Observation of structural and vascular features of retina and choroid in myopia using ultra-widefield SS-OCTA.

BACKGROUND: To find the relationship between the changes of retinal and choriodal structure/ vascular densities (VD) and the myopia progress. METHODS: 126 eyes of 126 age-matched young participants were divided into three groups: Emmetropia and Low Myopia (EaLM) (33 eyes), Moderate Myopia (MM) (39 eyes), and High Myopia (HM) (54 eyes). Fundus images measuring 12 × 12 mm were captured using ultra-widefield swept-source optical coherence tomography angiography (SS-OCTA). Each image was uniformly divided into nine regions: supra-temporal (ST), temporal (T), infra-temporal (IT), superior (S), central macular area (C), inferior (I), supra-nasal (SN), nasal (N), and infra-nasal (IN). Various structural parameters, including inner retina thickness (IRT), outer retina thickness (ORT), and choroid thickness (CT), were assessed, and the VD of the superficial capillary plexus (SCP), deep capillary plexus (DCP), choriocapillaries (CC), and choroid vessels (ChdV) were quantified. RESULTS: CT in upper fundus exhibited a significant reduction from EaLM to MM. Additionally, ORT (ST, S. SN, C, N, IT, I, IN), CT (ST, S, SN, T, C, N, IT, I, IN) and VDs of SCP (ST, S, C, I, IN), DCP (ST, S, T, C, I) and ChdV (T, N, I, IN) were statistically diminished in EaLM compared to HM. Furthermore, IRT (N), ORT (N, IN), CT (S, SN, T, C, IT, I) and VDs of SCP (I, IN) and DCP (I) exhibited significant decreases as MM progressed towards HM. Intriguingly, there was a notable increase in the VD of CC (ST, S, T, C, N) as myopia progressed from MM to HM. CONCLUSION: Significant changes in retinal and choroid structure and vascular density occur as moderate myopia advances to high myopia. Efforts to curb myopia progression to this stage are essential, as the failure to do so may lead to the development of corresponding retinopathy.

The Neuropeptide α-Melanocyte-Stimulating Hormone Prevents Persistent Corneal Edema following Injury.

Corneal endothelial cells (CEnCs) regulate corneal hydration and maintain tissue transparency through their barrier and pump function. However, these cells exhibit limited regenerative capacity following injury. Currently, corneal transplantation is the only established therapy for restoring endothelial function, and there are no pharmacologic interventions available for restoring endothelial function. This study investigated the efficacy of the neuropeptide α-melanocyte-stimulating hormone (α-MSH) in promoting endothelial regeneration during the critical window between ocular injury and the onset of endothelial decompensation using an established murine model of injury using transcorneal freezing. Local administration of α-MSH following injury prevented corneal edema and opacity, reduced leukocyte infiltration, and limited CEnC apoptosis while promoting their proliferation. These results suggest that α-MSH has a proregenerative and cytoprotective function on CEnCs and shows promise as a therapy for the prevention and management of corneal endothelial dysfunction.

The Autoimmune Rheumatic Disease Related Dry Eye and Its Association with Retinopathy.

Dry eye disease is a chronic disease of the ocular surface characterized by abnormal tear film composition, tear film instability, and ocular surface inflammation, affecting 5% to 50% of the population worldwide. Autoimmune rheumatic diseases (ARDs) are systemic disorders with multi-organ involvement, including the eye, and play a significant role in dry eye. To date, most studies have focused on Sjögren's syndrome (one of the ARDs) since it manifests as two of the most common symptoms-dry eyes and a dry mouth-and attracts physicians to explore the relationship between dry eye and ARDs. Many patients complained of dry eye related symptoms before they were diagnosed with ARDs, and ocular surface malaise is a sensitive indicator of the severity of ARDs. In addition, ARD related dry eye is also associated with some retinal diseases directly or indirectly, which are described in this review. This review also summarizes the incidence, epidemiological characteristics, pathogenesis, and accompanying ocular lesions of ARD's related dry eye, emphasizing the potential role of dry eye in recognition and monitoring among ARDs patients.

Corrigendum: Therapeutic potential of topical administration of acriflavine against hypoxia-inducible factors for corneal fibrosis.

[This corrects the article DOI: 10.3389/fphar.2022.996635.].

Perfluorodecalin-based oxygenated emulsion as a topical treatment for chemical burn to the eye.

