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.

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.

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.

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.

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.

The comparative safety of genipin versus UVA-riboflavin crosslinking of rabbit corneas.

PURPOSE: To investigate, after 24 h, the safety of genipin or ultraviolet A (UVA)-riboflavin crosslinking of keratocytes and endothelial cells. METHODS: Fifteen New Zealand white rabbits were selected and divided into a PBS group (five rabbits), a 0.2% genipin crosslinking (GP-CXL) group (five rabbits), and a UVA-riboflavin crosslinking (UVA-CXL) group (five rabbits). In the GP-CXL and PBS groups, 0.2% genipin or PBS was applied to the corneal surface of the right eyes. In the UVA-CXL group, a clinical crosslinking procedure was used. Before and after surgery, the operated eyes of each group were characterized with confocal microscopy, and the corneal buttons were excised for endothelium staining and electron microscopy. RESULTS: The corneal endothelial cell density of the GP-CXL, UVA-CLX, and PBS groups changed. There was a statistically significant difference in thickness and changes in corneal endothelial cell density between the UVA-CXL group and the PBS group (p<0.05), and between the UVA-CXL group and the GP-CXL group (p<0.05), but no statistically significant difference between the GP-CXL group and the PBS group. Confocal microscopy, transmission electron microscopy, and hematoxylin and eosin staining showed that there was keratocyte apoptosis in the anterior and middle stroma and endothelial cell damage in the UVA-CXL group. In the GP-CXL group, only active keratocytes were found and minimal endothelial cell damage. CONCLUSIONS: Treatment of rabbit corneas with 0.2% genipin showed minimal toxicity toward keratocytes and endothelial cells. Genipin is safer than UVA-CXL for crosslinking of thin corneas.

Arginase activity, urea, and hydroxyproline concentration are reduced in keratoconus keratocytes.

PURPOSE: Keratoconus (KC) is a disease characterized by thinning and deformation of the cornea, but its etiology remains unknown. Seventy percent of the corneal stroma consists of collagen, which is composed of three intertwined polypeptide chains with glycine-hydroxyproline-proline repeats along their sequence. Arginase is a cytoplasmatic enzyme and catalyzes the conversion of arginine to urea and ornithine, which serves as a precursor for the endogenous synthesis of proline and hydroxyproline. The purpose of this study was to analyze arginase activity, as well as collagen and urea formation in normal and KC-keratocytes and to determine the impact of urea on keratocyte viability and proliferation in vitro. METHODS: Primary human keratocytes were isolated by digestion in collagenase (1.0 mg/mL) from surgically removed corneas of eight keratoconus patients and eight normal human corneal buttons and cultured in DMEM/Ham's F12 medium supplemented with 5 % fetal calf serum. Arginase activity and urea concentration were measured in cell-lysates, hydroxyproline concentration in supernatant of cultured keratocytes using colorimetric assay. Cell viability and cell proliferation of cultured keratocytes were assessed after treatment with urea at concentrations up to10 mM for 24 h using assays for metabolic activity and DNA replication. RESULTS: Arginase activity and urea concentration in KC-keratocytes decreased by about 50 % compared to normal keratocytes (p = 0.003 and p = 0.008). Hydroxyproline synthesized by cultured KC-keratocytes was also approximately 50 % less compared to normal keratocytes (p = 0.02) and this difference decreased following treatment with 5.0 or 10.0 mM urea (p = 0.02; 0.03), without any change in cell viability (p > 0.09). However, the urea treatment increased modestly (by 20 %) the proliferation rate of KC-keratocytes (p = 0.04; 0.04; 0.04), without any effect on normal cultured keratocytes (p > 0.09). CONCLUSIONS: We identified suppressed arginase activity in the metabolic program of cultured keratoconus keratocytes. The level of urea, as one product of the enzyme arginase was also decreased. This results in impaired collagen synthesis, evidenced in the culture by reduced hydroxyproline concentration. In addition, our data showed that the other product of the arginase reaction, urea supports the proliferation of KC-keratocytes, without changes in their viability. The metabolic reprogramming of keratoconus keratocytes and its impact on development of a clinically detectable keratoconus disease has to be further analyzed.

Effects of 4-methylumbelliferone and high molecular weight hyaluronic acid on the inflammation of corneal stromal cells induced by LPS.

