Keratocyte Activation Research Articles

Corneal keratocytes, fibroblasts, and myofibroblasts exhibit distinct transcriptional profiles in vitro.

After stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. Primary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-β1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. Genes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-β1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. Overall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

A rare case of bilateral keratoconus and granular corneal dystrophy managed with contact lens

Keratoconus (KC) is a bilateral, non-inflammatory, progressive thinning disorder of the cornea that results in high irregular astigmatism, distortion, and poor vision. Granular corneal dystrophy (GCD) is an autosomal dominant stromal dystrophy with onset in childhood that involves the entire cornea. KC associated with GCD is a rare combination that could occur due to abnormal synthetic activity of keratocytes and degeneration of defective basal epithelial cells. This case report shows the management of KC associated with GCD – an uncommon condition.

Autoantibodies Which Bind to and Activate Keratinocytes in Systemic Sclerosis.

Systemic sclerosis (SSc) is a multisystem connective tissue disease characterised by pathological processes involving autoimmunity, vasculopathy and resultant extensive skin and organ fibrosis. Recent studies have demonstrated activation and aberrant wound healing responses in the epithelial layer of the skin in this disease, implicating the epithelial keratinocytes as a source of pro-fibrotic and inflammatory mediators. In this paper, we investigated the role of Immunoglobulin G (IgG) autoantibodies directed against epithelial cells, as potential initiators and propagators of pathological keratocyte activation and the ensuing SSc fibrotic cascade. A keratinocyte cell-based ELISA is used to evaluate the binding of SSc IgG. SSc skin biopsies were stained by immunofluorescence for the presence of IgG in the keratinocyte layer. Moreover, IgG purified from SSc sera was evaluated for the potential to activate keratinocytes in tissue culture and to induce TLR2 and 3 signalling in reporter cell lines. We demonstrate enhanced binding of SSc IgG to keratinocytes and the activation of these cells leading to the release of IL-1α, representing a potential initiating pathway in this disease.

The impact of UV cross-linking on corneal stromal cell migration, differentiation and patterning

Previous studies have demonstrated that UV cross-linking (CXL) increases stromal stiffness and produces alterations in extracellular matrix (ECM) microstructure. In order to investigate how CXL impacts both keratocyte differentiation and patterning within the stroma, and fibroblast migration and myofibroblast differentiation on top of the stroma, we combined CXL with superficial phototherapeutic keratectomy (PTK) in a rabbit model. Twenty-six rabbits underwent a 6 mm diameter, 70 μm deep phototherapeutic keratectomy (PTK) with an excimer laser to remove the epithelium and anterior basement membrane. In 14 rabbits, standard CXL was performed in the same eye immediately after PTK. Contralateral eyes served as controls. In vivo confocal microscopy through focusing (CMTF) was used to analyze corneal epithelial and stromal thickness, as well as stromal keratocyte activation and corneal haze. CMTF scans were collected pre-operatively, and from 7 to 120 days after the procedure. A subset of rabbits was sacrificed at each time point, and corneas were fixed and labeled in situ for multiphoton fluorescence microscopy and second harmonic generation imaging. In vivo and in situ imaging demonstrated that haze after PTK was primarily derived from a layer of myofibroblasts that formed on top of the native stroma. Over time, this fibrotic layer was remodeled into more transparent stromal lamellae, and quiescent cells replaced myofibroblasts. Migrating cells within the native stroma underneath the photoablated area were elongated, co-aligned with collagen, and lacked stress fibers. In contrast, following PTK + CXL, haze was derived primarily from highly reflective necrotic “ghost cells” in the anterior stroma, and fibrosis on top of the photoablated stroma was not observed at any time point evaluated. Cells formed clusters as they migrated into the cross-linked stromal tissue and expressed stress fibers; some cells at the edge of the CXL area also expressed α-SM actin, suggesting myofibroblast transformation. Stromal thickness increased significantly between 21 and 90 days after PTK + CXL (P < 0.001) and was over 35 μm higher than baseline at Day 90 (P < 0.05). Overall, these data suggest that cross-linking inhibits interlamellar cell movement, and that these changes lead to a disruption of normal keratocyte patterning and increased activation during stromal repopulation. Interestingly, CXL also prevents PTK-induced fibrosis on top of the stroma, and results in long term increases in stromal thickness in the rabbit model.

