Electrospun poly(l-lactide-co-dl-lactide) nanofibrous scaffold as substrate for ex vivo limbal epithelial cell cultivation.

Ultrathin electrospun poly (l-lactide-co-dl-lactide) nanofibrous membranes coated with fibronectin were explored as scaffolds for the ex vivo cultivation of limbal epithelial cells (LECs) for the treatment of limbal stem cell deficiency. The developed scaffolds were compared with the "gold-standard" fibrin gel. The resulting membranes composed of nanofibers possessed a very low thickness of 4 µm and allowed very good optical transparency in the wet state. The fibronectin-coated nanofibrous scaffolds demonstrated LEC expansion and successful cultivation similar to that on fibrin gel. Unlike the regular cobblestone epithelial cell morphology on the fibrin gel, the nanofibrous scaffold presented a mostly irregular epithelial morphology with a shift to a mesenchymal phenotype, as confirmed by the upregulation of profibroblastic genes: ACTA2 (p = 0.023), FBLN1 (p < 0.001), and THY1 (p < 0.001). Both culture conditions revealed comparable expression of stem cell markers, including KLF4, ΔNp63α and ABCG2, emphasizing the promise of polylactide-based nanofibrous membranes for further investigations.

Establishment of an LC-MS/MS method for quantification of lifitegrast in rabbit plasma and ocular tissues and its application to pharmacokinetic study.

Lifitegrast, a lymphocyte function-associated antigen 1 antagonist, was approved by the FDA for the treatment of dry eye disease. Cornea and conjunctiva have been reported to be the sites of action of lifitegrast. To investigate the pharmacokinetics of lifitegrast, a sensitive analytical method for the determination of lifitegrast in various biological matrices such as plasma and ocular tissues is required. However, only limited information about the analytical method for lifitegrast in biological samples is available. In the present study, we aimed to develop a new liquid chromatography-tandem mass spectrometry method for the determination of lifitegrast in rabbit plasma, cornea, conjunctiva, and sclera. Lifitegrast-d6 was used as an internal standard (IS). To prepare the biological samples, protein precipitation using acetonitrile was utilized. Analytes were separated from endogenous interferences on an Atlantis dC18 (5 µm, 2.1 × 150 mm), and a mixture of 0.1 % formic acid and acetonitrile was used as the mobile phase. The mass transition of precursor to product ion was monitored at 615.2 â†’ 145.0 for lifitegrast and 621.2 â†’ 145.1 for IS. The calibration curves were linear over the concentration range from 2 to 500 ng/mL for plasma and 5 to 500 ng/mL in ocular tissue homogenates. Intra- and inter-day accuracy ranged from 95.76 to 106.80 % in the plasma and 94.42 to 112.80 % in the ocular tissues. Precision was within 8.56 % in the plasma and 9.72 % in the ocular tissues. The short-term, long-term, auto-sampler, and freeze-thaw stabilities of lifitegrast were validated. The developed method was applied to a pharmacokinetic study of lifitegrast in rabbits. Following ophthalmic administration, only 3.26 % of administered lifitegrast was absorbed into the systemic circulation. Peak tissue concentrations were observed at 0.5 h after dosing, and topically administered lifitegrast was mainly distributed in the cornea and conjunctiva. The finding of this study is expected to be used in further pharmacokinetic studies and formulation development.

Distribution of the cysteinyl leukotriene system components in the human, rat and mouse eye.

