained their characteristic cobblestone morphology and expression of tight junction protein ZO-1 and pump protein Na+/K+ ATPase α1 after culture on collagen constructs for up to 14 days. Additionally, ultrastructural analysis suggested a well-integrated endothelial layer with tightly opposed cells and apical microvilli. Plastic compressed collagen is a superior biomaterial in terms of its speed and ease of production and its ability to be manipulated in a clinically relevant manner without breakage. This method provides expanded endothelial cells with a substrate that could be suitable for transplantation allowing one donor cornea to potentially treat multiple patients.

Effect of fibrin glue on the biomechanical properties of human Descemet's membrane.

BACKGROUND: Corneal transplantation has rapidly evolved from full-thickness penetrating keratoplasty (PK) to selective tissue corneal transplantation, where only the diseased portions of the patient's corneal tissue are replaced with healthy donor tissue. Descemet's membrane endothelial keratoplasty (DMEK) performed in patients with corneal endothelial dysfunction is one such example where only a single layer of endothelial cells with its basement membrane (10-15 µm in thickness), Descemet's membrane (DM) is replaced. It is challenging to replace this membrane due to its intrinsic property to roll in an aqueous environment. The main objective of this study was to determine the effects of fibrin glue (FG) on the biomechanical properties of DM using atomic force microscopy (AFM) and relates these properties to membrane folding propensity. METHODOLOGY/PRINCIPAL FINDINGS: Fibrin glue was sprayed using the EasySpray applicator system, and the biomechanical properties of human DM were determined by AFM. We studied the changes in the "rolling up" tendency of DM by examining the changes in the elasticity and flexural rigidity after the application of FG. Surface topography was assessed using scanning electron microscopy (SEM) and AFM imaging. Treatment with FG not only stabilized and stiffened DM but also led to a significant increase in hysteresis of the glue-treated membrane. In addition, flexural or bending rigidity values also increased in FG-treated membranes. CONCLUSIONS/SIGNIFICANCE: Our results suggest that fibrin glue provides rigidity to the DM/endothelial cell complex that may aid in subsequent manipulation by maintaining tissue integrity.

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.

Cornea lenticule viability and structural integrity after refractive lenticule extraction (ReLEx) and cryopreservation.

PURPOSE: To assess and compare keratocyte viability and collagen structure in cornea stroma lenticules collected immediately after refractive lenticule extraction (ReLEx) and one month after cryopreservation. METHODS: The fresh and cryopreserved human stroma lenticules procured after ReLEx were processed for ultrastructural analysis of keratocytes and collagen fibrils with transmission electron microscopy (TEM), apoptotic cell detection with deoxynucleotidyl transferase-mediated nick end labeling assay (TUNEL) assay, and cultured for keratocyte-specific gene expression analysis using reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The periphery of the lenticule had greater TUNEL-positive cells compared to the center of the lenticule in both fresh and cryopreserved groups. There was an increase in TUNEL-positive cells after cryopreservation, which was significantly higher in the center of the lenticule, but not in the periphery. TEM showed apoptotic, necrotic and viable quiescent keratocytes in fresh and cryopreserved lenticules. Collagen analysis with TEM showed a well preserved and well aligned structure in fresh and cryopreserved lenticules; without significant change in the total number of collagen fibrils but with an increased collagen fibril density (CFD) after cryopreservation. In vitro, isolated keratocytes derived from fresh and cryopreserved lenticules exhibited a typical fibroblastic phenotype. RT-PCR showed a positive gene expression for keratocan (KERA) and aldehyde dehydrogenase 3A1 (ALDH3A1) in cells isolated from fresh and cryopreserved lenticules. CONCLUSIONS: The stromal lenticules extracted from ReLEx surgery remain viable after cryopreservation. Although they showed a decrease in CFD, the collagen architecture was preserved and there was good cellular viability.

Biological and ultrastructural properties of acelagraft, a freeze-dried γ-irradiated human amniotic membrane.

OBJECTIVES: To compare the biological and ultrastructural properties of a commercially available decellularized and dehydrated human amniotic membrane (DDHAM) product with cryopreserved human amniotic membrane (CHAM) and to demonstrate the feasibility of DDHAM transplant in a case of chronic ocular surface disease. METHODS: Histologic examination, immunohistochemical examination for extracellular matrix molecules and growth factors, transmission and scanning electron microscopy, and atomic force microscopy were performed on both DDHAM and CHAM specimens. A DDHAM transplant was performed in a patient with chronic bullous keratopathy and ocular surface instability. RESULTS: Histologic examination and transmission electron microscopy revealed the disruption of the trilaminar structure of the basement membrane compared with CHAM, and immunohistochemical examination confirmed the loss of collagen IV and VII, laminin, and fibronectin in DDHAM. Lower levels of several growth factors were also seen in DDHAM compared with CHAM. Clinical transplant of DDHAM was, however, successful, with rapid reepithelialization. CONCLUSION: Significant differences in composition and ultrastructure exist between DDHAM and CHAM but do not appear to compromise cell survival in vivo. CLINICAL RELEVANCE: The ease of storage and handling of DDHAM make it potentially valuable in ocular surface surgery, but its biological properties have not been well characterized, and there are also few data on its clinical application.

