Precise longitudinal monitoring of corneal change through in vivo confocal microscopy in a rat dry eye disease model.

Purpose: While lacrimal gland removal is commonly used in animal models to replicate dry eye disease, research into systematically monitoring dry eye disease's longitudinal pathological changes is limited. In vivo confocal microscopy (Heidelberg Retina Tomograph 3 with a Rostock Cornea Module, Heidelberg Engineering Inc., Franklin, MA) can non-invasively reveal corneal histopathological structures. To monitor dry-eye-disease-related changes in corneal structures, we developed a precise monitoring method using in vivo confocal microscopy in a rat double lacrimal gland removal model. Methods: Five Sprague-Dawley rats (age 8-9 weeks, male) underwent double lacrimal gland removal. Modified Schirmer's tear test, blink tests, and in vivo confocal microscopy images were acquired pre-surgery and at 1, 2, and 4 weeks post-surgery. Three individual stromal nerves were selected per eye as guide images, and images of the corresponding sub-basal nerve plexus area were acquired via volume acquisition. The same area was re-imaged in subsequent weeks. Results: After double lacrimal gland removal, tear production was reduced by 60%, and the blink rate increased 10 times compared to pre-surgery. Starting from 1 week after surgery, in vivo confocal microscopy showed increased sub-basal nerve plexus nerve fiber density with inflammatory cell infiltration at the sub-basal nerve plexus layer and remained at an elevated level at 2 and 4 weeks post-surgery. Conclusions: We demonstrated that our precise monitoring method revealed detailed changes in the corneal nerves, the epithelium, and the stroma.

Induction of endothelial RAGE expression in pterygium.

PURPOSE: Chronic inflammation is a critical process in pterygium development and progression, including promotion of angiogenesis. Vascular endothelial cells (ECs) actively participate in and regulate inflammation. Pterygium research has uncovered multiple inflammatory cytokines that are upregulated, but there has been minimal focus on EC activation. The Receptor for Advanced Glycation Endproducts (RAGE), a major proinflammatory molecule expressed in the vascular endothelium and other cell types, is a major instigator of endothelial cell activation. In this study, we explored the hypothesis that RAGE is upregulated in ECs in pterygium. To this end, we examined RAGE expression and immunolocalization in human pterygium and normal conjunctival tissue, with a particular interest in assessing endothelial RAGE. METHODS: Pterygium specimens were obtained from 25 patients during surgery at the King Khaled Eye Specialist Hospital (KKESH). In the same patients, conjunctiva were obtained from the autograft during surgery. Tissue specimens were formalin-fixed and paraffin-embedded. Tissue sections were analyzed with immunohistochemistry with anti-RAGE antibody. Expression and localization of RAGE were evaluated in pterygium and corresponding conjunctiva. RESULTS: RAGE expression was detected in the vascular endothelium in all pterygium tissue specimens and most conjunctival specimens. Other cell types exhibited expression, notably epithelial cells, fibroblasts, and possibly macrophages. Strikingly, endothelial RAGE expression was increased in 19 of 25 pterygium tissue specimens, compared to the corresponding control conjunctiva. CONCLUSIONS: Our data reveal that RAGE expression is upregulated in vascular endothelial cells in pterygium. RAGE upregulation is an important mechanism by which endothelial cells amplify the overall inflammatory response, and suppression of RAGE has been shown to prevent the progression of some systemic disease processes in experimental models. This suggests that pharmacologic targeting of RAGE, which is already being attempted in clinical trials for some diseases, could be useful in treating pterygium.

Applications of nanobiotechnology in ophthalmology--Part I.

Much progress has been achieved in the field of nanotechnology and its applications in ophthalmology. It is evident that drug delivery, gene therapy, implantable devices and regenerative medicine are some of the key areas of active research. To the best of our knowledge, there is limited review work on this subject area in the current literature. To assist the interested clinicians and scientists, this bipartite commentary will focus the discussion on emerging researches in nano-ophthalmology and other enabling technologies that soon may be available in the clinician's armamentarium to maintain and restore eye sight. This installment will focus on recent discoveries in drug delivery, gene therapy, imaging and visual prostheses; the second installment will discuss the impact of nanotechnology on artificial environment, cell-nanostructure interaction, other enabling nano-ophthalmic technologies, and safety and biocompatibility of nanostructures. We will take this opportunity to introduce some exciting nano-ophthalmic applications under investigation in our laboratory. The accomplishments by the scientific community are tremendous and the future prospects are wide open.