The cysteinyl leukotrienes (CysLTs) have important functions in the regulation of inflammation and cellular stress. Blocking the CysLT receptors (CysLTRs) with specific antagonists is beneficial against progression of retinopathies (e.g. diabetic retinopathy, wet AMD). However, the exact cellular localization of the CysLTRs and their endogenous ligands in the eye have not been elucidated in detail yet. It is also not known whether the expression patterns differ between humans and animal models. Therefore, the present study aimed to describe and compare the distribution of two important enzymes in CysLT biosynthesis, 5-lipoxygenase (5-LOX) and 5-lipoxygenase-activating protein (FLAP), and of CysLTR1 and CysLTR2 in healthy human, rat and mouse eyes. Human donor eyes (n = 10) and eyes from adult Sprague Dawley rats (n = 5) and CD1 mice (n = 8) of both sexes were collected. The eyes were fixed in 4% paraformaldehyde and cross-sections were investigated by immunofluorescence with specific antibodies against 5-LOX, FLAP (human tissue only), CysLTR1 and CysLTR2. Flat-mounts of the human choroid were prepared and processed similarly. Expression patterns were assessed and semiquantitatively evaluated using a confocal fluorescence microscope (LSM710, Zeiss). We observed so far unreported expression sites for CysLT system components in various ocular tissues. Overall, we detected expression of 5-LOX, CysLTR1 and CysLTR2 in the human, rat and mouse cornea, conjunctiva, iris, lens, ciliary body, retina and choroid. Importantly, expression profiles of CysLTR1 and CysLTR2 were highly similar between human and rodent eyes. FLAP was expressed in all human ocular tissues except the lens. Largely weak immunoreactivity of FLAP and 5-LOX was observed in a few, yet unidentified, cells of diverse ocular tissues, indicating low levels of CysLT biosynthesis in healthy eyes. CysLTR1 was predominantly detected in ocular epithelial cells, supporting the involvement of CysLTR1 in stress and immune responses. CysLTR2 was predominantly expressed in neuronal structures, suggesting neuromodulatory roles of CysLTR2 in the eye and revealing disparate functions of CysLTRs in ocular tissues. Taken together, we provide a comprehensive protein expression atlas of CysLT system components in the human and rodent eye. While the current study is purely descriptive and therefore does not allow significant functional conclusions yet, it represents an important basis for future studies in diseased ocular tissues in which distribution patterns or expression levels of the CysLT system might be altered. Furthermore, this is the first comprehensive study to elucidate expression patterns of CysLT system components in human and animal models that will help to identify and understand functions of the system as well as mechanisms of action of potential CysLTR ligands in the eye.

Nanofibers in Ocular Drug Targeting and Tissue Engineering: Their Importance, Advantages, Advances, and Future Perspectives.

Nanofibers are frequently encountered in daily life as a modern material with a wide range of applications. The important advantages of production techniques, such as being easy, cost effective, and industrially applicable are important factors in the preference for nanofibers. Nanofibers, which have a broad scope of use in the field of health, are preferred both in drug delivery systems and tissue engineering. Due to the biocompatible materials used in their construction, they are also frequently preferred in ocular applications. The fact that they have a long drug release time as a drug delivery system and have been used in corneal tissue studies, which have been successfully developed in tissue engineering, stand out as important advantages of nanofibers. This review examines nanofibers, their production techniques and general information, nanofiber-based ocular drug delivery systems, and tissue engineering concepts in detail.

Phosphorylcholine-Based Contact Lenses for Sustained Release of Resveratrol: Design, Antioxidant and Antimicrobial Performances, and In Vivo Behavior.

