Induction of the integrated stress response in the rat cornea.

Keratoconus (KC), a progressive, degenerative corneal disease, represents the second leading indication for corneal transplantation globally. We have previously demonstrated that components of the Integrated Stress Response (ISR) are upregulated in human keratoconic donor tissue, and treatment of normal tissue with ISR agonists attenuates collagen production. With no consistently accepted animal models available for translational KC research, we sought to establish an in vivo model based on ISR activation to elucidate its role in the development of the KC phenotype. Four-week-old female SD rats were treated with topical SAL003 formulated as a nanosuspension or vehicle every 48 h for four doses. Animals were subject to monitoring for ocular inflammation and discomfort before being euthanized at 1, 14, or 28 days after treatment was withdrawn. Schirmer's tear test, intraocular pressure, and body weight measurements were obtained at baseline and prior to euthanasia. Globes were subject to routine histopathology, immunohistochemistry for ATF4, and qPCR for Col1a1 expression. ANOVAs and Student's t tests were used to assess statistical significance (α = 0.05). SAL003 treatment did not produce any adverse ocular or systemic phenotype but did result in decreased keratocyte density. Col1a1 transcripts were reduced, corresponding to nuclear ATF4 expression within the axial cornea. In vivo topical treatment with a gel-formulated ISR agonist recapitulates key features of the activated ISR including nuclear ATF4 expression and decreased extracellular matrix (ECM) production. Exogenous ISR agonists may present one approach to establishing a rodent model for keratoconus, a charge essential for future evaluations of pathogenesis and therapeutic interventions.

Effects of Mechanical Compression on Cell Morphology and Function in Human Corneal Fibroblasts.

Purpose: To explore the effect of mechanical compression (similar to that induced by eye rubbing) on cell morphology, proliferation, apoptosis, and extracellular matrix synthesis and degradation in human corneal fibroblasts.Materials and methods: Human corneal fibroblasts were isolated from corneal lenticule tissue of 10 patients after small incision lenticule extraction surgery. A system was established to mechanically compress corneal fibroblasts with pressure ranging from 0 to 524 Pa. Morphological changes, cell proliferation, apoptosis, and corneal matrix synthesis and degradation were examined using microscopy imaging, bromodeoxyuridine staining, flow cytometry, and qPCR analysis in human corneal fibroblasts after mechanical compression.Results: Human corneal fibroblasts showed short and thick cytoplasmic extensions, as well as a relatively low aspect ratio, suggesting significant morphological alterations caused by high levels of compressive stress. Mechanical compression inhibited cell proliferation and promoted cell apoptosis. Furthermore, compressive stress led to significant elevation in the expression of genes related to extracellular matrix degradation (matrix metalloproteinases MMP1 and MMP9) as early as 6 h after compression and moderate changes in the expression of tissue inhibitors of metalloproteinases. In addition, the mRNA expression levels of COL1A1, lumican, and vimentin were reduced 24 and 48 h after mechanical compression.Conclusions: Mechanical compression alters cell morphology, inhibits proliferation, induces apoptosis, upregulates genes related to extracellular matrix degradation, and downregulates corneal structural genes in human corneal fibroblasts. This study provides evidence that compressive stress significantly influences corneal keratocytes. Our findings suggest that this mechanical effect may be related to keratoconus associated with chronic eye rubbing.

Corneal Keratocyte Density and Corneal Nerves Are Reduced in Patients With Severe Obesity and Improve After Bariatric Surgery.