Reproducibility of tear meniscus measurement by Fourier-domain optical coherence tomography: a pilot study.

BACKGROUND AND OBJECTIVE: To study the reproducibility of tear meniscus measurement with high-speed high-resolution Fourier-domain optical coherence tomography (FD-OCT). PATIENTS AND METHODS: Twenty normal participants were enrolled in this prospective study. The lower tear meniscus in the right eye of each subject was imaged by vertical scans centered on the inferior cornea and the lower eyelid using an FD-OCT system (RTVue; Optovue, Inc., Fremont, CA) with a corneal adaptor. The system performs 26,000 axial scans per second and has a 5-micron axial resolution. Each subject was examined at two visits 30 to 60 days apart. Each eye was scanned twice on each visit. The scans were taken 2 seconds after a blink. The lower meniscus height, depth, and cornea-meniscus angle were measured with a computer caliper. The cross-sectional area was calculated using a two-triangle approximation. RESULTS: The between-visits coefficient of variation was 17.5%, 18.0%, 35.5%, and 12.2% for meniscus height, depth, area, and angle, respectively. The intraclass correlations for these parameters were 0.605, 0.558, 0.567, and 0.367, respectively. CONCLUSION: FD-OCT measures lower tear meniscus dimensions and area with higher between-visits reproducibility than previous OCT instruments. FD-OCT may be a useful way to measure dry eye severity and treatment effectiveness.

Subconjunctival dendrimer-drug therapy for the treatment of dry eye in a rabbit model of induced autoimmune dacryoadenitis.

PURPOSE: To investigate the efficacy of a single subconjunctival injection of dendrimer-dexamethasone conjugate in a rabbit model of induced autoimmune dacryoadenitis (AID). METHODS: Dendrimer biodistribution after subconjunctival injection in AID animals was evaluated using Cy5-labelled dendrimer (D-Cy5) and confocal microscopy. Diseased animals were treated with free dexamethasone (Free-Dex), dendrimer-dexamethasone (D-Dex), or saline via a single subconjunctival injection. The efficacy was evaluated using various clinical evaluations, such as Schirmer's test, tear breakup time (TBUT), and fluorescein and rose Bengal staining. Histopathology was evaluated by H&E staining and immunostaining. Levels of inflammatory cytokines and aquaporin proteins in the LGs were determined by real-time PCR. RESULTS: Subconjunctivally administered dendrimers selectively localized in the inflamed LGs, and were taken up by the infiltrating cells. At two weeks post single dose-treatment, the D-Dex group showed improved clinical evaluations. No significant changes were observed in other groups. H&E staining demonstrated less inflammatory cell infiltration and fewer atrophic acini in D-Dex group, compared to those treated with saline or Free-Dex. Immunohistochemistry demonstrated that the intensity of CD-18 (+) and RTLA (+) was weaker in LGs in the D-Dex group than in other treatment groups. Pro-inflammatory gene expression levels of MMP9, IL6, IL8, and TNFα were significantly decreased in the D-Dex group compared to the Free-Dex and saline group. CONCLUSIONS: The dendrimer exhibits pathology-dependent biodistribution in the inflamed LGs. Subconjunctivally administered D-Dex suppressed LG inflammation, leading to partial recovery of LG function with clinical improvement in induced AID. Sjögren's patients may benefit from this targeted nanomedicine approach.

Identification of Notch-1 expression in the limbal basal epithelium.