Evidence of Disruption in Neural Regeneration in Dry Eye Secondary to Rheumatoid Arthritis.

The purpose of our study was to analyze abnormal neural regeneration activity in the cornea through means of confocal microscopy in rheumatoid arthritis patients with concomitant dry eye disease. We examined 40 rheumatoid arthritis patients with variable severity and 44 volunteer age- and gender-matched healthy control subjects. We found that all examined parameters were significantly lower (p < 0.05) in rheumatoid arthritis patients as opposed to the control samples: namely, the number of fibers, the total length of the nerves, the number of branch points on the main fibers and the total nerve-fiber area. We examined further variables, such as age, sex and the duration of rheumatoid arthritis. Interestingly, we could not find a correlation between the above variables and abnormal neural structural changes in the cornea. We interpreted these findings via implementing our hypotheses. Correspondingly, one neuroimmunological link between dry eye and rheumatoid arthritis could be through the chronic Piezo2 channelopathy-induced K2P-TASK1 signaling axis. This could accelerate neuroimmune-induced sensitization on the spinal level in this autoimmune disease, with Langerhans-cell activation in the cornea and theorized downregulated Piezo1 channels in these cells. Even more importantly, suggested principal primary-damage-associated corneal keratocyte activation could be accompanied by upregulation of Piezo1. Both activation processes on the periphery would skew the plasticity of the Th17/Treg ratio, resulting in Th17/Treg imbalance in dry eye, secondary to rheumatoid arthritis. Hence, chronic somatosensory-terminal Piezo2 channelopathy-induced impaired Piezo2-Piezo1 crosstalk could result in a mixed picture of disrupted functional regeneration but upregulated morphological regeneration activity of these somatosensory axons in the cornea, providing the demonstrated abnormal neural corneal morphology.

Small-aperture intracorneal inlay implantation in emmetropic presbyopic patients: a systematic review.

Small-aperture corneal inlays, commonly known as KAMRA, are tiny optical devices inserted in the corneal stroma aiming to gain near vision in patients with presbyopia. The purpose of this study was to systematically review case series of small-aperture corneal inlays performed in presbyopic emmetropic patients and to evaluate the visual outcomes of this procedure. This systematic review included 18 articles published between 2011 and 2018, overall studying 2724 eyes from 2691 participants. The mean longest follow-up was 19 months. Results showed that 78.5% of eyes reported an uncorrected near visual acuity of 20/32 or better and 90.50% of eyes achieved an uncorrected distance visual acuity of 20/25 or better. All patients experienced an improvement in uncorrected near visual acuity with a patient satisfaction ranging between 60% and 90%. The highlighted complications were keratocyte activation leading to corneal stromal haze, epithelial growth, iron deposits and poor distance visual acuity. Explantation was carried out in 101 eyes (3.7%) due to distance vision blurriness, development of epithelial microcysts, incorrect implant placement or hyperopic shift changes. KAMRA demonstrated high efficacy. However, safety and satisfaction rates remain unclear. Despite the low explantation rates reported in the literature, some complications were permanent. The results and conclusions should be taken with caution due to the conflict of interest stated in the reviewed articles.

In vivo Confocal Microscopic Evaluation of Corneal Changes in Acute Endothelial Rejection.