Purpose: Obesity is associated with peripheral neuropathy, which bariatric surgery may ameliorate. The aim of this study was to assess whether corneal confocal microscopy can show a change in corneal nerve morphology and keratocyte density in subjects with severe obesity after bariatric surgery. Methods: Twenty obese patients with diabetes (n = 13) and without diabetes (n = 7) underwent assessment of hemoglobin A1c (HbA1c), lipids, IL-6, highly sensitive C-reactive protein (hsCRP), and corneal confocal microscopy before and 12 months after bariatric surgery. Corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), and keratocyte density (KD) from the anterior, middle, and posterior stroma were quantified. Twenty-two controls underwent assessment at baseline only. Results: CNFL (P < 0.001), CNBD (P < 0.05), and anterior (P < 0.001), middle (P < 0.001), and posterior (P < 0.001) keratocyte densities were significantly lower in obese patients compared to controls, and anterior keratocyte density (AKD) correlated with CNFL. Twelve months after bariatric surgery, there were significant improvements in body mass index (BMI; P < 0.001), HDL cholesterol (P < 0.05), hsCRP (P < 0.001), and IL-6 (P < 0.01). There were significant increases in CNFD (P < 0.05), CNBD (P < 0.05), CNFL (P < 0.05), and anterior (P < 0.05) and middle (P < 0.001) keratocyte densities. The increase in AKD correlated with a decrease in BMI (r = -0.55, P < 0.05) and triglycerides (r = -0.85, P < 0.001). There were no significant correlations between the change in keratocyte densities and corneal nerve fiber or other neuropathy measures. Conclusions: Corneal confocal microscopy demonstrates early small fiber damage and reduced keratocyte density in obese patients. Bariatric surgery leads to weight reduction and improvement in lipids and inflammation and an improvement in keratocyte density and corneal nerve regeneration.

Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo.

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFß) signaling. Initiation of the TGFß signaling cascade results from binding of TGFß to the labile type I TGFß receptor (TGFßRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFß signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence. In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFß1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed. In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFß1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls. Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

Fibroblastic and bone marrow-derived cellularity in the corneal stroma.

The unwounded, normal corneal stroma is a relatively simple, avascular tissue populated with quiescent keratocytes, along with corneal nerves and a few resident dendritic and monocyte/macrophage cells. In the past, the resting keratocytes were thought of as a homogenous cellular population, but recent work has shown local variations in vimentin and nestin expression, and responsiveness to transforming growth factor (TGF)-ß1. Studies have also supported there being "stromal stem cells" in localized areas. After corneal wounding, depending on the site and severity of injury, profound changes in stromal cellularity occur. Anterior or posterior injuries to the epithelium or endothelium, respectively, trigger apoptosis of adjacent keratocytes. Many contiguous keratocytes transition to keratocan-negative corneal fibroblasts that are proliferative and produce limited amounts of disorganized extracellular matrix components. Simultaneously, large numbers of bone marrow-derived cells, including monocytes, neutrophils, fibrocytes and lymphocytes, invade the stroma from the limbal blood vessels. Ongoing adequate levels of TGFß1, TGFß2 and platelet-derived growth factor (PDGF) from epithelium, tears, endothelium and aqueous humor that penetrate defective or absent epithelial barrier function (EBF) and epithelial basement membrane (EBM) and/or Descemet's basement membrane (DBM) drive corneal fibroblasts and fibrocytes to differentiate into alpha-smooth muscle actin (SMA)-positive myofibroblasts. If the EBF, EBM and/or DBM are repaired or replaced in a timely manner, typically measured in weeks, then corneal fibroblast and fibrocyte progeny, deprived of requisite levels of TGFß1 and TGFß2, undergo apoptosis or revert to their precursor cell-types. If the EBF, EBM and/or DBM are not repaired or replaced, stromal levels of TGFß1 and TGFß2 remain elevated, and mature myofibroblasts are generated from corneal fibroblasts and fibrocyte precursors that produce prodigious amounts of disordered extracellular matrix materials associated with scarring fibrosis. This fibrotic stromal matrix persists, at least until the EBF, EBM and/or DBM are regenerated or replaced, and keratocytes remove and reorganize the affected stromal matrix.

In Vivo Confocal Microscopy of Stromal Lenticule Addition Keratoplasty for Advanced Keratoconus.