PURPOSE: To determine whether Notch-1, a ligand-activated transmembrane receptor known to maintain cells in an undifferentiated state, primarily progenitor cells in other systems, could be used as a stem cell marker in human limbal epithelium. METHODS: Human corneoscleral tissues obtained from the Doheny Eye & Tissue Transplant Bank were prepared for cross section and whole mount analysis. Tissue for whole mount was incubated in dispase; the epithelial sheet was removed and fixed in 4% paraformaldehyde. Sections and whole mount were stained with antibodies against Notch-1, Notch-2, beta-1 integrin, alpha-6, and the G2 subtype member of the ATP binding cassette transporter (ABCG2). Specificity of the Notch-1 antibody was determined by western blot analysis with Cos-7 cells transfected with Notch-1. Explant culture was performed and only primary cultures were used in this experiment. RESULTS: Notch-1 was found to be expressed in the limbal basal region where stem cells reside. Notch-1 antigenicity was more pronounced in cell clusters, mainly in the palisades of Vogt. The central cornea was almost devoid of Notch-1. The intensity of Notch-1 staining in cultured cells from the limbal explants was high in only a few cells. The Notch-1 signal was diminished in dividing cells. Expression in cultured cells was more cytoplasmic; few cells showed additional nuclear staining. The Notch-1-stained whole mount showed only a few cells in the limbal region. A 300 kDa and a 110 kDa band confirmed the specificity of the antibody in Cos-7 cells transfected with Notch-1. Double staining for ABCG2 and Notch-1 showed some ABCG2-positive cells co-expressing Notch-1 in the limbal basal epithelium, indicating that Notch-1-expressing cells might be a unique subpopulation of cells with stem cell properties. CONCLUSIONS: Immunofluorescence data shows that Notch-1 could be a possible marker for the stem cells in the limbal basal epithelium. Further studies and characterization of the Notch pathway in corneal development will provide valuable clues for the identification of stem cells.

Tissue-engineered tear secretory system: functional lacrimal gland acinar cells cultured on matrix protein-coated substrata.

Dry eye is a general term that refers to a myriad of ophthalmic disorders resulting in the inadequate wetting of the corneal surface by the tear film. Dry eyes are typically treated by the application of artificial tears. However, patients with lacrimal insufficiencies such as Stevens-Johnson syndrome, chemical and thermal injuries, or ocular cicatricial pemphigoid have very limited options because of the short duration and action of lubricating agents. As a therapeutic strategy, we are working to develop a bioengineered tear secretory system for such patients. This article describes the growth and physiological properties of purified rabbit lacrimal gland acinar cells (pLGACs) on several matrix protein-coated polymers such as silicone, collagen I, copolymers of poly-D,L-lactide-co-glycolide (PLGA; 85:15 and 50:50), poly-L-lactic acid (PLLA), and Thermanox plastic cell culture coverslips. Monolayers of acinar cells were established on all of the polymeric substrata. An assay of beta-hexosaminidase activity in the supernatant medium showed significant increases in protein secretion, following stimulation with 100 microM carbachol on matrix protein-coated and uncoated polymers such as silicone, PLGA 85:15, and PLLA. Our study demonstrates that PLLA supported the morphological and physiological properties of purified rabbit lacrimal gland epithelial cells more successfully than the others.

Optical coherence tomography of corneal and scleral melts.

Corneal and scleral melts can be difficult to assess by slit-lamp due to the overlying opacity. The authors demonstrate the role of optical coherence tomography (OCT) in the diagnosis and management of two cases of corneal and scleral melt. A high-speed anterior segment OCT system operating at a 1310-nm wavelength was used. Cross-sectional OCT images showed the depth and extent of the melt. OCT images were obtained through an opaque pannus in one case and through a calcium plaque in the other. OCT images at the follow-up examination revealed a thin fluid space between the amniotic graft and cornea and its subsequent resolution in the first case and the fits of an epicardial graft and a subsequent clear lamellar corneal graft in the second case. OCT images allow physicians to assess melts through opaque media and subsequent graft integration after repair.

Chondroitin Sulfate-Based Biocompatible Crosslinker Restores Corneal Mechanics and Collagen Alignment.