PURPOSE: To investigate the effects of hyaluronic acid (HA) on the inflammation of corneal fibroblasts induced by lipopolysaccharide (LPS). METHODS: Primary rabbit corneal keratocytes were isolated with collagenase. The keratocytes were cultured in a serum-containing medium to induce corneal fibroblasts, which represented the wound repair phenotype of corneal keratocytes. Corneal fibroblasts were treated with LPS with or without 4-methylumbelliferone (4-MU) / high molecular weight hyaluronic acid (HMWHA). The gene expression was evaluated via real-time PCR, immunofluorescence, and western blot. The release of inflammatory cytokines and HA was determined by ELISA. RESULTS: Three types of hyaluronan synthase (HAS) were detected in corneal fibroblasts. LPS stimulation caused the up-regulation of HAS1 and HAS2 expression in corneal fibroblasts. LPS-induced HAS2 expression was significantly inhibited by 4-MU, and accompanied by decreased HA release by the corneal fibroblasts. In the corneal fibroblasts, 4-MU reduced the LPS-stimulated up-regulation of inflammatory cytokines including IL-1, IL-6, IL-8, TNF-α, and also attenuated the LPS-induced up-regulation of inflammatory related receptors including TLR2, TLR4, CD44, and CXCR1. HMWHA treatment resulted in a significant decline in the expression of IL-6, IL-8, TLR4, and CXCR1 responded to LPS stimulation. Consistent with mRNA expression of level, the up-regulation of the release of IL-6 and IL-8 induced by LPS in corneal fibroblasts was significantly attenuated by 4-MU and HMWHA. The LPS-induced expression of IL-8 and its receptor CXCR1 at both the mRNA and protein level were significantly attenuated by 4-MU and HMWHA. CONCLUSION: The inhibitor of HA synthesis 4-MU, and HMWHA successfully reduced LPS-induced inflammation in corneal fibroblasts. The mechanism might be via the inhibition of LPS-induced TLR4 up-regulation.

New histopathologic and ultrastructural findings in Reis-Bücklers corneal dystrophy caused by the Arg124Leu mutation of TGFBI gene.

BACKGROUND: Reis-Bücklers corneal dystrophy (RBCD) was consistently reported as a corneal dystrophy only affected Bowman's layer and superficial corneal stroma, and superficial keratectomy was a recommendation surgery for treatment in literatures. The study reported new histopathological and ultrastructural findings in RBCD caused by the Arg124Leu mutation of transforming growth factor induced (TGFBI) gene in a four-generation Chinese pedigree. METHODS: Subjects including eight patients and seven unaffected family members received slit-lamp biomicroscopy and photography. DNA was obtained from all subjects, and exons 4 and 11 to 14 of TGFBI gene were analyzed by polymerase chain reaction and the products were sequenced. Anterior segment optical coherence tomography (AS OCT) and in vivo confocal microscopy were conducted for ten eyes of five patients. Based on the results of AS OCT and in vivo confocal microscopy, deep anterior lamellar keratoplasty (DLKP) using cryopreserved donor cornea was applied for four eyes of four patients. Four lamellar dystrophic corneal buttons were studied by light and transmission electron microscopy, and TGFBI immunohistochemistry. RESULTS: Eight patients had typical clinical manifestations of RBCD presenting recurrent painful corneal erosion starting in their early first decades, along with age-dependent progressive geographic corneal opacities. TGFBI sequencing revealed a heterozygous mutation, Arg124Leu in all eight patients. Anterior segment optical coherence tomography and in vivo confocal microscopy showed the dystrophic deposits involved not only in subepithelial and superficial stroma, but also in mid- or posterior stroma in four examined advanced eyes. Light microscopy showed Bowman's layer was absent, replaced by abnormal deposits stain bright red with Masson's trichrome. In superficial cornea, the deposits stacked and produced three to five continuous bands parallel to the corneal collagen lamellae. In mid- to posterior stroma, numerous granular or dot- like aggregates were heavily scattered, and most of them presented around the nuclei of stromal keratocytes. Transmission electron microscopy revealed the multiple electron-dense rod-shaped deposits aggregated and formed a characteristic pattern of three to five continuous bands in superficial cornea, which were similar to those seen under light microscopy. In mid- to posterior stroma, clusters of rod-shaped bodies were scattered extracellular or intracellular of the stromal keratocytes between the stromal lamellae suggesting the close relationship between mutated proteins and keratocyte. CONCLUSIONS: The study offer evidences indicating DLKP is a viable treatment option for advanced RBCD to avoid recurrence, and the mutated TGFBIp in dystrophic corneas are of keratocytes origin.