PURPOSE: To investigate the in vivo corneal microscopic changes after femtosecond laser-assisted stromal lenticule addition keratoplasty in keratoconus by means of in vivo confocal microscopy. METHODS: Patients affected by advanced keratoconus were included in the study. Negative meniscus-shaped stromal lenticules, produced with a femtosecond laser (VisuMax; Carl Zeiss Meditec) from eye bank corneas were transplanted into a stromal pocket dissected in the recipient cornea at a depth of 120 µm. In vivo confocal microscopy was performed during the 12-month follow-up to investigate changes of the corneal and lenticule structure. RESULTS: Ten patients were enrolled in the study. No changes of the dendritic cell population were documented during the follow-up period. Mild edema and stromal keratocyte activation gradually decreased during the first month. Subbasal nerve density returned to preoperative values after 6 months. Donor-recipient interfaces appeared hyperreflective but gradually improved over time with significantly reduced reflectivity after 3 months. No evidence of stromal inflammatory cell migration or matrix opacification was observed. Endothelial and keratocyte density remained stable over time. A variable degree of stromal radially distributed folds, not visible on biomicroscopy, was observed in the lenticule and in the posterior recipient stroma. CONCLUSIONS: Stromal lenticule addition keratoplasty produces transitory nerve plexus density reduction and minor inflammatory reaction that rapidly decreases during the first month. Donor-recipient interface reflectivity is comparable to a femtosecond laser refractive procedure with no sign of stromal opacification or stromal rejection in 1 year of follow-up. [J Refract Surg. 2020;36(8):544-550.].

Multimodal imaging of pre-Descemet corneal dystrophy.

PURPOSE: The aim of this study was to assess structural and histological changes associated with pre-Descemet corneal dystrophy with multimodal in vivo imaging. METHODS: Retrospective case series including eight corneas from four unrelated male patients with pre-Descemet corneal dystrophy characterized by the presence of punctiform gray opacities located just anterior to the Descemet membrane at slit-lamp examination of both eyes. In vivo confocal microscopy images were obtained in the central, paracentral, and peripheral corneal zones from the superficial epithelial cell layer down to the corneal endothelium in both eyes. Spectral domain optical coherence tomography scans (central and limbal zones) and mapping of both corneas were acquired. RESULTS: Diffuse small extracellular stromal deposits, presence of enlarged hyperreflective keratocytes in the posterior stroma with either hyperreflective or hyporeflective intracellular dots, and presence of activated keratocytes in the very anterior stroma were observed in all corneas with in vivo confocal microscopy. Spectral domain optical coherence tomography scans showed a hyperreflective line anterior to Descemet's membrane running from limbus to limbus and associated with a second thinner hyperreflective line just beneath Bowman's layer. Fine hyperreflective particles were observed in the posterior, mid, and anterior stroma on optical coherence tomography scans. CONCLUSION: The clinical presentation and structural anomalies found in isolated sporadic pre-Descemet corneal dystrophy are in favor of a degenerative process affecting corneal keratocytes with no epithelial or endothelial involvement. The maximum damage is found just anterior to the Descemet membrane resulting in pre-Descemet membrane location of stromal opacities. Multimodal imaging of cornea reveals that the disorder affects the whole stroma and it permits better understanding of pre-Descemet corneal dystrophy pathophysiology together with ascertained diagnosis.

Histological Corneal Alterations in Keratoconus After Crosslinking-Expansion of Findings.

PURPOSE: To investigate histopathologic, immunohistochemical, and electron microscopic findings in 8 keratoplasty specimens with a history of corneal collagen crosslinking (CXL) for keratoconus. Five new (hitherto unreported) and 3 previously published specimens were analyzed. METHODS: Corneal buttons of 8 keratoconus corneas (5-114 months after CXL) were compared with 5 keratoconus specimens without CXL and 5 normal corneas for morphological alterations. Corneal buttons were evaluated by light microscopy and immunohistochemistry using antibodies against CD34, PGP 9.5, nestin, telomerase reverse transcriptase, and Ki67 as well as by transmission electron microscopy. RESULTS: Keratoconus corneas after CXL showed a significant keratocyte loss (except 1 specimen with an increased keratocyte number), whereas keratoconus corneas without CXL revealed a higher keratocyte density compared with healthy controls. Keratocyte loss could be clinically correlated with corneal opacification and corneal perforation. In corneas after CXL, the remaining keratocytes appeared more polymorphic and revealed a different expression of surface markers similar to keratocytes in corneal scars. The presence of proteoglycans, nerves, and endothelial cells was unaffected by CXL. CONCLUSIONS: CXL may cause permanent keratocyte loss or repopulation of altered keratocytes, resulting in clinical complications such as corneal opacification or perforation. Despite its good safety profile and high effectiveness in progressive keratoconus, CXL should be performed in accordance with current guidelines strictly adhering to protocol and safety standards.