Purpose:To evaluate the microstructural corneal changes during acute endothelial graft rejection and following treatment using in vivo confocal microscopy (IVCM).Methods:Patients with a clinical diagnosis of severe acute endothelial graft rejection following penetrating keratoplasty were included in this study. IVCM was performed on the 1st day the patient presented with rejection signs and at the time of clinical resolution.Results:Twenty-three patients were included in this study. Inflammatory cells appeared as dendritic cells (DCs) and less frequently, as non-DCs in basal epithelial and subbasal areas. Activated keratocytes (AKs) (type 1: large cells with visible cytoplasmic processes; type 2: elongated and spindle-shaped keratocytes) were visible in acute phase. Following resolution, type 1 AKs considerably reduced, but type 2 cells were more often persisted. Multiple types of keratic precipitates (KPs) were also visible in acute phase which resolved following resolution of rejection.Conclusions:Acute graft rejection was associated with an increase in the number of DCs, activation of keratocytes, and aggregation of various types of KPs. Inflammatory process subsided in almost all cases, but the IVCM changes did not return to normal early after clinical resolution of rejection.

The effect of biodegradable silk fibroin-based scaffolds containing glial cell line-derived neurotrophic factor (GDNF) on the corneal regeneration process

Corneal injury due to ocular trauma or infection is one of the most challenging vision impairing pathologies. The aim of the work was to study the effect of biodegradable silk fibroin-based scaffolds containing GDNF on the corneal regeneration process. During cultivate the highest keratocytes proliferative activity was registered with scaffolds containing 250 ng/ml and 500 ng/ml GDNF. In mice with an experimental model of epithelial-stromal damage to the cornea, silk fibroin-based scaffolds containing GDNF in various concentrations were used (in groups 1, 2 and 3 silk fibroin-based scaffolds containing GDNF in a concentration of 50 ng/ml, 250 ng/ml and 500 ng/ml, respectively; in group 4 - silk fibroin-based scaffolds without GDNF; in group 5 - a solution of GDNF with concentration of 500 ng/ml; group 6- control). The area of the corneal epithelial defect in groups 2, 3, and 5 was less than in the other groups. The most pronounced positive immunohistochemical reaction with antibodies to Bcl2, Bax, phosphoERK1/2 and phospho-JNK1/2, Ki67, Gap43 was observed in groups 2 and 3. Thus, silk fibroin-based scaffolds with GDNF stimulate the epithelialization process, proliferative activity of epithelial cells and keratocytes, accelerate the formation of the stromal nerve plexus and exhibit anti-apoptotic activity.

Biology of peripheral ulcerative keratitis

Peripheral ulcerative keratitis (PUK) is a progressive peripheral thinning of the corneal stroma caused by proinflammatory mediators' release from corneal limbal vasculitis. The clinical presentation is an epithelial defect with a crescent-shaped stromal inflammation. Its exact pathophysiologic mechanisms of PUK remain partially understood, but the overall understanding of the fundamental processes that mediate and effect corneal immunity has continued to expand over the past 25 years.The unique anatomical and physiological characteristics of the periphery in relation to collagen bundles and peripheral corneal vascular arch contribute to the occurrence of this type of ulcer in this region, in addition to the concentration of complement and immunoglobulins. There is a relevant participation of the adjacent conjunctiva. Both cell-mediated immunity and humoral immunity are implicated in the pathogenesis of PUK, and the postulated mechanisms are autoimmune reactions to corneal antigens, deposition of circulating immune complexes and hypersensitivity reactions to foreign antigens. These immunocomplexes are deposited in limbic vessels resulting in the activation of the classical pathway of the complement system and, consequently, in the chemotaxis of inflammatory cells and in the release of several pro-inflammatory cytokines, which allow the production and release of matrix metalloproteinases. The release of inflammatory cytokines by infiltrating cells may induce keratocyte activation, which could then generate more release of a variety of cytokines, such as the neutrophil calgranulin C, thus facilitating an autoimmune response to the protein and precipitating an antibody- and cell-mediated hyperimmune reaction in the peripheral cornea.