Design of advanced contact lenses (CLs) demands materials that are safe and comfortable for the wearers and that preserve the normal eye microbiota, avoiding chronic inflammation and biofilm development. This work aimed to combine the natural antibiofouling phosphorylcholine and the antioxidant and prebiotic resveratrol as integral components of CLs that may have the additional performance of preventing oxidative-stress related eye diseases. Different from previous uses of 2-methacryloyloxyethyl phosphorylcholine (MPC) as coating, we explored the feasibility of adding MPC at high proportions as a comonomer of 2-hydroxyethyl methacrylate (HEMA)-based hydrogels while still allowing for the loading of the hydrophobic resveratrol. Homogeneous distribution of MPC along the hydrogel depth (confirmed by Raman spectroscopy) notably increased solvent uptake and the proportion of free water while it decreased Young's modulus. Relevantly, MPC did not hinder the uptake of resveratrol by CLs (>10 mg/g), which indeed showed network/water partition coefficients of >100. Protocols for CLs sterilization and loading of resveratrol under aseptic conditions were implemented, and the effects of tear proteins on resveratrol release rate were investigated. CLs sustained resveratrol release for more than 24 h in vitro, and sorption of albumin onto the hydrogel, although attenuated by MPC, slowed down the release. The combination of MPC and resveratrol reduced P. aeruginosa and S. aureus growth as tested in a novel hydrogel disk-agar interface biofilm growth setup. The developed CLs showed excellent anti-inflammatory properties and biocompatibility in in ovo and rabbit tests and provided higher and more prolonged levels of resveratrol in tear fluid, which favored resveratrol biodistribution in anterior and posterior eye segments compared to eye drops. Correlations between the release profiles of resveratrol in vitro and in vivo were assessed. Relevantly, the CLs preserved the antioxidant properties of resveratrol during the entire 8 h of wearing. In sum, CLs prepared with high proportion in MPC may help address safety and comfort requirements while having drug releasing capabilities.

Simultaneous Determination of Moxifloxacin Hydrochloride and Dexamethasone Sodium Phosphate in Rabbit Ocular Tissues and Plasma by LC-MS/MS: Application for Pharmacokinetics Studies.

Treatment of ocular infection involves pharmacotherapy with steroids and antibiotic drops, such as moxifloxacin hydrochloride (MFH) and dexamethasone sodium phosphate (DSP). To characterize the pharmacokinetics of these two compounds, we performed and validated a liquid chromatography-mass spectrometry (LC-MS/MS) method to quantify them in rabbit ocular tissues and plasma. We used protein precipitation to extract the compounds. The analyte and internal standard (IS) were separated using a Shim-pack Scepter C18 column. The mobile phase was composed of 0.1% formic acid water (A) and methanol (B). MFH and DSP were detected using positive ion electrostatic ionization (ESI) in multiple reaction monitoring mode (MRM). The calibration curves for both compounds showed good linearity over concentrations ranging from 0.5 to 200 ng/mL in rabbit ocular tissues and plasma. The lower limit of quantification for both MFH and DSP was 0.5 ng/mL. We validated this method for selectivity, linearity (r2 > 0.99), precision, accuracy, matrix effects, and stability. Thus, we used this method to assess the pharmacokinetic (PK) characteristics of MFH and DSP in rabbit ocular tissues and plasma after single doses. Our results indicate that this method can be used for the simultaneous analysis of moxifloxacin hydrochloride and dexamethasone sodium phosphate in clinical samples.

Pharmacokinetics of Sirolimus in a Novel Liposome Delivery System in Selected Ocular Tissues and Plasma Following a Single Subconjunctival Injection in Dutch Belted Rabbits.

Purpose: To determine the pharmacokinetics of a proprietary liposomal sirolimus (LS) formulation in ocular tissues and plasma following a single subconjunctival (SCJ) injection in Dutch belted rabbits (DBR). Analytical methods for detection of LS in plasma, aqueous humor (AH), vitreous humor (VH), retina, combined retina/choroid/retinal pigment epithelium, sclera, and iris/ciliary body were developed to examine samples. Methods: Thirty male DBR were subconjunctivally injected in both eyes with 0.1 mL of LS of 1,000 µg/mL. At selected times post-injection, ocular tissues and whole blood samples were obtained. Sirolimus concentrations were measured using liquid chromatography/tandem mass spectrometry. Results: No LS was detected in serum or AH at any time. All other examined ocular tissues had quantifiable amounts of LS at all times. LS levels were highest in sclera and lowest in VH, suggesting LS followed the supraciliary and suprachoroidal spaces to reach the posterior segment. Vitreous peak of sirolimus levels occurred at 2 h, and the sclera adjacent to the injection peaked at both 2 and 96 h. LS levels in remaining ocular tissues peaked at 6 h and decreased with time, persisting at presumed therapeutic levels on day 22. Conclusions: LS can quickly diffuse into posterior intraocular tissues after SCJ injection without reaching quantifiable levels in AH or serum in DBR. Peak levels occurred in posterior intraocular tissues at 6 h and persisted in all tissues after 3 weeks. SCJ LS in DBR is safe, has a stable pharmacokinetic profile, and should be considered for further study in human trials for autoimmune ophthalmopathies.

