Rabbit Corneal Epithelial Cells Research Articles

Deoxynivalenol Damages Corneal Epithelial Cells and Exacerbates Inflammatory Response in Fungal Keratitis.

Fungal keratitis (FK) is a kind of infectious keratopathy with a high rate of blindness worldwide. Deoxynivalenol (DON) has been proven to have multiple toxic effects on humans and animals. The aim of this study was to explore a possible pathogenic role of DON in FK. We first made an animal model of FK in New Zealand white rabbits, and then attempted to detect DON in a culture medium in which Fusarium solani had been grown and also in the corneal tissue of the animal model of Fusarium solani keratitis. Next, a model of DON damage in human corneal epithelial cells (HCECs) was constructed to evaluate effects of DON on the activity, migration ability, cell cycle, and apoptosis in the HCECs. Then, putative the toxic damaging effects of DON on rabbit corneal epithelial cells and the impact of the repair cycle were studied. The expression levels of inflammatory factors in the corneas of the animal model and in the model of DON-damaged HCECs were measured. The Fusarium solani strain used in this study appeared to have the potential to produce DON, since DON was detected in the corneal tissue of rabbits which had been inoculated with this Fusarium solani strain. DON was found to alter the morphology of HCECs, to reduce the activity and to inhibit the proliferation and migration of HCECs. DON also induced the apoptosis and S-phase arrest of HCECs. In addition, DON was found to damage rabbit corneal epithelial cells, to prolong the corneal epithelial regeneration cycle, and to be associated with the upregulated expression of inflammatory factors in HCECs and rabbit corneas. DON appears to have a toxic damaging effect on HCECs in FK, and to induce the expression of inflammatory factors, leading to the exacerbation of keratitis and the formation of new blood vessels. Future studies will explore the possibility of developing a test to detect DON in ophthalmic settings to aid the rapid diagnosis of FK, and to develop DON neutralizers and adsorbents which have the potential to improve keratocyte status, inhibit apoptosis, and alleviate inflammation, therein providing new thinking for therapy of clinical FK.

Formulating Resveratrol and Melatonin Self-Nanoemulsifying Drug Delivery Systems (SNEDDS) for Ocular Administration Using Design of Experiments.

Recent studies have demonstrated that Sirtuin-1 (SIRT-1)-activating molecules exert a protective role in degenerative ocular diseases. However, these molecules hardly reach the back of the eye due to poor solubility in aqueous environments and low bioavailability after topical application on the eye's surface. Such hindrances, combined with stability issues, call for the need for innovative delivery strategies. Within this context, the development of self-nanoemulsifying drug delivery systems (SNEDDS) for SIRT-1 delivery can represent a promising approach. The aim of the work was to design and optimize SNEDDS for the ocular delivery of two natural SIRT-1 agonists, resveratrol (RSV) and melatonin (MEL), with potential implications for treating diabetic retinopathy. Pre-formulation studies were performed by a Design of Experiment (DoE) approach to construct the ternary phase diagram. The optimization phase was carried out using Response Surface Methodology (RSM). Four types of SNEDDS consisting of different surfactants (Tween® 80, Tween® 20, Solutol® HS15, and Cremophor® EL) were optimized to achieve the best physico-chemical parameters for ocular application. Stability tests indicated that SNEDDS produced with Tween® 80 was the formulation that best preserved the stability of molecules, and so it was, therefore, selected for further technological studies. The optimized formulation was prepared with Capryol® PGMC, Tween® 80, and Transcutol® P and loaded with RSV or MEL. The SNEDDS were evaluated for other parameters, such as the mean size (found to be ˂50 nm), size homogeneity (PDI < 0.2), emulsion time (around 40 s), transparency, drug content (>90%), mucoadhesion strength, in vitro drug release, pH and osmolarity, stability to dilution, and cloud point. Finally, an in vitro evaluation was performed on a rabbit corneal epithelial cell line (SIRC) to assess their cytocompatibility. The overall results suggest that SNEDDS can be used as promising nanocarriers for the ocular drug delivery of RSV and MEL.