Purpose: To evaluate the crosslinking effect of functionalized chondroitin sulfate (CS) in an ex vivo rabbit cornea model. Methods: Chondroitin sulfate molecules were chemically modified with the N-hydroxysuccinimide (NHS) group. Enucleated rabbit eyes were crosslinked with 2, 5, or 10 mg/mL CS-NHS solution for 30 or 60 minutes. The CS-NHS penetration, corneal swelling ratio, Young's modulus, and ultrastructure of the crosslinked corneas were characterized. In addition, rabbit corneas were further treated with a collagenase-chondroitinase solution to create an ex vivo keratoconus (KC)-like model. The KC model corneas were crosslinked with a standard riboflavin-ultraviolet (UV) method or alternatively with CS-NHS. Corneal mechanics, ultrastructure, and keratocyte gene expression were evaluated after UV and CS-NHS crosslinking. Results: CS-NHS effectively penetrated into the corneal stroma within 60 minutes of treatment initiation. CS-NHS crosslinking reduced the swelling ratio by 35%, increased Young's modulus by 20%, and increased collagen fibril diameter and density. CS-NHS crosslinking improved corneal mechanics of KC model corneas to levels comparable to those with UV crosslinking. Moreover, CS-NHS crosslinking demonstrated significant downregulation of proinflammatory gene expression of keratocytes, indicating a potential protective effect imparted by CS-NHS during crosslinking. Conclusions: Our results demonstrated that CS-NHS can reinforce normal and KC model corneal mechanics, and restore collagen density and alignment in KC model corneas without causing extensive keratocyte apoptosis and proinflammatory gene upregulation. Therefore, CS-NHS crosslinking can potentially provide an effective, safe, and biocompatible means of corneal reinforcement.

Esterified estrogens combined with methyltestosterone raise intraocular pressure in postmenopausal women.

PURPOSE: To investigate the effect of esterified estrogens combined with methyltestosterone (EECM) (Estratest, Solvay, Pharmaceuticals, Inc, Baudette, Minnesota, USA) on intraocular pressure (IOP) in postmenopausal women. DESIGN: Observational case series. METHODS: The IOP of 13 consecutive postmenopausal women with dry eye syndrome were recorded before and during EECM therapy (1.25 mg of esterified estrogens and 2.5 mg of methyltestosterone for several months). RESULTS: The mean IOP increased from a baseline of 15.0 mm Hg before treatment to 18.2 mm Hg on EECM therapy (P < .0001) after a median duration of 11.3 months (range, 0.9 to 24 months). The increase in IOP was statistically significant at the 0.05 level of significance within three months and continued over 12 months. Two patients whose pressures increased (>4 mm Hg) returned to baseline levels after EECM was discontinued. CONCLUSIONS: Esterified estrogens combined with methyltestosterone produce a clinically significant increase in IOP in postmenopausal women with dry eye syndrome.

Dysfunctional tear syndrome: a Delphi approach to treatment recommendations.

PURPOSE: To develop current treatment recommendations for dry eye disease from consensus of expert advice. METHODS: Of 25 preselected international specialists on dry eye, 17 agreed to participate in a modified, 2-round Delphi panel approach. Based on available literature and standards of care, a survey was presented to each panelist. A two-thirds majority was used for consensus building from responses obtained. Treatment algorithms were created. Treatment recommendations for different types and severity levels of dry eye disease were the main outcome. RESULTS: A new term for dry eye disease was proposed: dysfunctional tear syndrome (DTS). Treatment recommendations were based primarily on patient symptoms and signs. Available diagnostic tests were considered of secondary importance in guiding therapy. Development of algorithms was based on the presence or absence of lid margin disease and disturbances of tear distribution and clearance. Disease severity was considered the most important factor for treatment decision-making and was categorized into 4 levels. Severity was assessed on the basis of tear substitute requirements, symptoms of ocular discomfort, and visual disturbance. Clinical signs present in lids, tear film, conjunctiva, and cornea were also used for categorization of severity. Consensus was reached on treatment algorithms for DTS with and without concurrent lid disease. CONCLUSION: Panelist opinion relied on symptoms and signs (not tests) for selection of treatment strategies. Therapy is chosen to match disease severity and presence versus absence of lid margin disease or tear distribution and clearance disturbances.