Ectopic ocular tissue in testicular teratoma: A case report and review of the literature.

INTRODUCTION AND IMPORTANCE: Teratoma is a common neoplasm in prepubertal and post-pubertal periods. It consists of various types of tissues arising from different germinal layers, endoderm, mesoderm, and ectoderm. Ectopic ocular tissue is a rare phenomenon, with only few reported cases in other locations. CASE PRESENTATION: This is a 10-month-old boy who presented with a painless scrotal mass. Following orchidectomy, the excised mass confirmed the presence of uveal and retinal tissues originating in a benign testicular teratoma by histopathological examination. DISCUSSION: Choroidal and retinal tissue are the most frequently encountered ectopic ocular tissue, while the least observed tissue is the lens. Most of the reported cases of ectopic ocular tissue present in ovarian teratomas. The only 2 previously reported cases of ocular-like tissue in testicular teratoma lack well-defined medullary epithelium, uveal, and retinal tissue as in our case. CONCLUSION: To our knowledge, developing ocular tissue in a testicular teratoma is extremely rare. Herein we report a unique case with mature defined ocular tissue within a testicular teratoma in an infant, which should not be overlooked.

Borrelia Ocular Infection: A Case Report and a Systematic Review of Published Cases.

INTRODUCTION: Lyme borreliosis can cause many diverse manifestations, also ocular disease where the diagnosis of ocular borreliosis is challenging. The primary aim was to report on the evidence of Borrelia spirochetes in the ocular tissue in presumed ocular borreliosis. METHODS: A systematic review of pathological eye conditions was performed where Borrelia has been suspected in relevant ocular tissue, together with a case report of diagnosed uveitis with polymerase chain reaction (PCR)-confirmed Borrelia afzelii in the vitreous. The evidence for clinical and laboratory diagnosis was evaluated systematically. As a secondary aim, the treatment of ocular Borrelia infection was also evaluated for confirmed cases. RESULTS: Thirteen includable studies were found, and after the removal of case duplicates, eleven unique cases were extracted. Apart from the present case report, 4 other cases reported strong evidence for the detection of B. spirochetes in ocular tissue. Four cases presented reasonable evidence for assumed detected Borrelia, while three additional cases showed only weak diagnostic credibility that Borrelia was detected. CONCLUSION: This systematic review, including all reported cases and our case report, supports evidence of ocular infection of Borrelia species. Furthermore, in case of suspicion of infection and seronegativity, it is justified to look for Borrelia in eye tissue samples. In addition, microscopy without using PCR is not sufficient to confirm the diagnosis of borreliosis on ocular tissue. In the articles studied, there was no unambiguous recommendation of treatment.

Extranodal natural killer/T-cell lymphoma nasal type with extensive ocular tissue involvement: a case report.

BACKGROUND: To report a rare case of extranodal natural killer/T-cell lymphoma (ENKTL), nasal type related to extensive ocular tissue, including conjunctiva, ciliary body, vitreous and retina. CASE PRESENTATION: A 52-year-old woman who had been treated by radiotherapy for ENKTL, nasal type in the right nasal cavity presented with a dramatic deterioration of vision in right eye. Physical and accessory examination showed extensive ocular tissue related, including conjunctiva, ciliary body, vitreous and retina. Vitreous specimen and conjunctiva biopsy revealed the presence of ENKTL, nasal type in the right eye. She was treated with systemic and ophthalmic chemotherapy, her ocular symptoms significantly improved, and systemic condition remained stable 7 months after the diagnosis. CONCLUSIONS: Extranodal natural killer/T-cell lymphoma, nasal type is an aggressive disease and may relate extensive ocular tissue and course dramatic vision deterioration. It is important to observe ocular related and begin aggressive combined therapy as early as possible after diagnosis.