Protective effect of pituitary adenylate cyclase activating polypeptide in diabetic keratopathy

Diabetic keratopathy (DK) is the major complication of the cornea characterizing diabetes-affected patients. This ocular pathology is correlated with the hyperglycemic state leading to delayed corneal wound healing and recurrent corneal ulcers. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread distribution throughout the body, and exerting cytoprotective effects in the neural and non-neuronal parts of the eye, including the cornea. The purpose of the present study was to investigate whether changes in PACAP expression can concur for delayed epithelial wound healing in diabetic cornea and whether the protective effect of the peptide could be mediated through the activation of the EGFR signaling pathway, which has been reported to be impaired in DK. Expression and distribution of PACAP, PAC1R, and EGFR were investigated through immunohistochemistry analysis in the cornea of normal and diabetic rats. The role of the peptide on wound healing during DK was evaluated in an in vitro model represented by rabbit corneal epithelial cells grown in high glucose conditions. Western blotting and immunofluorescence analysis were used to examine the ability of PACAP to induce the activation of the EGFR/ERK1/2 signaling pathway. Our results showed that in diabetic cornea the expression of PACAP, PAC1R, and EGFR is drastically reduced. The treatment with PACAP via PAC1R activation enhanced cell viability and corneal epithelium wound healing in cells grown under high glucose conditions. Furthermore, both EGFR and ERK1/2 signaling was induced upon the peptide treatment. Overall, our results showed the trophic efficiency of PACAP for enhancing the corneal epithelium re-epithelialization suggesting that the peptide could be beneficially valuable as a treatment for DK.

Injectable hydrogels derived from marine polysaccharides as cell carriers for large corneal epithelial defects

Injectable hydrogels have been employed for sutureless repair of corneal epithelial defects, which can perfectly fit the defect sites and minimize the associated discomfort. However, numerous hydrogels are ineffective in treating large corneal epithelial defects and still suffer from poor biocompatibility or weak applicability when used as cell carriers. Herein, hydroxypropyl chitin/carboxymethyl chitosan (HPCT/CMCS) temperature-sensitive hydrogels are fabricated, and their physicochemical properties and suitability for corneal epithelial repair are investigated. The results demonstrate that HPCT/CMCS hydrogels have excellent temperature sensitivity between 20 and 25 °C and a transparency of over 80 %. Besides, HPCT/CMCS hydrogels can promote cell proliferation and facilitate cell migration of primary rabbit corneal epithelial cells (CEpCs). A rabbit large corneal epithelial defect model (6 mm) is established, and CEpCs are transplanted into defect sites by HPCT/CMCS hydrogels. The results suggest that HPCT/CMCS/CEpCs significantly enhance the repair of large corneal epithelial defects with a healing rate of 99.6 % on day 8, while reducing inflammatory responses and scarring formation. Furthermore, HPCT/CMCS/CEpCs can contribute to the reconstruction of damaged tissues and the recovery of functional capacities. Overall, HPCT/CMCS hydrogels may be a feasible corneal cell carrier material and can provide an alternative approach to large corneal epithelial defects.

Synergistic Effect of Substance P with Insulin and Insulin-Like Growth Factor-I on Epithelial Migration of the Transformed Human Corneal Epithelial Cells (SV-40)

On the cornea, daily epithelial resurfacing is a critical process to prevent the loss of normal function, ocular morbidity, corneal structure, and vision loss. There are several components to wound healing, including cellular attachment, migration, and growth. To develop a treatment for corneal epithelial healing, we studied the effect of substance P (SP) on corneal epithelial cell migration using a cell culture system of either transformed human corneal epithelial cells (SV-40), or rabbit corneal epithelial cells (SIRC). We investigated the effect of SP with insulin and insulin-like growth factor-I (IGF-1). We found that SP is synergistic with insulin and IGF-1 on the stimulation of transformed human corneal epithelial migration in a cell culture system, as well as in rabbit SIRC cells. The addition of either SP, insulin, or IGF-1 alone did not greatly affect epithelial migration, while the combination of SP and insulin or SP and IGF-1 markedly stimulated epithelial migration in a dose-dependent manner. The synergistic effects of SP with insulin and SP with IGF-1 were inhibited by the addition of the SP-specific inhibitor (CP96345). However, the effect of insulin and IGF-1 alone were not inhibited by the NK-1-specific inhibitor (CP96345). Our results are consistent with the need for the involvement of the neuropeptide SP in corneal epithelial wound healing of diabetic corneas where nerve-ending dropout occurs. Additionally, almost identical results were obtained with human and rabbit corneal epithelial cells. These results suggest that the maintenance of the normal integrity of the corneal epithelium might be regulated by both humoral and neural factors.

MiRNA 24-3p-rich exosomes functionalized DEGMA-modified hyaluronic acid hydrogels for corneal epithelial healing.