Potential Effect of Plasma Rich in Growth Factors-Endoret in Stromal Wound Healing in Additive Surgery.

PURPOSE: To compare the clinical and histological outcomes after intrastromal corneal ring segment (ICRS) implantation with and without plasma rich in growth factors (PRGF) in an experimental animal model. MATERIALS AND METHODS: First, the toxicity of PRGF was tested in hen's keratocyte cultures. Then, an animal model with 18 hens was randomly divided into 2 groups. In the first group, one ICRS was implanted in each eye (ICRS group). In the second group, the ICRS was firstly immersed 30 min in PRGF-Endoret solution, then implanted and, finally, PRGF-Endoret was inoculated into the channel (PRGF-ICRS group). Animals of each group were also separated into 3 groups regarding the time they were sacrificed, and corneal tissue was fixed for histological analysis at 2, 7 and 30 days. Cell death was detected by terminal uridine nick end labelling (TUNEL) assay. Proliferation was labelled by 5-bromo-2-deoxyuridine (BrdU) incorporation and myofibroblast differentiation by alpha-smooth muscle actin (αSMA) immunodetection. Clinical examination, analyzing epithelial wound closure, deposits and stromal haze, was carried out at the different study times. RESULTS: No toxic effect was observed by PRGF in hen stromal cell cultures. Clinically, in PRGF-ICRS corneas at 7 days, there were more deposits with higher intensity than in ICRS group. Histologically, at day 2 there was less epithelial damage over the segment in the PRGF-ICRS group, corneal oedema around the segment disappeared earlier and, at day 7, there was also double the number of cells around the segment than in the ICRS group displaying different morphologies. The number of TUNEL-positive cells was statistically higher in the PRGF-ICRS group at 7 and 30 days, and the number of BrdU-positive cells was statistically higher at all analyzed times. However, there were no differences in the number of αSMA-positive cells at 30 days between both groups. CONCLUSIONS: The ICRS immersion in PRGF-Endoret prior and after to its corneal implantation, in an experimental animal model, enhances clinical deposits and histological cell turnover without increasing myofibroblast differentiation reducing stromal wound-healing time after surgery.

Cellular and molecular assessment of rose bengal photodynamic antimicrobial therapy on keratocytes, corneal endothelium and limbal stem cell niche.

Rose Bengal Photodynamic Antimicrobial Therapy (RB-PDAT) is a novel potential treatment for progressive infectious keratitis. The principle behind this therapy is using Rose Bengal as a photosensitizer that can be activated by green light and results in the production of oxygen free radicals which in turn eradicate the microorganism. Given RB-PDAT's mechanism of action and the potential cytotoxic effects, concerns regarding the safety of this technique have arisen. The purpose of this study was to evaluate the effect of RB-PDAT on keratocytes, while focusing on the safety profile that the photo-chemical reaction has on the limbal stem cell (LSC) niche and endothelial cell layer of the treated cornea. To perform RB-PDAT, Rose Bengal solution (0.1% RB in BSS) was applied to the right cornea of rabbits for 30 min and then irradiated by a custom-made green LED light source (525 nm, 6 mW/cm2) for 15 min (5.4 J/cm2). Three rabbits were sacrificed and enucleated after 24 h for evaluation. TUNEL assay and immunohistochemistry for endothelium and limbal stem cell viability were performed on whole mounts and frozen sections in treated and control eyes. LSC of both eyes were isolated and cultured to perform MTT viability and proliferation, and scratch wound healing assays under time-lapse microscopy. Interestingly, while Rose Bengal dye penetration was superficial, yet associated cellular apoptosis was evidenced in up to 1/3 of the stromal thickness on frozen sections. TUNEL assay on whole mounts showed no endothelial cell death following treatment. Immunohistochemistry on frozen sections of LSC displayed no structural difference between treated and non-treated eyes. There was no difference in LSC proliferation rates and scratch wound healing assay demonstrated adequate cell migration from treated and non-treated eyes. The current study suggests that even though penetration of the RB dye has been shown to be limited, oxidative stress produced by RB-PDAT can reach deeper into the corneal stroma. Nevertheless, our results show that performing RB-PDAT is safe on the corneal endothelium and has no effect on LSC viability or function.