Effect of intracameral injection of fibrin tissue sealant on the rabbit anterior segment.

PURPOSE: To investigate the effect of intracameral injection of fibrin tissue sealant on the anterior segment structures in a rabbit model. METHODS: One eye of 10 rabbits received an intracameral injection of fibrin tissue sealant with a thrombin concentration of 500 IU (TISSEEL), and the fellow eye received an intracameral injection of balanced salt solution as a control. The rabbits were followed up with serial slit-lamp examinations, photography, high resolution anterior segment optical coherence tomography scans with pachymetry measurement, and intraocular pressure (IOP) monitoring until complete dissolution of the fibrin sealant. Corneal endothelial cell viability was evaluated using live/dead cell assays. Apoptosis of the cornea and trabecular meshwork were evaluated using TUNEL assays. Ultra-structural examinations of the cornea and trabecular meshwork were performed using electron microscopy. Histology of the trabecular meshwork and iris were analyzed using light microscopy. RESULTS: The quantity of the intracameral fibrin sealant was shown to be significantly correlated with increased IOP and pachymetry post-operatively. Complete dissolution of the fibrin sealant occurred between 15 and 30 days. Live/dead cell assays showed no decrease in viability of the corneal endothelium, and TUNEL assays showed no increase in apoptosis of the corneal epithelium, stroma, endothelium, or trabecular meshwork in the eyes with the fibrin sealant. Light and electron microscopy of the anterior segment structures were unremarkable. CONCLUSION: The intracameral use of fibrin glue was associated with a transient increase in IOP and pachymetry. However, there was no evidence of toxicity or structural damage to the corneal endothelium, trabecular meshwork, or iris.

SPARC deficiency results in improved surgical survival in a novel mouse model of glaucoma filtration surgery.

Glaucoma is a disease frequently associated with elevated intraocular pressure that can be alleviated by filtration surgery. However, the post-operative subconjunctival scarring response which blocks filtration efficiency is a major hurdle to the achievement of long-term surgical success. Current application of anti-proliferatives to modulate the scarring response is not ideal as these often give rise to sight-threatening complications. SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein involved in extracellular matrix (ECM) production and organization. In this study, we investigated post-operative surgical wound survival in an experimental glaucoma filtration model in SPARC-null mice. Loss of SPARC resulted in a marked (87.5%) surgical wound survival rate compared to 0% in wild-type (WT) counterparts. The larger SPARC-null wounds implied that aqueous filtration through the subconjunctival space was more efficient in comparison to WT wounds. The pronounced increase in both surgical survival and filtration efficiency was associated with a less collagenous ECM, smaller collagen fibril diameter, and a loosely-organized subconjunctival matrix in the SPARC-null wounds. In contrast, WT wounds exhibited a densely packed collagenous ECM with no evidence of filtration capacity. Immunolocalization assays confirmed the accumulation of ECM proteins in the WT but not in the SPARC-null wounds. The observations in vivo were corroborated by complementary data performed on WT and SPARC-null conjunctival fibroblasts in vitro. These findings indicate that depletion of SPARC bestows an inherent change in post-operative ECM remodeling to favor wound maintenance. The evidence presented in this report is strongly supportive for the targeting of SPARC to increase the success of glaucoma filtration surgery.

Effect of dispase denudation on amniotic membrane.

PURPOSE: To describe the cellular components, biochemical composition, and membrane surface characteristics of denuded human amniotic membrane (DHAM) treated with Dispase II. METHODS: DHAM was incubated with Dispase II (1.2 U/ml) for 30 min, 60 min, or 120 min. This was followed by gentle scraping to remove any remaining epithelial cells using a cell scraper. Histology, immunohistochemistry for extracellular matrix molecules and growth factors, and transmission (TEM) and scanning electron microscopy (SEM) were performed to assess the effects of increasing durations of incubation on DHAM structure. RESULTS: Dispase II treatment was associated with the digestion of several ECM molecules, particularly those in the basement membrane including collagen VI, fibronectin, and laminin. FGF-2 and PDGF-B expression were unaffected by Dispase II, but TGF-alpha, TGF-beta1, TGF-beta 2R, PDGF-A, VEGF, and EGFR expression were all reduced by Dispase II incubation. TEM confirmed the disruption of DHAM ultrastructure with increasing duration of Dispase II incubation, beginning with disruption of the basal lamina and progressing to loosening of the stromal collagen network as well. CONCLUSIONS: The use of Dispase II in the preparation of DHAM causes significant changes to the ultrastructure of the membrane, particularly the BM. Prolonged incubation with dispase may cause significant disruption in DHAM structure which may affect cell growth in cultured explants.