The damage of corneal epithelium may lead to the formation of irreversible corneal opacities and even blindness. The migration rate of corneal epithelial cells directly affects corneal repair. Here, we explored ocu-microRNA 24-3p (miRNA 24-3p) that can promote rabbit corneal epithelial cells migration and cornea repair. Exosomes, an excellent transport carrier, were exacted from adipose derived mesenchymal stem cells for loading with miRNA 24-3p to prepare miRNA 24-3p-rich exosomes (Exos-miRNA 24-3p). It can accelerate corneal epithelial migration in vitro and in vivo. For application in cornea alkali burns, we further modified hyaluronic acid with di(ethylene glycol) monomethyl ether methacrylate (DEGMA) to obtain a thermosensitive hydrogel, also reported a thermosensitive DEGMA-modified hyaluronic acid hydrogel (THH) for the controlled release of Exos-miRNA 24-3p. It formed a highly uniform and clear thin layer on the ocular surface to resist clearance from blinking and extended the drug-ocular-epithelium contact time. The use of THH-3/Exos-miRNA 24-3p for 28 days after alkali burn injury accelerated corneal epithelial defect healing and epithelial maturation. It also reduced corneal stromal fibrosis and macrophage activation. MiRNA 24-3p-rich exosomes functionalized DEGMA-modified hyaluronic acid hydrogel as a multilevel delivery strategy has a potential use for cell-free therapy of corneal epithelial regeneration.

Toxicity of simultaneous intrastromal and intracameral injection of voriconazole on corneal endothelium in a rabbit model

Purpose To investigate the toxicity of repeated simultaneous intrastromal and intracameral injections of voriconazole in corneal endothelial cells in a rabbit model. Methods Thirty-six eyes of 18 New Zealand white rabbits (six eyes per group) were divided into 6 groups according to the concentration of voriconazole (Group A, 0%; Group B, 0.05%; Group C, 0.1%; Group D, 0.25%; Group E, 0.5%; Group F, 1%). A combination of intrastromal and intracameral voriconazole injections were administrated to the eyes of each group three times on days 0, 3, and 7. Corneal clouding grades and central corneal thickness (CCT) were examined on days 0, 3, 7, 10, and 14. The endothelial cell counts (ECC) were measured on days 0 and 14. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed on day 14. Results Group F (1%) showed more severe corneal clouding than the other groups (Groups A–E) from day 7 (p < 0.05, respectively). There were no significant differences in CCT and ECC among the six groups at any time point (p > 0.05, respectively). SEM revealed blurring of the cell border and loss of microvilli at concentrations ≥0.25% (Groups D–F). TEM revealed microstructural changes in endothelial cells at concentrations ≥0.1% (Groups C–F), and multiple vacuoles were observed at a concentration of 1% voriconazole (Group F). Conclusions Repeated simultaneous intrastromal and intracameral voriconazole injections at a concentration of 0.1% or higher induced microstructural endothelial damage in rabbit corneal endothelial cells.

The effect of EDTA solution on corneal endothelial cells in rabbits

Corneal disease threatens vision globally. Among corneal diseases, calcific band keratopathy has severe effects on vision owing to its unique location. Currently, ethylene diamine tetraacetic acid (EDTA) chelation remains the most important treatment. However, only the safety of low-dose topical EDTA eye drops is well established in humans. Therefore, the purpose of this study was to determine the safe dose range of EDTA for calcific band keratopathy surgery and its toxic effects on rabbit eyes. Rabbits were administered different doses of EDTA solutions (0.50, 0.20, 0.10, 0.05, and 0.01 M) for twenty minutes. In day seven, the rabbits were euthanized and pathological examination was performed for cornea. We found severe corneal edema in 0.50 M group, while milder edema in lower-concentration treated groups. Followed by corneal thickness measurement, the measured values increase to the peak in post-operative three day (0.20 M group) or one day (lower-concentration groups), then decreased. Groups comparison shown significant difference between BSS control group and higher concentration groups (0.20 M and 0.10 M) (P < 0.001) in observation period, but no significance was observed between low concentration and control group in the day seven after surgery (P > 0.05). Confocal microscopy examination suggested, the number of corneal endothelial cells significantly decreased from 3428.6 ± 180.3 cells/mm2 to 2808 ± 80.6 cells/mm2 in the 0.50 M group, while the lower-concentration groups showed lesser toxic effects on corneal endothelial cells. Finally, our histological examination demonstrated inflammation in each experimental group and dose-dependent, compared with control group. Our study found 0.05 M and 0.01 M EDTA solutions had no obvious toxic effect on the corneal endothelium compared with higher concentration. However, further study of EDTA side effect by clinical trials, and therapeutic effect observation with different concentration are necessary.