Tissue engineering: current and future approaches to ocular surface reconstruction.

Although cells have been cultured outside the body for many years, research has only recently begun to develop complex three-dimensional tissue constructs that will, ideally, mature into fully functional tissues and organs. Tissue engineering is an emerging field in the area of biotechnology that combines the principles and methods of life sciences with those of engineering for the purpose of regenerating, repairing, or replacing diseased tissues. In this review, we describe the recent advances and current development of tissue engineering approaches as related to the ocular surface system, which comprises the three main integrated tissue units: conjunctiva, cornea and lacrimal glands.

Comparison of 20% sulfur hexafluoride with air for intraocular tamponade in Descemet membrane endothelial keratoplasty (DMEK).

PURPOSE:: To compare the effect of 20% sulfur hexafluoride (SF6) with that of air on graft detachment rates for intraocular tamponade in Descemet membrane endothelial keratoplasty (DMEK). METHODS:: Forty-two eyes of patients who underwent DMEK by a single surgeon (A.S.J.) at Wilmer Eye Institute between January 2012 and 2014 were identified; 21 received air for intraocular tamponade and the next consecutive 21 received SF6. The main outcome measure was the graft detachment rate; univariate and multivariate analyses were performed. RESULTS:: The graft detachment rate was 67% in the air group and 19% in the SF6 group (p<0.05). No complete graft detachments occurred, and all partial detachments underwent intervention with injection of intraocular air. The percentages of eyes with 20/25 or better vision were not different between the groups (67% vs. 71%). Univariate analysis showed significantly higher detachment rates with air tamponade (OR, 8.50; p<0.005) and larger donor graft size (OR, 14.96; p<0.05). Multivariate analysis with gas but not graft size included showed that gas was an independent statistically significant predictor of outcome (OR, 6.65; p<0.05). When graft size was included as a covariate, gas was no longer a statistically significant predictor of detachment but maintained OR of 7.81 (p=0.063) similar to the results of univariate and multivariate analyses without graft size. CONCLUSION:: In comparison with air, graft detachment rates for intraocular tamponade in DMEK were significantly reduced by 20% SF6.

Three-Dimensional Culture of Functional Adult Rabbit Lacrimal Gland Epithelial Cells on Decellularized Scaffold.

Aqueous tear-deficient dry eye disease is a multifactorial chronic disorder, in which the lacrimal gland fails to produce enough tears to maintain a healthy ocular surface. Some severe cases may develop corneal damage and significant vision loss. Treatment primarily involves palliation using ocular surface lubricants, but can only provide temporary relief. Construction of a bioengineered lacrimal gland having functional secretory epithelial cells is a potentially promising option for providing long-term relief to severe dry eye patients. Using sphere-forming culture techniques, we cultured adult rabbit lacrimal gland progenitor cells and prepared a lacrimal gland scaffold by decellularization. When progenitor cells were seeded onto the decellularized scaffold, they formed duct- and acinar-like structures in the three-dimensional culture system. Lacrimal gland epithelial cells showed good cell viability, cell differentiation, and secretory function in decellularized lacrimal gland matrix, as indicated by morphology, immunostaining, and ß-hexosaminidase secretion assay. This study demonstrated the potential suitability of utilizing tissue-specific progenitor cells and a tissue-derived bioscaffold for lacrimal gland restoration.

Combined esterified estrogen and methyltestosterone treatment for dry eye syndrome in postmenopausal women.

PURPOSE: To determine whether systemic replacement with combined esterified estrogen (EE) and methyltestosterone (MT) (EE + MT) would reduce symptoms and promote clinical improvement in postmenopausal women with dry eye syndrome (DES). DESIGN: Retrospective, noncomparative, interventional case series. METHODS: Investigators reviewed the charts of 11 postmenopausal women treated within the last 3 years with EE + MT. RESULTS: The mean patient age was 65.2 years (standard deviation [SD] 11.4, range 48-84 years). The mean treatment duration was 12.2 months (SD 6.2 months, range 4-24 months). Ten (91%) of 11 patients reported improvement in dry eye symptoms while receiving treatment. For these 10, relief occurred after an average of 4.1 months of treatment (SD 3.2, range, 1-9 months). CONCLUSIONS: Treatment with EE + MT may be efficacious for DES of various etiologies. A randomized placebo-controlled trial is planned to further evaluate these encouraging findings.