Longitudinal Changes in Corneal Cell and Nerve Fiber Morphology in Young Patients with Type 1 Diabetes with and without Diabetic Retinopathy: A 2-Year Follow-up Study.

Purpose: We have previously used in vivo corneal confocal microscopy (IVCCM) to demonstrate significant alterations in the corneal epithelial cells, stromal keratocytes, and subbasal nerves in young patients with type 1 diabetes mellitis (T1DM), especially those with diabetic retinopathy (DR). We have evaluated the change in corneal cellular and subbasal nerve morphology over 2 years in young patients with T1DM with or without DR. Methods: A total of 19 patients with T1DM, without (n = 12) and with (n = 7) DR and 19 age- and sex-matched healthy control subjects underwent quantification of corneal cellular and subbasal nerve plexus morphology by using IVCCM at baseline and after 2 years. Results: There was no significant change in corneal basal epithelial, posterior stromal keratocyte, or endothelial cell densities over 2 years. However, there was a significant reduction in corneal nerve branch (P = 0.03) and total nerve branch density (P = 0.04) in patients without DR and a significant reduction in corneal nerve fibre density (P = 0.004) in those with DR. Conclusions: IVCCM can detect a progressive loss of corneal nerve fibers in young patients with T1DM and may allow the identification of individuals at risk of neuropathy progression for more active risk factor reduction.

Quantitative Analysis of the Corneal Collagen Distribution after In Vivo Cross-Linking with Second Harmonic Microscopy.

Corneal cross-linking (CXL) is a surgical procedure able to modify corneal biomechanics and stabilize keratoconus progression. Although it is known that CXL produces changes in corneal collagen distribution, these are still a topic of discussion. Here we quantitatively compare the corneal stroma architecture between two animal models four weeks after in vivo conventional CXL treatment, with second harmonic generation (SHG) imaging microscopy and the structure tensor (ST). The healing stage and the stroma recovery were also analyzed by means of histological sections. Results show that the CXL effects depend on the initial arrangement of the corneal collagen. While the treatment increases the order in corneas with a low level of initial organization, corneas presenting a fairly regular pattern are hardly affected. Histological samples showed active keratocytes in anterior and middle stroma, what means that the recovery is still in progress. The combination of SHG imaging and the ST is able to objectively discriminate the changes suffered by the collagen arrangement after the CXL treatment, whose effectiveness depends on the initial organization of the collagen fibers within the corneal stroma.

Influence of Biochemical Cues in Human Corneal Stromal Cell Phenotype.

PURPOSE: To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration. METHODS: Proliferation was assessed by PrestoBlue™, morphology was monitored by phase contrast microscopy, phenotype was analyzed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay. RESULTS: Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-ß1) and 3 (TGF-ß3) promoted the differentiation toward myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression. CONCLUSIONS: Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.

No Relation Between the Severity of Corneal Nerve, Epithelial, and Keratocyte Cell Morphology With Measures of Dry Eye Disease in Type 1 Diabetes.

Purpose: Patients with diabetes have a propensity to develop dry eye symptoms (DES), with reduced tear secretion and corneal sensitivity. The underlying pathologic basis of DES was explored in patients with Type 1 diabetes. Methods: Forty-two patients with Type 1 diabetes mellitus (T1DM) (age: 49.21 ± 2.53 years, duration of diabetes: 29.98 ± 2.64 years) and 25 control subjects (age: 48.70 ± 2.84 years) underwent assessment of DES using a validated dry eye questionnaire, and tear stability and tear production were assessed using tear breakup time (TBUT) and Schirmer's test, respectively. Corneal confocal microscopy was undertaken to quantify corneal nerve fiber density (CNFD), branch density (CNBD), fiber length (CNFL), keratocyte density (KD), and corneal epithelial basal cell (CEBC) density and area. Results: The prevalence of DES was significantly higher (P = 0.03), and TBUT (P = 0.006), corneal sensation (P < 0.0001), CNFD (P = 0.001), CNBD (P = 0.001), CNFL (P = 0.003), and KD (P = 0.04) were significantly lower in patients with T1DM compared to control subjects. However, these measures did not differ significantly between T1DM patients with and without dry eye. There was no correlation between DES and TBUT or corneal nerve keratocyte and CEBC morphology. Conclusions: DES and TBUT are significantly increased in patients with T1DM, but are not related to corneal nerve, basal epithelial, or keratocyte cell morphology.