Material properties and effect of preconditioning of human sclera, optic nerve, and optic nerve sheath.

The optic nerve (ON) is a recently recognized tractional load on the eye during larger horizontal eye rotations. In order to understand the mechanical behavior of the eye during adduction, it is necessary to characterize material properties of the sclera, ON, and in particular its sheath. We performed tensile loading of specimens taken from fresh postmortem human eyes to characterize the range of variation in their biomechanical properties and determine the effect of preconditioning. We fitted reduced polynomial hyperelastic models to represent the nonlinear tensile behavior of the anterior, equatorial, posterior, and peripapillary sclera, as well as the ON and its sheath. For comparison, we analyzed tangent moduli in low and high strain regions to represent stiffness. Scleral stiffness generally decreased from anterior to posterior ocular regions. The ON had the lowest tangent modulus, but was surrounded by a much stiffer sheath. The low-strain hyperelastic behaviors of adjacent anatomical regions of the ON, ON sheath, and posterior sclera were similar as appropriate to avoid discontinuities at their boundaries. Regional stiffnesses within individual eyes were moderately correlated, implying that mechanical properties in one region of an eye do not reliably reflect properties of another region of that eye, and that potentially pathological combinations could occur in an eye if regional properties are discrepant. Preconditioning modestly stiffened ocular tissues, except peripapillary sclera that softened. The nonlinear mechanical behavior of posterior ocular tissues permits their stresses to match closely at low strains, although progressively increasing strain causes particularly great stress in the peripapillary region.

Towards safer non-volatile tissue fixatives: Evaluation of choline-based ionic liquids for fixing ocular tissues.

Volatile organic chemicals (VOCs) are routinely used for processing biological tissue samples in clinical laboratories. Recognizing their serious health and environmental impacts, a few non-volatile green solvents (choline based ionic liquids, ILs) were evaluated as tissue fixatives here. Microscopic evaluation of histo-morphology, fixation and staining quality, and macromolecular integrity (DNA and proteins) were assessed in human eye tissues (sclera, choroid, retinal layers and retinal pigmented epithelium, eyelid and orbit) after IL-fixation. Formalin-fixed tissues were used as standard reference. Microscopic examination revealed favorable histomorphology, tissue fixation and staining characteristics in most tissues immersed in ILs. Time taken to fix, and stability over a period of time (24 h, 48 h, 1 week, 1 month) was also recorded. Electrophoretic analysis revealed stability of cellular proteins and nucleic acids in IL-fixed scleral tissues. Heterogeneity in tissue fixation property relative to the type of ocular tissue, duration of fixation and storage, warrant further design and optimization of ILs to fix biological tissues. The simple cholinium salts based ILs tested here show favorable potential for tissue fixation application, and as an alternative approach to the use of VOCs, towards sustainable biomedical practice.

Effect of purine-rich box1 on proliferation of fibroblasts.

Fibroblasts are pleomorphic cells that have a multi-directional effect on organ morphogenesis, tissue homeostasis, and immune response. In fibrotic diseases, fibroblasts synthesize large amounts of extracellular matrix (ECM), leading to scarring and organ failure. Purine-rich box1 (PU.1) is a specific transcription factor of hematopoietic cell and belongs to the E26 transformation specificity (ETS) family. Recently, it was found that the transcription factor PU.1 is an important regulatory factor of the profibrotic gene expression program. TGF-ß had been proved to play an important role in many ocular tissue fibrosis diseases, and up-regulated the expression of PU.1 in fibroblasts producing ECM in a Smad-3 dependent manner. We explore the effect of PU.1 on fibrosis of different ocular tissues from this perspective. This article reviews the role of PU.1 and its effects on fibrosis of ocular tissue and other tissues.

Quantitative Proteomic Profiling of Murine Ocular Tissue and the Extracellular Environment.