Protecting the Ocular Surface at the Time of Cataract Surgery: Intracameral Mydriatic and Anaesthetic Combination Versus A Standard Topical Protocol

IntroductionAn intracameral mydriatic and anaesthetic combination has been approved for injection into the anterior chamber in order to provide rapid and stable mydriasis and sustained intraocular anaesthesia during cataract surgery.MethodsIn this prospective study, conducted at the Eye Clinic, University of Florence, Italy, we compared phacoemulsification using the standard mydriatic-anaesthetic eye-drop protocol with that using the standard protocol in terms of corneal changes, ocular surface parameters and visual quality. Sixty patients (60 eyes) were included in the study, with 30 eyes receiving Mydrane®, a novel injectable intracameral solution, during phacoemulsification (Mydrane protocol, MP) and 30 eyes receiving the standard mydriatic-anaesthetic eye drops (standard protocol, SP). The following parameters were assessed using in vivo confocal microscopy (IVCM): central corneal thickness (CCT); flare and cells in the aqueous humor (Flare); keratocyte activation (KA), Langerhans’ cell density (LCD), nerve fibre density (NFD) and endothelial cell density (ECD). The Ocular Surface Disease Index (OSDI) score, tear breakup time (TBUT) and Schirmer’s test I (STI) were also evaluated. The Optical Scattering Index (OSI) and its standard deviation (OSI-SD) were assessed using the Optical Quality Analysing System (Visiometrics SL, Terrassa, Spain).ResultsIn the MP group, CCT, Flare, KA and LCD values returned to baseline values within 15 postoperative days. The mean ECD and NFD decreased significantly in both groups from baseline at all follow-up assessments, with no statistically significant difference between groups. TBUT returned to the preoperative level at postoperative day 15 in the MP group. OSDI scores and STI were significantly worse in both groups at all follow-up assessments compared to baseline. At postoperative day 15 OSI and OSI-SD values were significantly better in the MP group than in the SP group.ConclusionsThe use of Mydrane during cataract surgery showed a good safety profile and few toxic side effects, ensuring better optical quality and tear film stability.

ECM Stiffness Controls the Activation and Contractility of Corneal Keratocytes in Response to TGF-β1

After surgery or traumatic injury, corneal wound healing can cause a scarring response that stiffens the tissue and impairs ocular function. This fibrosis is caused in part by the activation of corneal keratocytes from a native mechanically quiescent state to an activated myofibroblastic state. This transformation is tied to signaling downstream of transforming growth factor-β1 (TGF-β1). Here, to better understand how biochemical and biophysical cues interact to regulate keratocyte activation and contractility, we cultured primary rabbit corneal keratocytes on flexible substrata of varying stiffness in the presence (or absence) of TGF-β1. Time-lapse fluorescence microscopy was used to assess changes in keratocyte morphology, as well as to quantify the dynamic traction stresses exerted by cells under different experimental conditions. In other experiments, keratocytes were fixed after 5 days of culture and stained for markers of both contractility and myofibroblastic activation. Treatment with TGF-β1 elicited distinct phenotypes on substrata of different stiffnesses. Cells on soft (1 kPa) gels formed fewer stress fibers and retained a more dendritic morphology, indicative of a quiescent keratocyte phenotype. Keratocytes cultured on stiff (10 kPa) gels or collagen-coated glass coverslips, however, had broad morphologies, formed abundant stress fibers, exhibited greater levels of α-smooth muscle actin (α-SMA) expression, and exerted larger traction forces. Confocal images of phospho-myosin light chain (pMLC) immunofluorescence, moreover, revealed stiffness-dependent differences in the subcellular distribution of actomyosin contractility, with pMLC localized at the tips of thin cellular processes in mechanically quiescent cells. Importantly, keratocytes cultured in the absence of TGF-β1 showed no stiffness-dependent differences in α-SMA immunofluorescence, suggesting that a stiff microenvironment alone is insufficient to induce myofibroblastic activation. Taken together, these data suggest that changes in ECM stiffness can modulate the morphology, cytoskeletal organization, and subcellular pattern of force generation in corneal keratocytes treated with TGF-β1.