Elucidating the Corneal Endothelial Cell Proliferation Capacity through an Interspecies Transcriptome Comparison.

The regenerative capacity of corneal endothelial cells (CECs) differs between species; in bigger mammals, CECs are arrested in a non-proliferative state. Damage to these cells can compromise their function causing corneal opacity. Corneal transplantation is the current treatment for the recovery of clear eyesight, but the donor tissue demand is higher than the availability and there is a need to develop novel treatments. Interestingly, rabbit CECs retain a high proliferative profile and can repopulate the endothelium. There is a lack of fundamental knowledge to explain these differences. Gaining information on their transcriptomic variances could allowthe identification of CEC proliferation drivers. In this study, human, sheep, and rabbit CECs are analyzed at the transcriptomic level. To understand the differences across each species, a pipeline for the analysis of pathways with different activities is generated. The results reveal that 52 pathways have different activity when comparing species with non-proliferative CECs (human and sheep) to species with proliferative CECs (rabbit). The results show that Notch and TGF-β pathways have increased activity in species with non-proliferative CECs, which might be associated with their low proliferation. Overall, this study illustrates transcriptomic pathway-level differences that can provide leads to develop novel therapies to regenerate the corneal endothelium.

Biomimetic Convex Implant for Corneal Regeneration Through 3D Printing.

Blindness caused by corneal damage affects millions of people worldwide, and this number continues to rise. However, rapid epithelization and a stable epithelium process are the two biggest challenges for traditional corneal materials. These processes are related to corneal curvature, which is an important factor in determination of the corneal healing process and epithelial behavior during corneal damage. In this study, smooth 3D-printed convex corneal implants based on gelatin methacrylate and collagen are generated. As epithelium distribution and adhesion vary in different regions of the natural cornea, this work separates the surfaces into four regions and studies how cells sense topological cues on curvature. It is found that rabbit corneal epithelial cells (RCECs) seeded on steeper slope gradient surfaces on convex structures result in more aligned cell organization and tighter cell-substrate adhesion, which can also be verified through finite element simulation and signaling pathway analysis. In vivo transplantation of convex implants result in a better fit with adjacent tissue and stronger cell adhesion than flat implants, thereby accelerating corneal epithelialization and promoting collagen fibers and neural regeneration within 180 days. Taken together, printed convex corneal implants that facilitate corneal regeneration may offer a translational strategy for the treatment of corneal damage.

Corneal wound healing and nerve regeneration by novel ophthalmic formulations based on cross-linked sodium hyaluronate, taurine, vitamin B6, and vitamin B12.

Introduction: To evaluate the pharmacological profile of ocular formulations based on cross-linked sodium hyaluronate (CL-SH), taurine (Tau), vitamin B6 (Vit B6) and vitamin B12 (Vit B12) using in vitro and in vivo paradigms. Methods: Rabbit corneal epithelial cells were used to assess wound healing and reactive oxygen species (ROS) formation by scratch assay and oxidative stress (0.3mM H2O2; 30min), respectively with or without ocular formulations exposure. In vivo studies were carried out on albino rabbits to evaluate corneal nerve regeneration and corneal wound healing with or without treatment with six different formulations. Animals were anesthetized, the corneal epithelium was removed, and formulations were topically administered (30μL/eye; 3 times/day for 6days). Slit-lamp observation was carried out at different time points. After 6days the animals were killed, and corneas were collected to evaluate corneal re-innervation by immunohistochemistry of selective neuronal marker β-III tubulin. Results: Formulations containing the concentrations 0.16% or 0.32% of cross-linked sodium hyaluronate, taurine, vitamin B6 and vitamin B12 accelerated corneal wound healing. Cells exposed to H2O2 led to significant (p < 0.05) increase of reactive oxygen species concentration that was significantly (p < 0.05) counteract by formulations containing cross-linked sodium hyaluronate (0.32%) and taurine with or without vitamins. The extent of re-innervation, in terms of β-III tubulin staining, was 5-fold greater (p < 0.01) in the eye of rabbits treated with formulation containing 0.32% cross-linked sodium hyaluronate, taurine, vitamins (RenerviX®) compared with the control group (no treatment). Furthermore, re-innervation elicited by RenerviX® was significantly greater (p < 0.01) compared with the group treated with the formulation containing 0.32% cross-linked sodium hyaluronate and taurine without vitamins, and with the group treated with the formulation containing 0.5% linear sodium hyaluronate (SH), taurine, and vitamin B12, respectively. Discussion: In conclusion, among the formulations tested, the new ophthalmic gel RenerviX® was able to contrast oxidative stress, to accelerate corneal re-epithelization and to promote nerve regeneration.