Tissue reaction after intrastromal corneal ring implantation in an experimental animal model.

PURPOSE: To evaluate corneal wound healing in the hen animal model after additive surgery with an intracorneal ring segment (ICRS). METHODS: We implanted one ICRS in each eye of 76 hens. In control group 1 (n = 22 hens), the stromal channel was prepared but no ICRS was inserted. In control group 2 (n = 2 hens), no surgery was performed. Animals were randomly separated into groups and euthanized after clinical follow-up of 4 and 12 hours, 1, 2, 3, and 7 days, and 1, 2, 3, 4, and 6 months. Corneas were stained with hematoxylin-eosin. Apoptosis was measured by terminal uridine nick end-labeling assays. Cell proliferation and myofibroblast-like differentiation were assayed by BrdU and α-smooth muscle actin immunofluorescence microscopy. Stromal matrix changes were documented by electron microscopy. RESULTS: Epithelial and stromal cell apoptosis around the ICRS-implanted and control group 1 eyes peaked at 12 hours, but continued for 72 hours. In ICRS-implanted eyes, epithelial and stromal proliferation was present at 12 and 24 hours, respectively, and peaked at 7 days and 72 hours, respectively. Some proliferation in the ICRS-implanted group continued through the 6-month follow-up, and myofibroblast-like cells differentiated one to three months after ICRS implantation. The segments rotated within the stroma as the limbal inferior angle approached the epithelium. CONCLUSIONS: Wound healing after ICRS implantation in hen corneas was similar to that of other corneal surgical wounds in stages. However, there were some specific features related to the small size of the epithelial wound and the device permanently implanted inside the cornea.

Dehydroxymethylepoxyquinomicin, a novel nuclear factor-κB inhibitor, reduces chemokines and adhesion molecule expression induced by IL-1ß in human corneal fibroblasts.

PURPOSE: Dehydroxymethylepoxyquinomicin (DHMEQ) is derived from the antibiotic, epoxyquinomicin C, and is a novel low molecular weight nuclear factor-κB (NF-κB) inhibitor. We investigated the effects of DHMEQ on the expression of chemokines and the intercellular adhesion molecule (ICAM)-1 induced by proinflammatory cytokines in cultures of the human corneal fibroblasts (HCFs). METHODS: The cytotoxicity of DHMEQ on cultured HCFs was evaluated by cell proliferation assays. Cultures were exposed to interleukin (IL)-1ß, and the production of IL-8 and monocyte chemoattractant protein (MCP)-1 was assessed by enzyme-linked immunosorbent assay. The degree of expression of ICAM-1 was measured by flow cytometry. The translocation of NF-κB p65 into the nucleus of HCFs was assessed by immunocytochemistry. RESULTS: DHMEQ was not toxic to cultured HCFs at doses up to 10 µg/ml. DHMEQ significantly suppressed the production of both IL-8 and MCP-1 in IL-1ß-stimulated HCFs. In addition, DHMEQ down-regulated ICAM-1 expression in IL-1ß-stimulated HCFs in a dose-dependent manner. DHMEQ inhibited the IL-1ß-induced nuclear accumulation of p65, a component of NF-κB, in HCFs. CONCLUSIONS: The suppression of inflammatory chemokines IL-8 and MCP-1 and inhibition of the expression of ICAM-1 in cultured HCFs by DHMEQ indicates that DHMEQ may have a therapeutic potential for treating ICAM-1 and chemokine-mediated corneal inflammatory disorders.

Early effects of corneal collagen cross-linking by iontophoresis in ex vivo human corneas.