Mass spectrometry-based proteomics provides a robust and reliable method for detecting and quantifying changes in protein abundance among samples, including cells, tissues, organs, and supernatants. Physical damage or inflammation can compromise the ocular surface permitting colonization by bacterial pathogens, commonly Pseudomonas aeruginosa, and the formation of biofilms. The interplay between P. aeruginosa and the immune system at the site of infection defines the host's ability to defend against bacterial invasion and promote clearance of infection. Profiling of the ocular tissue following infection describes the nature of the host innate immune response and specifically the presence and abundance of neutrophil-associated proteins to neutralize the bacterial biofilm. Moreover, detection of unique proteins produced by P. aeruginosa enable identification of the bacterial species and may serve as a diagnostic approach in a clinical setting. Given the emergence and prevalence of antimicrobial resistant bacterial strains, the ability to rapidly diagnose a bacterial infection promoting quick and accurate treatment will reduce selective pressure towards resistance. Furthermore, the ability to define differences in the host immune response towards bacterial invasion enhances our understanding of innate immune system regulation at the ocular surface. Here, we describe murine ocular infection and sample collection, as well as outline protocols for protein extraction and mass spectrometry profiling from corneal tissue and extracellular environment (eye wash) samples. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Murine model of ocular infection Basic Protocol 2: Murine model sample collection Basic Protocol 3: Protein extraction from eye wash Basic Protocol 4: Protein extraction from corneal tissue Basic Protocol 5: Mass spectrometry-based proteomics and bioinformatics from eye wash and corneal tissue samples.

Ocular Tissue for Research in Australia: Strategies for Potential Research Utility of Surplus and Transplant-Ineligible Deceased Donations.

A 2016 Price Waterhouse Cooper Report, commissioned by the Australian Commonwealth Government's Organ and Tissue Authority, indicated that Australia had been meeting its human ocular tissue for transplant needs. It further suggested that Australia should consider exportation as a management strategy for excess tissue. Although we do not seek to discuss how the Price Waterhouse Cooper Report determined that need was being met, nor the potential value of exportation in this article, we propose that Ocular Tissue for Research (OTR), and particularly identification of donors for research, and timely access to fresh domestic tissue, be considered as an alternate or simultaneous surplus management strategy. A robust OTR system could provide long-term domestic support and investment into research and development of therapies in Australia. Such a system would also provide a meaningful donation option for those otherwise unable to donate for transplant. This article attempts to document, for the first time to our knowledge, the current recovery and distribution processes of deceased OTR in Australia. It maps the process steps, identifies the stakeholders and needs, discusses the limitations and barriers, and proposes key policy and practice reform strategies that may assist in improving access to OTR. Translational Relevance: To improve and increase access to human ocular tissue for research, and in turn, advance vision science and clinical application.

Comparative Assessment of Distribution Characteristics and Ocular Pharmacokinetics of Norvancomycin Between Continuous Topical Ocular Instillation and Hourly Administration of Eye Drop.