Thrombospondin-1 induction and VEGF reduction by proteasome inhibition

The present study focuses on investigating the expression of thrombospondin-1 (TSP-1), a natural inhibitor of neovascularization. Immunofluorescent staining was used to detect the expression of TSP-1 in rabbit corneal tissue with vascularization induced by limbectomy. TSP-1 was detected in healthy and Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) grafted rabbit corneas. TSP-1 was not detected in diseased corneas. Rabbit and human primary oral mucosal and corneal epithelial cells were cultured and treated with proteasome inhibitor (PI) in vitro. Changes in the expression of TSP-1, HIF-1 alpha and 2 alpha, VEGF-A, and VEGF receptor were analyzed by Western blotting. Neovascularization developed in rabbits’ corneas as early as 1 month after limbectomy and was stable for at least 3 months. HIF-1 alpha and VEGF-A expression was reduced in CAOMECS grafted corneas, as compared to sham corneas. While TSP-1 expression was decreased in injured corneas, it was expressed in CAOMECS grafted corneas, but still less expressed compared to healthy corneas. PI treatment, of human oral mucosal and corneal epithelial cells increased TSP-1 expression and reduced VEGF-A expression. The results showed that TSP-1 expression was lost in injured corneal surface and that CAOMECS grafting restored TSP-1 expression to certain extent. Proteasome inhibition treatment increased TSP-1 and decreased VEGF-A expression in human oral mucosal and corneal epithelial cells. The result suggests that corneal neovascularization could be managed with the inhibition of the proteasome after CAOMECS grafting and increase corneal transparency.

Nerve regeneration and corneal wound healing by ophthalmic formulations based on sodium hyaluronate, taurine, vitamin B6 and vitamin B12

AbstractPurpose: To evaluate the pharmacological profile of ocular formulations based on cross‐linked sodium hyaluronate (CL‐SH), taurine (Tau), Vit B6 and Vit B12 using in vitro and in vivo paradigms.Methods: Rabbit corneal epithelial cells (SIRC) were used to assess wound healing and ROS formation by scratch assay and oxidative stress (0.3 mM H2O2; 30 min), respectively w/ or w/o ocular formulations exposure. In vivo studies were carried out on albino rabbits to evaluate corneal nerve regeneration and corneal wound healing with or without treatment with six different formulations. Animals were anaesthetised, the corneal epithelium was removed, and formulations were topically administered (30 μl/eye; 3 times/day for 6 days). Slit‐lamp observation was carried out at the following day: T0, T2 and T5. After 6 days the animals were killed, and corneas were collected to evaluate corneal re‐innervation by immunohistochemistry of selective neuronal marker β‐III tubulin.Results: Formulations containing the concentrations 0.16% or 0.32% of CL‐SH, Tau, Vit B6 and B12 accelerated corneal wound healing. Cells exposed to H2O2 led to significant (p &lt; 0.001) increase of ROS concentration that was significantly (p &lt; 0.001) counteract by formulations containing CL‐SH (0.32%) and Tau w/ or w/o Vit B6 and B12. The extent of re‐innervation, in terms of β‐III tubulin staining, was 5‐fold greater (p &lt; 0.01) in the eye of rabbits treated with formulation containing 0.32% CL‐SH, Tau, Vit B6 and Vit B12 (RenerviX®) compared with the control group (no treatment). Furthermore, re‐innervation with RenerviX® group was significantly greater (p &lt; 0.01) also compared with the groups treated with the formulation containing 0.32% CL‐SH and Tau w/o vitamins and with the formulation containing 0.5% linear SH, Tau, and Vit B12.Conclusions: Among all formulations tested, the new ophthalmic gel RenerviX® was able to contrast oxidative stress, to accelerate corneal re‐epithelization and to promote nerve regeneration.