PURPOSE: The purpose was to investigate the early modifications induced by collagen cross-linking by iontophoresis of riboflavin (ionto-CXL) in ex vivo human corneas by evaluating different protocols of UVA irradiation. METHODS: In this experimental study 46 ex vivo human corneas obtained from the Eye Bank of Mestre (Italy) were divided in different groups: six were utilized as control (CTL); eight were treated with ionto-CXL at 3 mW/cm(2) power for 30 min (I-3); eight were treated with ionto-CXL at 10 mW/cm(2) for 9 min (I-10); eight were treated with iontophoretic delivery of riboflavin only (I-0); eight were treated with the standard CXL at 3 mW/cm(2) for 30 min (S-3); and eight were treated with CXL at 10 mW/cm(2) for 9 min (S-10). All samples were evaluated by haematoxylin-eosin staining and immunohistochemical analysis using different markers (Connexin 43, CD34, Collagen I, TUNEL assay). Western blot analysis, utilizing Bax and Ki67 primary antibodies, for detection of keratocyte apoptosis and proliferation, respectively, was also performed. RESULTS: No endothelial damage was evidenced in the treated groups. In I-10 corneas the epithelial layers were not always well-preserved. Anterior stroma showed an uneven distribution and numerical reduction of keratocytes as well as increased apoptosis; a reduced subepithelial interweaving of collagen I fibers was observed. In S-3 and S-10 the changes induced by treatments were similar to I-10. I-3 and I-0 showed no significant changes with respect to the control group. CONCLUSIONS: In the ionto-CXL at 10 mW/cm(2) group occurred the main morphological and biomolecular changes. This experimental study suggests that iontophoresis can be considered a non-invasive potential delivery tool for riboflavin penetration in corneal stroma during CXL.

Corneal Confocal Microscopy in Dry Eye Treated with Corticosteroids.

PURPOSE: To evaluate, by in vivo laser scanning confocal microscopy (LSCM), the corneal findings in moderate-to-severe dry eye patients before and after treatment with topical corticosteroid and to associate the confocal findings to the clinical response. METHODS: Fifty eyes of 50 patients with moderate-to-severe dry eye were included in this open-label, masked study. Exclusion criteria were any systemic or ocular condition (other than dry eye) and any systemic or topical treatment (except artificial tears), ongoing or performed in the previous 3 months, with known effect on the ocular surface. All patients were treated with loteprednol etabonate ophthalmic suspension 0.5% qid for 4 weeks. Baseline and follow-up (day 30 ± 2) visits included Ocular Surface Disease Index (OSDI) questionnaire, full eye examination, and central cornea LSCM. We compared data obtained before and after treatment and looked for associations between baseline data and steroid-induced changes. Based on the previously validated OSDI Minimal Clinically Important Difference, we reanalyzed the baseline findings comparing those patients clinically improved after steroids to patients not clinically improved after steroids. RESULTS: Ocular Surface Disease Index score and LSCM dendritic cell density (DCD) significantly decreased after treatment. Baseline DCD correlated with both OSDI and DCD steroid-related changes (r = -0.44, p < 0.05 and r = -0.70, p < 0.01, respectively; Spearman) and was significantly higher in patients clinically improved after steroids than in patients not clinically improved after steroids (164.1 ± 109.2 vs. 72.4 ± 45.5 cells/mm2, p < 0.01; independent samples t test). CONCLUSIONS: Laser scanning confocal microscopy examination of DCD allows detection of treatment-related inflammation changes and shows previously unknown associations between confocal finding and symptoms improvement after treatment. These promising preliminary data suggest the need for future studies testing the predictive value of DCD for a clinical response to topical corticosteroids.

Comparison of corneal keratocytes before and after corneal collagen cross-linking in keratoconus patients.

Keratoconus is a bilateral and asymmetric corneal ectasia. Cross-linking uses ultra-violate rays to enhance corneal tissue. The purpose of this study was to use in vivo confocal microscopy to quantitatively analyze microstructural changes over time, after corneal collagen cross-linking (CXL) in keratoconus patients. In this before-and-after study, a total of 45 keratoconic eyes were selected for cross-linking among patients referred to Al-Zahra ophthalmology clinic during 2013-2014. All patients underwent complete ophthalmologic examinations. Keratocytes and the present of activated keratocytes were calculated preoperatively and at 1, 3, 6, and 12 months postoperatively using confocal microscopy. One year after CXL, best corrected and best uncorrected visual acuity was increased significantly (p < 0.001). Spherical equivalent and spherical refractive errors reduced significantly (p < 0.001). The reduction in density of anterior and mid-stromal keratocytes was significant initially (p < 0.001). During follow-up, the density of keratocytes increased significantly in all layers reaching near normal values by 12 months. The percentage of activated keratocytes showed a significant increase 1 month after cross-linking (p < 0.001) albeit this percent reduced as the corneal healing proceeded by month 12. The endothelial cells showed no significant reduction during the follow-up. Collagen cross-linking-induced significant reduction in keratocyte density. The percent of activated keratocytes increased significantly after cross-linking which showed reduction with improvement of corneal healing. After collagen cross-linking, hyper-reflective structures were observed consistent with the stromal collagen structures. Further studies are needed to assess possible changes on corneal biomechanics. The consistency in corneal endothelium numbers proves the safety of this technique.