Thymosin beta 4 and the eye: the journey from bench to bedside.

INTRODUCTION: Thymosin beta 4 (Tß4) has important applications in ocular repair and Phase 3 clinical trials using Tß4 to treat dry eye and neurotrophic keratopathy are currently ongoing. These exciting clinical possibilities for Tß4 in the eye are the result of seminal basic scientific discoveries and contributions from so many talented investigators. Areas covered: My personal Tß4 journey began at the NIH in 1998 and propelled my career as a clinician scientist. As a tribute to the amazing individuals who have guided and supported me along with my brilliant colleagues and students who have contributed and collaborated with me over the years, this review will tell the cumulative story of how Tß4 became a major potential new therapy for corneal wound healing disorders. The journey has been marked by the thrilling exhilaration from fundamental breakthroughs in the laboratory and clinic, combined with the challenging and often harsh realities of submitting grants and obtaining funding. Expert opinion: The electrifying possibility of Tß4 as a revolutionary novel dry eye therapy is something that could have only been dreamed about just a few years ago. We believe that Tß4 eyedrops will help many patients suffering from several ocular surface related disorders.

Lithium chloride (LiCl) induced autophagy and downregulated expression of transforming growth factor ß-induced protein (TGFBI) in granular corneal dystrophy.

This study evaluated whether lithium chloride (LiCl) prevents cytoplasmic accumulation of mutant-transforming growth factor ß-induced protein (Mut-TGFBI) in granular corneal dystrophy (GCD) via activation of the autophagy pathway. Levels of TGFBI and microtubule-associated protein 1A/1B-light chain 3 (LC3) in 3 GCD patients and healthy controls were analyzed by immunohistochemistry (IHC) staining and Western blot. Primary corneal fibroblasts were isolated and transfected with wild type or mutant type TGFBI over-expressed vectors, and then treated with LiCl and/or autophagy inhibitor 3-methyladenine (3-MA). Then, levels of TGFBI, glycogen synthase kinase-3 (GSK-3) and LC3-I/-II were detected. Cell viability and transmission electron microscopy assay were also performed. Levels of TGFBI and LC3 were significantly increased in GCD patients. Over-expression of mutant type TGFBI inhibited cell viability and induced autophagy in corneal fibroblasts. LiCl downregulated the expression of TGFBI in mutant type TGFBI over-expressed cells in a dose- and time-dependent manner. LiCl enhanced autophagy in mutant type TGFBI over-expressed cells and recovered cell viability in those cells. However, the effects of LiCl were partly attenuated when autophagy was suppressed by 3-MA. To summarize, treatment with LiCl inhibited the expression of TGFBI and recovery the inhibitory of mutant type TGFBI in cell viability, at least part through enhancing of autophagy. These data strongly suggest that LiCl may be useful in the treatment of GCD.

Morphometric analysis of cornea in the Slc39a13/Zip13-knockout mice.

Ehlers-Danlos syndrome (EDS) is a group of hereditary diseases caused by mutation of extracellular matrix-related genes. Recently, spondylodysplastic EDS-Zip13 (spEDS-Zip13: OMIM 612350) was recognized as a new EDS type. This current study could reveal various morphometric differences of collagenous population in the proper substance of cornea between the wild type and spEDS-Zip13-knockout (Zip13-KO) mice. Blockade of Smad-signaling pathway might initiate these alterations. Predilected dissimilarity in level of transcriptional activity probably dictated morphology of keratocyte and shape and electron density of its nucleus. In addition, the imbalance of proteoglycans and glycosaminoglycans would also affect the diameter and arrangement of collagen fibrils. These findings would be considered as vulnerable characteristics of corneal stroma of the Zip13-KO mice.

The Effect of Antiamoebic Agents on Viability, Proliferation and Migration of Human Epithelial Cells, Keratocytes and Endothelial Cells, In Vitro.