Topical sevoflurane corneal toxicity: in vitro study

AbstractPurpose: The purpose of the study is to investigate the cytotoxic effect of sevoflurane (SF) and povidone‐iodine (PI) on cultured rabbit corneal epithelial cells in vitro.Methods: We evaluated the cytotoxic effect of a 5 min exposition of SF, PI and balanced saline solution (BSS) using an in vitro rabbit corneal cell culture model (Short‐Time Exposure Test Method). In order to quantify cytotoxicity MTT assay was employed. Repeated exposition of SF was evaluated as well as cell viability after different recovery times (30 min, 60 min and 1 h). Statistical results were obtained with Kruskal‐Wallis test and Conover post‐hoc test.Results: The median of cell viability of SF was 85.29%, PI 0.52% and BSS 97.29%, with statistically significant differences between them (p = 0.005). After a 5 minute recovery, the median of cell viability for SF was 87.77%, 74.48% after 30 minutes and 76.66% after 60 minutes. After a double exposition it was 70.59%, with no statistically significant differences between single and double exposition (p = 0.18).Conclusions: No corneal toxicity of SF was observed, in contrast with PI. Cellular damage did not change with longer recovery time or double exposition to SF.ReferencesSevoflurane (SF) has been proved recently to have analgesic/antimicrobial effects. (1, 2) We believe of interest to study the effect of SF in the ocular surface. As there is no literature about this topic, in vitro studies are required before in vivo tests.1. Chu CC, Wu SZ, Su WL, Shieh JP, Kao CH, Ho ST, et al. Subcutaneous injection of inhaled anaesthetics produces cutaneous analgesia. Can J Anesth Can Anesth. 2008;55(5):290–4.2. Manuel Gerónimo Pardo, María Martínez Serrano, Ángel Martínez Monsalve, Juan Luis Rueda Martínez. Usos Alternativos del Sevoflurano: Efecto Analgésico Tópico. Rev Electrónica AnestesiaR [Internet]. 2012 [cited 2019 Apr 15];4(5). Available from: http://revistaanestesiar.org/index.php/rear/article/view/373

The Therapeutic Effects of a PEDF-Derived Short Peptide on Murine Experimental Dry Eye Involves Suppression of MMP-9 and Inflammation

PurposeTo evaluate the efficacy of a pigment epithelium-derived factor (PEDF)-derived short peptide 29-mer, on the treatment and prevention of experimental dry eye (EDE).MethodsC57BL/6 mice were housed in a low humidity controlled environment chamber for 14 days to induce EDE. The 29-mer was administered topically to their eyes, for treatment or dosing, from the point of housing in the controlled environment chamber. The efficacy of the 29-mer on EDE was evaluated in terms of corneal epithelial integrity, tear secretion, and the density of conjunctival goblet cells. PEDF and inflammatory factors, including tumor necrosis factor-α, IL-1β, IL-6, monocyte chemotactic protein (MCP)-1, matrix metalloproteinase-9, and macrophage infiltration, were examined by real-time polymerase chain reaction, Western blotting, and immunostaining. The involvement of the PEDF receptor/PNPLA2 on the 29-mer effects was evaluated by a specific inhibitor, atglistatin. Rabbit corneal epithelial cells were exposed to hyperosmotic medium to induce inflammatory responses.ResultsThe levels of PEDF protein increased in the corneal epithelium of EDE, compared with the nonstressed mice. The 29-mer showed a therapeutic effect on EDE and prevented the development of EDE, accompanied by amelioration of the inflammatory factors. The 29-mer effects of inflammatory relief were dramatically reversed by atglistatin. The 29-mer also suppressed the expression of matrix metalloproteinase-9 and proinflammatory cytokines in rabbit corneal epithelial cells induced by hyperosmolarity.ConclusionsThrough this animal study, we provide a proof of concept of the anti-inflammatory domain of PEDF having potential to treat dry eye disease.Translational RelevanceThis study shows the 29-mer has novel potential as an ophthalmic drop treatment for dry eye disease.

Effect of mechanical stretching and substrate stiffness on the morphology, cytoskeleton and nuclear shape of corneal endothelial cells