In vivo confocal microscopy analyses of corneal microstructural changes in a prospective study of collagen cross-linking in keratoconus.

PURPOSE: To use in vivo confocal microscopy (IVCM) to quantitatively analyze microstructural changes over time, after corneal collagen cross-linking for keratoconus. DESIGN: Prospective cohort study. PARTICIPANTS: A total of 38 eyes of 38 patients undergoing collagen cross-linking for keratoconus. METHODS: Prospective, clinical cohort study of corneal collagen cross-linking in progressive keratoconus. Laser scanning IVCM performed preoperatively and at 1, 3, 6, and 12 months postoperatively. MAIN OUTCOME MEASURES: Density of corneal sub-basal nerves, anterior and posterior keratocytes, and corneal endothelium. RESULTS: Compared with baseline values, the mean sub-basal nerve density decreased significantly at 1, 3, and 6 months postoperatively (P < 0.01); however, this returned to preoperative values at 12 months (P = 0.57). One month postoperatively, there was complete absence of keratocyte nuclei in 86% of corneas. Anterior stromal edema with hyper-reflective cytoplasm and extracellular lacunae in a honeycomb-like appearance was observed and persisted at 3 months postoperatively. Scattered, presumed fragmented keratocyte nuclei, were observed at 1 and 3 months, but by 6 months, keratocyte repopulation of the anterior stroma was apparent. Quantitative analysis confirmed a significant decrease in the mean anterior keratocyte density 1, 3, and 6 months postoperatively (P ≤ 0.01) with return to baseline values at 12 months postoperatively (P = 0.57). The demarcation between treated and untreated corneal stroma appeared as a region where normal keratocytes transitioned into elongated, hyper-reflective, needle-like structures and then into large hyper-reflective stromal bands. There was no significant change in posterior keratocyte density or endothelial density at any postoperative time point. CONCLUSIONS: This prospective IVCM study revealed complete loss of the sub-basal nerve plexus and loss of anterior stromal keratocytes in the early postoperative period, with complete regeneration of the sub-basal nerve plexus and keratocyte repopulation by 12 months postoperatively. The posterior stroma and corneal endothelium were unaffected.

Confocal microscopy evaluation of the corneal response following AcuFocus KAMRA inlay implantation.

PURPOSE: To describe the corneal appearance on confocal microscopy after AcuFocus KAMRA Inlay (AcuFocus, Inc., Irvine, CA) implantation and evaluate the visual acuity compared to the confocal microscopy data. METHODS: Twelve eyes of 12 patients implanted with one of three models of the AcuFocus KAMRA Inlay (ACI 7000, 7000T, and 7000PDT) were prospectively evaluated by confocal microscopy 6 months after implantation. Additionally, 4 eyes of 4 patients explanted during the follow-up period were evaluated. RESULTS: Among the eyes implanted, mean epithelial thickness was 54.6 ± 22 µm. The subbasal nerve plexus was detected in 10 patients. The corneal nerves per unit area were 2.73 ± 2.1 sprouts/mm(2). The branch pattern was found in 8 patients. The mean keratocyte density value was 540 ± 210 cells/mm(2). A low grade of keratocyte activation was found in all patients. Among the eyes explanted, the mean wound healing opacity was 1,092.75 ± 1,877.35 µm/pixel. CONCLUSIONS: The corneal tolerance to the KAMRA Inlay appeared to be good. The inlay modified the normal structure of the corneal layer, but it was not associated with severe complications of the eye. Keratocyte activation was the finding most associated with a negative visual outcome. Confocal microscopy can be useful to evaluate the long-term evolution of the corneal layer changes following KAMRA Inlay implantation.

Evaluation of corneal changes after conventional versus accelerated corneal cross-linking: a randomized controlled trial.