Comparison of donor insertion techniques for descemet stripping automated endothelial keratoplasty.

OBJECTIVE: To evaluate the difference in endothelial cell damage between 2 donor insertion techniques for Descemet stripping automated endothelial keratoplasty (DSAEK). DESIGN: Experimental study and prospective case series. Thirty donor corneas and 10 patients undergoing DSAEK with glide insertion were included. Donor cornea lenticules were prepared and a wet lab DSAEK model established. Donor lenticules were inserted either by a "taco" fold (n = 15) or glide insertion (n = 15). Endothelial cell damage was assessed by scanning electron microscopy (n = 20) and trypan blue exclusion (n = 10). Endothelial cell count was assessed by specular microscopy in the clinical patients. RESULTS: Endothelial cell viability and scanning electron microscopy demonstrated 2 different patterns of cell damage in either group. Cell viability and scanning electron microscopy showed there was mean cell damage of 9% and 9.2% , respectively, following glide insertion and 32% and 38%, respectively, following the taco-folded insertion (P = .004). The mean (SD) cell loss in the clinical patients following glide insertion was 25.3% (4.3%) at 6 months. CONCLUSION: Endothelial cell damage was higher in a wet lab model following taco-folded insertion compared with glide insertion. Initial clinical results with glide insertion showed satisfactory endothelial cell loss at 6 months. Clinical Relevance Folding of the corneal tissue during DSAEK causes more endothelial damage than glide insertion.

Analysis of conjunctival fibroblasts from a proband with Schnyder corneal dystrophy.

PURPOSE: To analyze for the presence of lipids in conjunctival fibroblasts of a patient with Schnyder corneal dystrophy (SCD). METHODS: A proband with SCD was identified, and the pedigree was examined. The proband underwent an automated lamellar therapeutic keratoplasty (ALTK). At the same time, the proband underwent a skin and conjunctival biopsy. Specimens were sent for histological and ultrastructural examination. Conjunctival fibroblasts were cultured from the biopsy specimen and stained with filipin. RESULTS: The proband showed no evidence of recurrence following the ALTK procedure. Histopathological examination showed no evidence of hydrophobic lipids in the conjunctival or dermal fibroblasts. Lipid particles were detected in the cornea. Ultrastructural examination showed no lipid particles in the conjunctival fibroblasts. Cultured fibroblasts showed no evidence of intracellular unesterified cholesterol unless low density lipoprotein (LDL) was added to the culture medium. CONCLUSIONS: There was no evidence of lipid deposition in conjunctival or skin fibroblasts in our patient with SCD. The evidence suggests local factors are responsible for the lipid deposition in the cornea.

Primary graft failure after Descemet-stripping automated endothelial keratoplasty: clinico-pathological study.

PURPOSE: Descemet-stripping automated endothelial keratoplasty (DSAEK) is a selective tissue corneal transplantation procedure in which only the diseased endothelium and the Descemet membrane is replaced. Refractive and visual results have been encouraging with this procedure, but higher rates of primary graft failure have been noted. We herein report histopathologic and ultrastructural changes in 2 cases of primary graft failure after DSAEK. We are not aware of these features being reported previously. METHODS: Two cases of primary graft failure after DSAEK. One patient underwent DSAEK for Fuchs endothelial dystrophy and the other for pseudophakic bullous keratopathy. Both DSAEK procedures were uneventful. Postoperatively, there was no graft dislocation, but 1 patient had a nasal Descemet detachment that was reapposed with intracameral air. One month postoperatively, there was no improvement in the vision, and both patients had pronounced swelling of the recipient and donor corneas. Both patients underwent graft exchange. Both the recipient and donor corneal edema resolved. RESULTS: Histopathologic evaluation showed marked corneal edema and loss of endothelial cells. Ultrastructural evaluation showed only remnant endothelial cell membranes present in 1 sample devoid of any intracellular contents. Transmission electron microscopy showed the presence of extensively damaged keratocytes in the deep posterior stroma and also in the interface at the graft-host junction. CONCLUSIONS: These cases show widespread and irrecoverable endothelial and keratocyte damage in patients with primary graft failure after DSAEK.