PURPOSE: To analyze the effect of diamidines (hexamidine-diisethionat (HD), propamidin-isethionate (PD), dibromopropamidine-diisethionat (DD)), and biguanides (polyhexamethylen biguanid (PHMB), chlorhexidine (CH)) on human corneal epithelial cell, keratocyte and endothelial cell viability, proliferation, and migration, in vitro. METHODS: For epithelial and endothelial cells a human cell line and for keratocytes primary cultures were used (n = 6 each). We used 3.9x10-4-0.1% HD, PD or DD, 3.9x10-4-0.0125% PD, 7.8x10-5-0.02% PHMB or CH concentration for 24 h to determine viability (Cell Proliferation Kit XTT), proliferation (Cell Proliferation ELISA BrdU kit), and migration using wound healing assay. Viability/proliferation/migration values of each drug were summarized as "area under curve" (AUC) together with a Mann-Whitney test. RESULTS: HCEC, keratocyte, and HCEC-12 viability AUC, comparing PD and PHMB (p ≤ 0.014 for all; PD better) or PD and HD (p ≤ 0.011 for all; PD better) differed significantly. Keratocyte and HCEC-12 viability AUC comparing CH and HD (p ≤ 0.027; CH better), HCEC-12 viability AUC comparing PD and HD (p = 0.005; PD better) and HCEC viability AUC comparing CH and PHMB (p = 0.014; CH better) differed significantly. HCEC proliferation AUC, comparing PD with PHMB, CH, DD, HD (p ≤ 0.016; PD worse for all) and keratocyte proliferation AUC, comparing PHMB with HD, PD (p = 0.004; p = 0.002; PHMB better for both), CH with HD, PD (p ≤ 0.001; CH better for both) and DD with PD (p = 0.043; DD better) differed significantly. Keratocyte migration AUC comparing PD with control, PHMB, CH, DD and HD differed significantly (p ≤ 0.012; PD worse for all). CONCLUSIONS: Propamidin-isethionate as diamidine and chlorhexidin as biguanide may be used clinically to reduce cytotoxicity of antiamoebic treatment on human corneal cells. Diamidines reduce proliferation of human epithelial cells and keratocytes more than biguanides and propamidin-isethionate reduces migration of keratocytes. Therefore, in spite of lower cytotoxicity, the inhibitory effect on proliferation and migration indicates that extended use of propamidin-isethionate should be avoided in patients.

Histopathological and Molecular Changes in the Rabbit Cornea From Arsenical Vesicant Lewisite Exposure.

Lewisite (LEW), a potent arsenical vesicating chemical warfare agent, poses a continuous risk of accidental exposure in addition to its feared use as a terrorist weapon. Ocular tissue is exquisitely sensitive to LEW and exposure can cause devastating corneal lesions. However, detailed pathogenesis of corneal injury and related mechanisms from LEW exposure that could help identify targeted therapies are not available. Using an established consistent and efficient exposure system, we evaluated the pathophysiology of the corneal injury in New Zealand white rabbits following LEW vapor exposure (at 0.2 mg/L dose) for 2.5 and 7.5 min, for up to 28 day post-exposure. LEW led to an increase in total corneal thickness starting at day 1 post-exposure and epithelial degradation starting at day 3 post-exposure, with maximal effect at day 7 postexposure followed by recovery at later time points. LEW also led to an increase in the number of blood vessels and inflammatory cells but a decrease in keratocytes with optimal effects at day 7 postexposure. A significant increase in epithelial-stromal separation was observed at days 7 and 14 post 7.5 min LEW exposure. LEW also caused an increase in the expression levels of cyclooxygenase-2, IL-8, vascular endothelial growth factor, and matrix metalloproteinase-9 at all the study time points indicating their involvement in LEW-induced inflammation, vesication, and neovascularization. The outcomes here provide valuable LEW-induced corneal injury endpoints at both lower and higher exposure durations in a relevant model system, which will be helpful to identify and screen therapies against LEW-induced corneal injury.

Queratitis por Nocardia farcinica en paciente inmunocompetente. Descripción del primer caso en España / Keratitis by Nocardia farcinica in immunocompetent patient. Description of the first case in Spain

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