BACKGROUND: The aim of this study was to compare the distribution characteristics and ocular pharmacokinetics of norvancomycin (NVCM) in ocular tissues of the anterior segment between continuous topical ocular instillation and hourly administration of eye drop in rabbits. METHODS: Sixty rabbits were randomly divided into two groups: continuous topical ocular instillation drug delivery (CTOIDD) group and eye drop (control) group. In the CTOIDD group, NVCM solution (50 mg/mL) was perfused to the ocular surface using the CTOIDD system at 2 mL/h up to 10 h and the same solution was administered at one drop (50 µL) per hour for 10 h in the control group. Animals (N=6 per time-point per group) were humanely killed at 2, 4, 6, 10, and 24 h to analyze their ocular tissues and plasma. The concentrations of NVCM in the conjunctiva, cornea, aqueous humour, iris, ciliary body and plasma were measured by HPLC with photodiode array detector. The pharmacokinetic parameters were calculated by Kinetica 5.1. RESULTS: The highest concentrations of NVCM for the CTOIDD group and control group were 2105.45±919.89 µg/g and 97.18±43.14 µg/g in cornea, 3033.92±1061.95 µg/g and 806.99±563.02 µg/g in conjunctiva, 1570.19±402.87 µg/g and 46.93±23.46 µg/g in iris, 181.94±47.11 µg/g and 15.38±4.00 µg/g in ciliary body, 29.78±4.90 µg/mL and 3.20±1.48 µg/mL in aqueous humour, and 26.89±5.57 µg/mL and 1.90±1.87 µg/mL in plasma, respectively. The mean NVCM levels significantly increased at all time-points in cornea, iris, and ciliary body (p<0.05) in the CTOIDD group. The AUC0-24 values in the CTOIDD group were 27,543.70 µg·h/g in cornea, 32,514.48 µg·h/g in conjunctiva, 8631.05 µg·h/g in iris, 2194.36 µg·h/g in ciliary body and 343.9 µg·h/mL in aqueous humour, which were higher than for the eye drop group in all tissues. CONCLUSION: Since continuous instillation of NVCM with CTOIDD could reach significantly higher concentrations and was sustained for a longer period compared with hourly administration of eye drop, CTOIDD administered NVCM could be a possible method to treat bacterial keratitis.

Early biological responses in ocular tissue after SMILE and LASIK surgery.

We studied the early protein profile in the ocular tissue extracted after LASIK and SMILE surgery. SMILE and LASIK was performed in contralateral eyes and stromal tissue samples were collected from 10 eyes of 5 donors. The stromal tissue samples were analyzed using label free quantification approach and ITRAQ labelling approach in LC-MS/MS. Combined functional analysis revealed many differentially expressed proteins which were involved in important biological processes. About 117 unique differentially expressed proteins were identified using two different proteomic approaches. Collagens, proteoglycans, corneal crystallins were enriched and showed differential expression in SMILE and LASIK as compared to the non-surgical control. Apart from these, 14-3-3 class of proteins, Lysozyme (LYZ), Macrophage Migratory Inhibitory Factor protein (MIF), Pigment Epithelial Derived Factor (PEDF) were differentially expressed when compared between LASIK and SMILE. Peroxiredoxin 1 (PRDX1) expression was found to be reduced in LASIK as compared to SMILE. The expression of Lysozyme C and Macrophage Migratory Inhibitory Factor inflammatory response was found to be less in SMILE as compared to LASIK. Western blot validation of specific markers such as Collagen IV (COL4), Keratocan (KERA), Lumican (LUM), Aldehyde dehydrogenase 3 A1 (ALDH3A1), Lysozyme C (LYZC) confirmed the differences in the protein levels observed in SMILE and LASIK operated tissues as compared to non-surgical controls. In conclusion, this study revealed the early molecular changes occurring in the cornea resulting from these two surgical procedures which may have implications on managing post-operative complications.

Mycobacterium tuberculosis does not show evidence of molecular DNA in human cadaveric ocular tissues in an endemic setting.

BACKGROUND: Mycobacterium tuberculosis (MTB) in latent infection has been demonstrated in pulmonary/extra-pulmonary locations (lung, spleen, liver, kidney, adipose tissue) in autopsy studies, but its presence in ocular tissues in the latent state is not known. METHODS: We conducted molecular and histopathological study of 100 cadaver eyes (50 humans) who died from causes other than tuberculosis (TB) (and were potential candidates for corneal transplantation) to detect MTB in ocular tissues in an endemic setting. After removal of the corneal button, an 8 to 10 mm block of tissue (choroid, retina and part of the vitreous) was excised from the remaining globe for DNA isolation. Gel-based IS6110 and devR3 polymerase chain reaction (PCR) assays were done, followed by real-time PCR using beta actin gene as an internal control. Sixteen randomly selected DNA samples were double checked using a commercial kit for MTB and non-tuberculous mycobacteria (NTM) DNA. The remaining larger part of the globe was subjected to histopathology. RESULTS: The mean age was 65.14 ± 18 years. All 100 samples were negative for both IS6110 and devR, and all 16 samples were negative with NTM MTB commercial kit. All samples were negative with Ziehl-Neelsen stain for acid fast bacilli and none showed any inflammation or granulomatous pathology. CONCLUSIONS: MTB could not be detected in human ocular tissues in latent state in India, a TB-endemic country. This may suggest the inability of MTB to seed ocular tissues in the latent state, unlike other organs which serve as reservoirs for the bacilli in the absence of manifest disease.