Due to the limited capacity of corneal endothelial cells (CECs) division, corneal endothelial diseases have become a great challenge. The cornea is subjected to various mechanical stimuli in vivo, which may have a positive or negative influence. Thus, it is significant to gain an insight into the mechanism of mechanobiology of CECs for seeking more possible treatment. The purpose of this study was to determine the impacts of mechanical stretch and substrate stiffness on the morphology and fundamental cell behavior of CECs. Rabbit corneal endothelial cells (RCECs) were subjected to a 5% mechanical stretch or cultured on substrates of different stiffness. The impacts of mechanical stimulus on cell area, aspect ratio, circularity, cell density, nuclear shape, cytoskeleton, and cell viability were investigated. The expressions of the corneal endothelium-related markers ZO-1 and Na+/K+-ATPase were also evaluated by confocal immunofluorescence microscopy in the stiffness group. Our results suggested that mechanical stretch promoted the rearrangement of the cytoskeleton while decreasing the cell circularity, nuclear area, and cell density as well as cell viability. RCECs cultured on 10 ​kPa substrates, which was close to the physiological stiffness of rabbit Descemet's membrane (DM), showed better cell morphology, more stable actin cytoskeleton assembly, and more robust expression of the functional marker compared with other softer or stiffer substrates. In summary, mechanical stretch and substrate stiffness have profound influences on the morphology and function of CECs, which may have implications for the understanding and possible treatment of corneal endothelial diseases.

Therapeutic effect of amniotic membrane-fibrin sealant cement on severe ocular surface alkali burn in rabbits

Objective To prepare a biologically active amniotic membrane powder and explore its preservation conditions, and to evaluate the efficacy of the amniotic membrane (AM)-fibrin sealant (FS) cement made from the amniotic powder on the rabbit severe ocular surface alkali burn model. Methods Experimental research. Fresh AM was air-dried, cooled with liquid nitrogen, ground into amniotic powder and sterilized by radiation. The expression of transformed growth factor, nerve growth factor receptor (NGFR), hepatocyte growth factor (HGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) after preparation and 10, 20 and 30 days after storage at room temperature, 4 ℃ and -20 ℃ was tested and compared with that in the fresh AM. The AM-FS cement containing different concentrations of amniotic powder and no amniotic powder was diluted. Rabbit corneal epithelial cells were cultured for 72 hours. The effects of different concentrations of amniotic powder on epithelial cell growth were observed by light microscopy, and the amniotic powder concentration with the largest absorbance value at 450 nm was selected for subsequent animal experiments. Thirty-two right eyes of 32 rabbits as the severe ocular surface alkali burn model were divided using the random counting method into the AM-FS cement group, fresh AM transplantation group, FS group and antibiotic control group (8 rabbits each group) and given different interventions. After weekly observation of corneal repair, hematoxylin and eosin staining, immunohistochemical staining of monocyte chemotaxis protein 1 (MCP-1)and vascular endothelial growth factor (VEGF) were performed and detected by light microscopy at 28 days. The logFC values of the growth factor or receptor expression difference ratio were corrected by BH; the data were analyzed by t-test and analysis of variance. Results: The expression of TGF in the amniotic membrane powder compared with the fresh amniotic membrane group (logFC=-0.11), and the expression of NGFR (HGF, EGF, bFGF) was higher than that of the fresh amniotic membrane group (logFC=-2.07, 0.72, 0.46, 2.62; P<0.05); the expression of HGF, bFGF and EGF in amniotic membrane powder stored for 10 days and 20 days were no lower than fresh amniotic membrane; at 30 days, the expression of growth factors or receptors except HGF and bFGF were decreased, and HGF, bFGF and EGF were no less than 4 ℃ and -20 ℃.The maximum A value was obtained for 0.25 mg/ml of the amniotic membrane powder after 72 hours of the CEC culture 0.98±0.05. The corneal recovery was better in the AM-FS and fresh amniotic membrane transplant groups, with corneal turbidity scores of 3.75±0.46 and 3.50±0.46, respectively, on 28 days, lower than antibiotics (4.29±0.45) (t=2.480, 3.629; P=0.019, 0.001). The corneal neovascular area in the antibiotic control group was compared with the other three groups (t=4.040, 4.339, 2.820; all P<0.001); the corneal neovascular area in the AM-FS group was (9.88±0.20) and (18.96±0.18) mm2 at 7 and 28 days. The corneal neovascularization area at 7 and 28 days in the fresh AM group [(9.54±0.22) and (18.08±0.96) mm2] was smaller than the AM-FS group (t=3.085, 3.017, P=0.005, 0.005). Despite the tiny statistical difference (0.34, 0.88), there was no clinical difference. Hematoxylin and eosin staining showed corneal structures were intact in the AM-FS and fresh AM groups, the epithelial arrangement became normal, and the corneal healing was superior to the FS and antibiotic control groups. Immunohistochemistry showed that the positive expression of VEGF in the fresh AM group was weaker than that in the remaining three groups. MCP-1 was expressed to a similar extent in the AM-FS and fresh AM groups. Conclusions: The active cytokine had high expression and stable properties at room temperature. The AM-FS cement containing 0.25 mg/ml amniotic powder can promote the repair of corneal epithelium, reduce inflammatory reaction and corneal neovascularization after alkali burning in rabbit eyes.