Femtosecond laser-assisted cataract surgery in eyes with shallow anterior chamber depth: comparison with conventional phacoemulsification.

To compare the postoperative outcomes between femtosecond laser-assisted cataract surgery (FLACS) and conventional phacoemulsification in eyes with shallow anterior chamber depth (ACD). Eye Clinic, NEUROFARBA Department, University of Florence, Italy. Prospective case series. Forty eyes of 40 patients with senile cataract and true ACD less than 2.00 mm underwent FLACS (femto group, n = 20) or manual phacoemulsification (phacoemulsification group, n = 20). Preoperatively and 1 week and 1 month and 6 months postoperatively, central corneal thickness (CCT) and endothelial cell density were evaluated; basal epithelial cell (BEC) and Langerhans dendritic cell (LDC) densities and keratocyte activation were assessed using in vivo corneal confocal microscopy. Intraoperative parameters such as cumulative dissipated energy (CDE) and ultrasound (US) power were recorded. Endothelial cell loss (ECL) was significantly lower in the femto group at all timepoints (P ≤ .001). In the phacoemulsification group, the CCT was significantly higher 1 week (P < .001) and 1 month (P < .001) postoperatively than preoperatively; conversely, in the femto group, it was higher only after 1 week (P < .001). BECs and LDCs significantly increased at 1 postoperative week (P < .001), returning to preoperative values after 1 month in both groups. Keratocyte activation remained significantly higher at 1 postoperative month only in the phacoemulsification group (P = .005). CDE and US power were lower in the femto group (P = .017 and P = .001, respectively); they were correlated with ECL (r = 0.662, P = .000; r = 0.389, P = .013). In eyes with shallow ACD, FLACS was a safe and an effective technique, significantly reducing the postoperative ECL and corneal inflammation compared with conventional phacoemulsification.

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

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. 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. 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. 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.].

Stability of freeze-dried plasma rich in growth factors eye drops stored for 3 months at different temperature conditions.

The purpose of this study was to analyze the biological content and activity of freeze-dried plasma rich in growth factors eye drops after their storage at 4°C and at room temperature for 3 months with respect to fresh samples (time 0). Plasma rich in growth factors was obtained after blood centrifugation from three healthy donors. After platelet activation, the obtained plasma rich in growth factors eye drops were lyophilized alone or in combination with lyoprotectant (trehalose), then they were stored for 3 months at room temperature or at 4°C. Several growth factors were analyzed at each storage time and condition. Furthermore, the proliferative and migratory potential of freeze-dried plasma rich in growth factors eye drops kept for 3 months at different temperature conditions was evaluated on primary human keratocytes. The different growth factors analyzed maintained their levels at each time and storage condition. Freeze-dried plasma rich in growth factors eye drops stored at room temperature or 4°C for 3 months showed no significant differences on the proliferative activity of keratocytes in comparison with fresh samples. However, the number of migratory human keratocytes increased significantly after treatment with lyophilized plasma rich in growth factors eye drops kept for 3 months compared to those obtained at time 0. No significant differences were observed between the freeze-dried plasma rich in growth factors eye drops whether mixed or not with lyoprotectant. Freeze-dried plasma rich in growth factors eye drops preserve the main growth factors and their biological activity after storage at room temperature or 4°C for up to 3 months. Lyophilized plasma rich in growth factors eye drops conserve their biological features even without the use of lyoprotectants for at least 3 months.