Fiber Diameter Differentially Regulates Function of Retinal Pigment and Corneal Epithelial Cells on Nanofibrous Tissue Scaffolds.

Biomaterials have significant functions as tissue scaffolds to support cells for regeneration. Nanofibrous scaffolds which mimic the architecture of the extracellular matrix are well suited to support epithelial cells for ocular tissue engineering. This study aimed at investigating the role of scaffold architecture, if any, on the response of ocular epithelial cells. Thus, we have cultured two different types of ocular epithelial cells on nanofibrous scaffolds of two different diameters to evaluate their generic and cell-specific properties. Human adult retinal pigment epithelial (ARPE-19) and human corneal epithelial (HCE-T) cells were cultured on poly(ε-caprolactone) (PCL) nanofibers of different diameters, nominally 500 and 1300 nm. Moduli of the fiber mats were marginally different at 7.4 and 11.1 kPa for 500 and 1300 nm diameter, respectively. The molecular changes in the cells in response to the different fibers were analyzed by qRT-PCR, Western blot, immunofluorescence, ELISA, flow cytometry, MTT assay, and SEM to assess properties such as proliferation, apoptosis, membrane potential, epithelial-mesenchymal transition, stem cell population, VEGF-A secretion, differentiation, and metabolic status of the cells. HCE-T cells revealed characteristic morphology along with higher expression of proliferation, differentiation, and lower apoptotic markers when cultured on PCL nanofibers of 500 nm. However, on nanofibers of 1300 nm, the cells showed higher expression of the corneal stem/progenitor as well as pluripotent stem cell markers. ARPE-19 cells exhibited characteristic hexagonal morphology with elevated expression levels of proliferative markers, phagocytic activity, and lower apoptosis levels. However, on 500 nm nanofibers, they expressed higher levels of pluripotent markers and secretion of VEGF-A. These findings demonstrate that the response can differ markedly from scaffold architecture even if derived from the same tissue and originating from the same germ layer. Furthermore, it paves the way for a target specific outcome and, thereby, for personalized translational medicine.

A multilayered sheet-type device capable of sustained drug release and deployment control.

Minimally invasive delivery of a sustained drug release device to the body is a promising approach for treating chronic conditions such as retinal diseases. Herein, we describe a sheet-type device capable of sustained drug release and deployment control after being applied to the body through a small opened hole via a syringe-type injector. Such device consists of a four-layered structure of thin photopolymerized sheets, which are in turn made of different ratios of a mixture of polyethylene glycol dimethacrylate (PEGDM) and triethylene glycol dimethacrylate (TEGDM). A layer containing a model drug, i.e., fluorescein, was sandwiched between a controlled release and guard layer to achieve sustained unidirectional drug release. A deployment layer was then attached onto the guard layer to control the curvature of the device following deployment. The sheet-type device was sufficiently flexible to be rolled up and could be inserted into a syringe-type injector. When the device was injected into the subconjunctival space of a rabbit eye through a small opened hole, it unfolded to fit the eyeball curvature. Moreover, homogenates of the choroid/retinal pigment epithelium (RPE) as well as the retina exhibited fluorescence during 4 weeks after implantation, confirming that the drug could be delivered to the retina by using the device. This developed sheet-type device offers the possibility of achieving minimally invasive transplantation into diseased tissues and organs, and could provide improved therapeutic modalities as well as reduce possible side effects.