Temperature effects on the disappearance and reappearance of corneal-endothelium primary cilia.

To elucidate the specific functions of the primary cilia in corneal endothelial cells (CECs) by investigating the histological changes of corneal endothelium exposed at low temperature. Experimental study. This study involved corneas freshly obtained from Japanese white rabbits preserved in Optisol™-GS (Bausch & Lomb) corneal storage medium at 4°C for 0, 1, and 7days. Corneas preserved for 7days were also incubated at 37°C in culture media for an additional 2days. A rabbit CEC line was also preserved in Optisol™-GS at 4°C for 0 and 1day. The corneal endothelium specimens and CECs were then assessed by immunostaining and scanning electron-microscopy (SEM). Immediately post isolation, the CECs of the specimens showed positive immunostaining for primary cilia (i.e., approximately 20%) via anti-acetylated alpha Tubulin antibody and SEM observation. Primary cilia were found to have attenuated/disappeared on the corneal endothelium specimens preserved for 1 or 7days at 4°C. After an additional 2-day incubation at 37°C, primary cilia reappeared on the corneal endothelium specimens (approximately 20%). The disappearance of cilia during the preservation period was also observed in the immortalized CECs. The findings in this study using rabbit corneas indicate that the primary cilia of corneal endothelium preserved at low temperature disappeared, then reappeared after returning to body temperature, suggesting that temperature has a direct effect on the primary cilia of corneal endothelium.

Lysozyme Protects Against Severe Acute Respiratory Syndrome Coronavirus 2 Infection and Inflammation in Human Corneal Epithelial Cells.

PurposeThe purpose of this study was to investigate the effects of lysozyme, an antimicrobial enzyme found in tears that protects the eye against pathogens, on pseudotyped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through corneal epithelial cells.MethodsThe expression of the angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease (TMPRSS2) in human corneal epithelial cells (HCECs) was measured by RT-PCR and Western blotting. The altered expression of the pro-inflammatory molecules induced by spike protein and lysozyme was analyzed by RT-PCR. Cell toxicity was tested by CCK8 assay. The cell entry of SAR-CoV-2 in HCECs and primary rabbit corneal epithelial cells (RbCECs) was detected by luciferase assay.ResultsACE2 and TMPRSS2 were highly expressed in HCECs. The spike proteins of SARS-CoV-2 stimulated a robust inflammatory response in HCECs, characterized by increased secretion of pro-inflammatory molecules, including IL-6, TNF-α, iNOS, and MCP-1, and pretreatment with lysozyme in HCECs markedly decreased the production of proinflammatory molecules induced by spike proteins. In addition, the inflammatory cytokine TNF-α enhanced the entry of SARS-CoV-2 into HCECs, which can be mitigated by pretreatment with lysozyme.ConclusionsIn this study, we analyzed the susceptibility of human corneal epithelial cells to SARS-CoV-2 infection and suggested the protective effects of lysozyme on SARS-CoV-2 infection.

Fluctuations in Corneal Endothelial LAP2 Expression Levels Correlate with Passage Dependent Declines in Their Cell Proliferative Activity.

The corneal endothelium is the inner corneal mono-layered epithelium, fundamental for preserving corneal hydration and transparency. However, molecular mechanisms that regulate corneal endothelial cells (CEnCs), in particular regarding their proliferative capacity, have been only partially elucidated. CEnCs are quiescent in vivo and they easily undergo endothelial to mesenchymal transition (EnMT) in vitro. This study aims to analyze CEnCs behavior and expression in vitro, either in sub-confluent growing (S) or confluent (C) CEnCs cultures. Primary rabbit and human CEnCs were cultured and used for RT-PCR, immunofluorescence or western blot analysis. These methods allowed identifying a novel molecular marker, LAP2, that is upregulated in S while downregulated in C human or rabbit CEnCs. Those results were observed for several subsequent passages in culture and this, together with the correlation between ki67 and LAP2 expression, suggested LAP2 as a novel possible indicator for culture ageing. Finally, treatment with FGF and TGFβ in rCEnCs highlighted how LAP2 can vary as the cells regulate their proliferative state. In conclusion, we have identified a novel marker for CEnCs, LAP2, that regulates its expression depending on the cells sub/confluent state and that correlates with CEnCs proliferation.