PURPOSE: To evaluate the outcome of accelerated and conventional corneal cross-linking (CXL) procedures regarding their effect on morphological and optical properties of the cornea. METHODS: One hundred fifty-three eyes of 153 patients were evaluated before and during a 15-month follow-up period after CXL (76 eyes in the conventional group and 77 eyes in the accelerated group). Measured variables include corrected distance visual acuity (CDVA), uncorrected distance visual acuity (UDVA), refraction, maximum keratometry, endothelial cell density, anterior and posterior stromal keratocyte density, and subbasal nerve density. All variables were compared between the two study groups. RESULTS: Cylindrical and spherical components of refraction improved significantly during 15 months of follow-up. No difference was observed between the two study groups. UDVA and CDVA improved in the same manner, with no intergroup differences. Endothelial cell density did not change significantly during the follow-up period in either group. K-max increased slightly in the first month of the follow-up, but started to decrease at postoperative visits without any significant difference in the two groups. Anterior stromal keratocyte density and subbasal nerve density decreased significantly in both groups 1 month postoperatively. Both variables had a more significant decrease in the conventional group at all visits before the 1-year visit. At the final 15-month visit, there were no significant differences in any value between the two groups. CONCLUSIONS: Accelerated and conventional CXL seem to have a comparable and acceptable effect on keratoconus in the short-term follow-up period.

Whole exome sequencing identifies a mutation for a novel form of corneal intraepithelial dyskeratosis.

BACKGROUND: Corneal intraepithelial dyskeratosis is an extremely rare condition. The classical form, affecting Native American Haliwa-Saponi tribe members, is called hereditary benign intraepithelial dyskeratosis (HBID). Herein, we present a new form of corneal intraepithelial dyskeratosis for which we identified the causative gene by using deep sequencing technology. METHODS AND RESULTS: A seven member Caucasian French family with two corneal intraepithelial dyskeratosis affected individuals (6-year-old proband and his mother) was ascertained. The proband presented with bilateral complete corneal opacification and dyskeratosis. Palmoplantar hyperkeratosis and laryngeal dyskeratosis were associated with the phenotype. Histopathology studies of cornea and vocal cord biopsies showed dyskeratotic keratinisation. Quantitative PCR ruled out 4q35 duplication, classically described in HBID cases. Next generation sequencing with mean coverage of 50× using the Illumina Hi Seq and whole exome capture processing was performed. Sequence reads were aligned, and screened for single nucleotide variants and insertion/deletion calls. In-house pipeline filtering analyses and comparisons with available databases were performed. A novel missense mutation M77T was discovered for the gene NLRP1 which maps to chromosome 17p13.2. This was a de novo mutation in the proband's mother, following segregation in the family, and not found in 738 control DNA samples. NLRP1 expression was determined in adult corneal epithelium. The amino acid change was found to destabilise significantly the protein structure. CONCLUSIONS: We describe a new corneal intraepithelial dyskeratosis and how we identified its causative gene. The NLRP1 gene product is implicated in inflammation, autoimmune disorders, and caspase mediated apoptosis. NLRP1 polymorphisms are associated with various diseases.

Preliminary study of the association between corneal histocytological changes and surgically induced astigmatism after phacoemulsification.

BACKGROUND: Surgically induced astigmatism (SIA) was one of the factors that influences the desirable refractive outcome, and it was related to the length, type, location, structure of the incision and to the suture closure technique, etc. The aim was to evaluate the association of corneal histocytological changes with SIA after phacoemulsification. METHODS: The study enrolled 68 cases of cataract patient (68 eyes). Corneal histocytological parameters at corneal incision, central cornea and contralateral incision obtained by confocal microscope through focusing (CMTF) were compared preoperatively and 1 week, 2 weeks, 1 month, 3 months and 6 months postoperatively. These biometric parameters included the endothelial cell density, keratocyte density of posterior stromal layer, and the morphological changes. SIA was calculated by Jaffe's vector analysis. RESULTS: 1 From preoperatively to 1 week, 2 weeks, 1 month, 3 months and 6 months postoperatively, the endothelail cell density was decreased significantly (p < 0.05). Keratocyte density of posterior stroma layer was increased significantly only at 1 week, 2 weeks, 1 month, 3 months postoperatively (p <0.05), but not statistically significant (p = 0.173) at 6 months postoperatively compared to preoperative values. 2 The histocytological observations indicated that the morphology changed significantly postoperatively at the corneal incision, including the cell absent area, wave-like area, dot-like and mass-like hyperreflection, stripe-like absent area, in the endothelial layer, and the keratocyte activation, microfolds, irregular hyporeflective or hyperreflective belt, and a little dot-like hyperreflection in the posterior stroma layer. 3 The reduction of the endothelial cell density at the corneal incision at 1 week, 2 weeks, 1 month postoperatively, were positively correlated with SIA (P1 week = 0.003, P2 weeks = 0.003, P1 month = 0.032), while others were not associated with SIA statistically. CONCLUSIONS: The reduction of endothelail cell density and the histocytological changes at the corneal incision were associated with SIA. The underlining mechanism needs further study.