Protective Effects of Soluble Collagen during Ultraviolet-A Crosslinking on Enzyme-Mediated Corneal Ectatic Models.

Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.

Dry eye disease: A review of diagnostic approaches and treatments.

Dry eye (DE) is a common ocular disease that results in eye discomfort, visual disturbance and substantially affects the quality of life. It has a multifactorial etiology involving tear film instability, increased osmolarity of the tear film and inflammation of the ocular surface with potential damage to the ocular surface. This review discusses the classification, diagnostic approaches and treatments of DE.

Stem cell-based therapy for treating limbal stem cells deficiency: A review of different strategies.

The self renewal capability of limbal epithelial stem (LEST) cells is fundamental to the maintenance and healing of corneal epithelium. Limbal stem cell deficiency (LSCD), due to dysfunction or loss of LEST cells, therefore presents as persistent epithelial defects, corneal vascularization, conjunctivalization etc. Stem cell-based therapy, in its simplest form - limbal autograft, has been used successfully for more than a decade. For bilateral LSCD, similar approaches with limbal allografts have been unsuccessful largely due to strong immune rejection. Therefore, as an alternate strategy for treating bilateral LSCD, ex vivo expansion of the remaining LEST cells or autologous stem cells sourced from other potential sites is being explored. Different culture systems (with and without xenobiotic supplements) using substrates like amniotic membrane or fibrin gels have been used successfully for ex vivo LEST cell maintenance and reproduction by imitating the stem cell niche. This paper is organized into sections reviewing the LEST cells, LSCD and various stem cell-based approaches for treating LSCD and discussing future direction and challenges.

Ocular surface rehabilitation: Application of human amniotic membrane in high-risk penetrating keratoplasties.

BACKGROUND: Human amniotic membrane is a versatile tool for management of ocular surface disorders. This study evaluates the effect of cryopreserved human amniotic membrane (hAM) on one-year survival of penetrating keratoplasties (PKP) in high-risk recipients. METHOD: This is a retrospective noncomparative cohort study of 58 consecutive eyes undergoing PKP with concurrent placement of a self-retained cryopreserved hAM (PROKERA®) at a tertiary care center from January 2009 to July 2010. RESULTS: Mean patient age was 66.7 ± 17.2 years and 30 (54%) were males. 51 eyes were pseudophakic and one aphakic. 27 eyes were glaucomatous; 24 had glaucoma drainage device and 2 had previous endocyclophotocoagulation. 12 patients had PKP for the first time and 46 had repeat PKP (average number of prior PKP = 1.63 ± 1.1, range: 1-5). Risk factors for graft failure included repeat PKP (79.3%), corneal neovascularization (51.7%), preexisting glaucoma (46.6%), and presence of anterior synechiae (37.9%). Both First Transplant and Repeat Transplant groups had similar survival rates until 6 months after transplant (75% vs 74%, odds ratio = 1.06, p = 1.00). At 12 months, First Transplant group showed a better survival rate (67% vs 43%, odds ratio = 2.60, p = 0.20). Eyes with >3 risk factors had a higher graft failure rate (odds ratio = 5.81, p = 0.003). CONCLUSION: Survey of the literature suggests that high-risk PKP with concurrent hAM placement demonstrate comparable graft survival. Presence of multiple risk factors is associated with poor survival.

Strategies for local gene therapy of corneal allograft rejection.

Penetrating keratoplasty is the most common type of tissue transplant in humans. Irreversible immune rejection leads to loss of vision and graft failure. This complex immune response further predisposes future corneal transplants to rejection and failure. A diverse armamentarium of surgical and pharmacologic tools is available to improve graft survival. In this review, we will discuss the various gene therapeutic strategies aimed at potentiating the anterior chamber-associated immune deviation to extend graft survival.