Diamond Burr Polishing of Bowman’s Layer in the Treatment of Subepithelial Fibrosis after Radial Keratotomy (Clinical Case)

Radial keratotomy (RK) was widely used at the end of the last century for refractive purposes. However, ophthalmologists are increasingly faced with negative long-term consequences after RK now. A relatively rare complication after RK is subepithelial fibrosis (according to some data, it occurs in 2.7 % of cases), which may be the result of abnormal activation or proliferation of stromal keratocytes (stromal fibroblasts) after surgical or accidental injury of the Bowman membrane. Phototherapeutic keratectomy and manual mechanical removal by the “scraper” are used for the removal of fibrous tissue, as well as a combination of these methods with mitomycin. Since laser energy can provoke a recurrence of fibrosis in the future due to the activation of stromal keratocytes and using “scraper” is not always possible to remove fibrotic tissue and the use of mitomycin is associated with a number of complications, the question of finding new methods for its removal with minimal risk of complications arises. The method of diamond burr polishing of the cornea, which developed by us for treating the syndrome of recurrent cornea erosion, was, in our opinion, the most optimal for this purpose. The method of diamond burr polishing of the cornea in this clinical case made it possible to remove the epithelial fibrosis zone without damaging the Bowman’s layer, thereby avoiding the recurrence of fibrosis in the postoperative period (the follow-up period was 2 years). The depth of the location pathological tissue location was assessed using the OCT of the cornea. The absence of recurrence is also confirmed biomicroscopically and with the OCT of the cornea. So, the method of diamond burr polishing of the Bowman’s layer can be a method of choice for the treatment of subepithelial fibrosis for patients after keratorefractive operations, being highly effective, accessible, easy to perform.

An in vivo confocal, prospective, masked, 36 months study on glaucoma patients medically treated with preservative-free or preserved monotherapy

The aim of this study was to evaluate the in vivo effects at 3 years of preservative-free tafluprost on corneal health. It was a prospective, masked, study on consecutive patients with a new prescription of preservative-free (PF) tafluprost (naïve-N or switched-S, 44 and 14 patients), and preserved (P) bimatoprost 0.003% or travoprost 0.004% (P-group, 35 patients). A complete ophthalmic examination and an in vivo corneal confocal microscopy evaluation were performed at baseline and every 6 months for 3 years. Ninety-three patients were enrolled, clinical parameters were similar in the groups at baseline, apart from intraocular pressure (IOP) which was lower in the S-group (p = 0.012). Both at baseline and over time, confocal microscopy parameters had different trends. At baseline, keratocyte activation was similar in the three groups (p = 0.43) but over the next months naïve patients treated with PF-tafluprost presented a significant (p = 0.004) reduction in keratocyte activation. Sub-basal nerves tended to increase in patients switched to PF-tafluprost (p = 0.07) while were stable in the other two groups (p = 0.11 in PF and 0.40 in P group). Grade of tortuosity was stable over time in the three groups. Beading-like formations were stable over time for the P- and the PF-group, while significantly increased in the S-group (p = 0.027). Endothelial density values were statistically different at baseline (p = 0.007), they decreased both in PF-group and in S-group (p = 0.048 and 0.001, respectively), while increased in P-group (p = 0.006). Our study is the first to show that a PF-tafluprost formulation does not significantly alter the corneal structures as examined by confocal microscopy after 36 months of topical daily therapy, while improving corneal alterations due to chronic preserved therapies.

Expression of visual system homeobox 1 in human keratoconus.

To investigate the expression of visual system homeobox 1 (VSX1) and myofibroblast marker alpha smooth muscle actin (α-SMA) in keratoconus (KC). Thirty corneal tissue were collected from KC patients after corneal transplantation and 15 normal donor corneas were obtained. All corneal tissues divided into 4 parts for different detections. Scanning electron microscopy was used to observe the ultrastructure of the specimens. VSX1 and α-SMA localization in cornea tissues was detected using immunofluorescence histochemistry. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot were performed to analyze the expression level of VSX1 and α-SMA. Compared to normal cornea tissue, the collagen fibers in KC stroma were distortional and attenuated and keratocytes were abnormally changed. VSX1 and α-SMA located in the corneal stroma. The mRNA and protein expression level of VSX1 in KC were about 3 times as high as that of normal tissue (P<0.001). α-SMA was hardly expressed in the normal corneas, however, its expression in the KC was about 1.5 times higher than that of the normal corneas (P<0.0001). Compared with normal corneal the expression of VSX1 and α-SMA in KC both increased. VSX1 is related to the activation of keratocytes and involved in the pathogenesis of keratoconus.

Assessment of Human Corneas Prior to Transplantation Using High-Resolution Two-Photon Imaging.

The purpose of this study was to evaluate the feasibility of using two-photon imaging (TPI) to assess the condition of human corneas for transplantation. Human corneas were imaged after different storage times: short-term (STS), medium-term (MTS), and long-term (LTS) storage. A high-resolution, custom-built 5-dimensional multiphoton microscope with 12-fs pulsed laser excitation was used for image acquisition. Optical discrimination between different corneal layers and sublayers based on their morphologic characteristics revealed by two-photon autofluorescence (AF) is possible. Furthermore, all layers were characterized based on AF lifetimes to gain information on metabolic activities of cells. The NAD(P)H free to protein-bound ratio (a1/a2) of epithelial cells increased significantly in both MTS and LTS corneas compared with STS corneas. In endothelial cells, NAD(P)H a1/a2 was significantly increased in MTS samples. For keratocytes, the NAD(P)H a1/a2 decreased significantly with storage time. This could indicate that the metabolic activity of the epithelial and endothelial cells reduces, whereas the activity of keratocytes increases with storage time. The analysis of the stroma SHG images indicated that the organization of collagen fibers decreases with storage time. The feasibility of measuring the endothelial cell density (ECD) using TPI was demonstrated. An ECD of 1461 ± 190 cells/mm2 was obtained for MTS samples based on TPI. TPI can provide information not accessible by current clinical methods, such as the cells' metabolic state and structural organization of the stroma, with subcellular resolution. Thus, it may improve the screening process of corneas prior to transplantation and might help to optimize the storage conditions.

Development of gelatin/ascorbic acid cryogels for potential use in corneal stromal tissue engineering.

Multifunctional cryogel material can offer an ideal hospitable environment for cell-mediated tissue reconstruction. To our knowledge, this is the first report describing the use of gelatin/ascorbic acid (AA) cryogels as keratocyte carriers for corneal stromal tissue engineering. The AA loading during cryogel fabrication is found to have a significant effect on cross-linking degree and pore dimension, mechanical and biological stability, ocular biocompatibility, cell culture performance, and cytoprotective activity, giving comprehensive insight into fine-tuning the structure-property-function interrelationships of keratocyte carrier material. Using an alkali burn-induced animal model, we present evidence that with the optimum AA loading into cryogel materials, intrastromally implanted cell/carrier constructs exhibited better capability to enhance tissue matrix regeneration and transparency maintenance as well as to mitigate corneal damage.

Aberrant Patterns of Key Epithelial Basement Membrane Components in Keratoconus.

In the cornea, the epithelial basement membrane (BM) plays an important role in maintaining corneal integrity and homeostasis. Aberrations in this vital structure are associated with several corneal pathologies including keratoconus. The aim of this study was to investigate the expression of key structural components of the epithelial BM in keratoconic corneas and to identify and describe any aberrant patterns. Immunohistochemical labeling of key BM components including fibronectin, laminin, and type IV and VII collagen was performed in healthy and keratoconic corneas. Clear changes in the BM components in the keratoconic corneas were seen with the key structural components either being absent or forming a discontinuous pattern. Another aberrant pattern, the expression of BM proteins, particularly fibronectin, laminin, and type IV collagen, in the anterior stroma of keratoconic corneas was also observed. These results indicate the activation of keratocytes into the fibroblast and myofibroblast wound phenotypes and the potential source of corneal scarring commonly observed in keratoconic corneas. Our data also support the hypothesis of dysregulated collagen synthesis and breakdown in the keratoconic cornea, in particular, the BM, and suggest a role for the BM in initiation and progression of keratoconus.