Chemical injuries to the eye are emergencies with limited acute treatment options other than prompt irrigation and can cause permanent vision loss. We developed a perfluorodecalin-based supersaturated oxygen emulsion (SSOE) to topically deliver high concentration of oxygen to the eye. SSOE is manufactured in hyperbaric conditions and stored in a ready-to-use canister. Upon dispensation, SSOE rapidly raises partial oxygen pressure 3 times over atmospheric level. SSOE is biocompatible with human corneal cells and safe on mouse eyes in vivo. A single topical application of SSOE to the eye after alkali injury significantly promotes corneal epithelial wound healing, decreases anterior chamber exudation, and reduces optical opacity and cataract formation in mice. SSOE treatment reduces intraocular hypoxia, cell death, leukocyte infiltration, production of inflammatory mediators, and hypoxia-inducible factor 1-alpha signaling, thus hastening recovery of normal tissue integrity during the wound healing process. Here, we show that SSOE is an effective topical therapeutic in the acute treatment of ocular chemical injuries.

Therapeutic potential of topical administration of acriflavine against hypoxia-inducible factors for corneal fibrosis.

Transdifferentiation of keratocytes into fibroblasts or further into myofibroblasts, which produced denser and more disorganized extracellular matrix, is the major cause of corneal fibrosis and scarring, leading to corneal blindness. TGF-ß1 is the critical cytokine for the myofibroblast's transdifferentiation and survival. Hypoxia Inducible Factor (HIF) was found to play an important role in promoting fibrosis in lung, kidney, and dermal tissues recently. Our preliminary study demonstrated that topical administration of the acriflavine (ACF), a drug inhibiting HIF dimerization, delayed corneal opacity and neovascularization after the alkali burn. To know whether ACF could prevent corneal fibrosis and improve corneal transparency, we created a mouse mechanical corneal injury model and found that topical administration of ACF significantly inhibited corneal fibrosis at day 14 post-injury. The reduction of myofibroblast marker α-SMA, and fibronectin, one of the disorganized extracellular matrix molecules, in the corneal stroma were confirmed by the examination of immunohistochemistry and real-time PCR. Furthermore, the ACF inhibited the expression of α-SMA and fibronectin in both TGF-ß1 stimulated or unstimulated fibroblasts in vitro. This effect was based on the inhibition of HIF signal pathways since the levels of the HIF-1α downstream genes including Slc2a1, Bnip3 and VEGFA were downregulated. To our knowledge, this is the first time to implicate that HIFs might be a new treatment target for controlling corneal fibrosis in mechanical corneal injuries.

Therapeutic efficacy of topical blockade of substance P in experimental allergic red eye.

PURPOSE: Allergic conjunctivitis is the most common cause leading to ocular redness (OR). Herein, using an animal model of allergic OR, we evaluated the therapeutic efficacy of topical blockade of substance P (SP) in treating red eye. METHODS: Allergic OR was induced in guinea pigs with topical histamine. Ocular SP was blocked using a specific SP receptor (neurokinin-1 receptor, NK1R) antagonist, L-703,606, via topical application 10 min before or 10 min after histamine instillation. Animal eyes were examined and a series of images were taken for up to 60 min post-OR induction. The severity of redness was analyzed using the quantitative ocular redness index (ORI). At the end of clinical examination, conjunctival tissues were collected for histological examination of conjunctival blood vessels and infiltrating eosinophils and neutrophils. In addition, SP concentration was quantified in the tear fluid and expression levels of inflammatory cytokines were assessed in the conjunctival tissues. RESULTS: Topical histamine application successfully induced red eye, evidenced by the significantly increased ORI during the observation period, with peak values at 10 min, along with significantly increased levels of SP in the tears. Topical treatment with L-703,606, either before histamine application or at the time of peak ORI, effectively reduced ORI and suppressed conjunctival blood vessel dilation, along with decreased eosinophil and neutrophil infiltration, and inflammatory cytokine expression in the conjunctiva, as well as reduced SP levels in the tears. CONCLUSIONS: Topical blockade of SP effectively prevents and treats allergy-related ocular redness by suppressing blood vessel dilation and allergic inflammation.

Promotion of corneal angiogenesis by sensory neuron-derived calcitonin gene-related peptide.

The normal cornea has no blood vessels but has abundant innervation. There is emerging evidence that sensory nerves, originated from the trigeminal ganglion (TG) neurons, play a key role in corneal angiogenesis. In the current study, we examined the role of TG sensory neuron-derived calcitonin gene-related peptide (CGRP) in promoting corneal neovascularization (CNV). We found that CGRP was expressed in the TG and cultured TG neurons. In the cornea, minimal CGRP mRNA was detected and CGRP immunohistochemical staining was exclusively co-localized with corneal nerves, suggesting corneal nerves are likely the source of CGRP in the cornea. In response to intrastromal suture placement and neovascularization in the cornea, CGRP expression was increased in the TG. In addition, we showed that CGRP was potently pro-angiogenic, leading to vascular endothelial cell (VEC) proliferation, migration, and tube formation in vitro and corneal hemangiogenesis and lymphangiogenesis in vivo. In a co-culture system of TG neurons and VEC, blocking CGRP signaling in the conditioned media of TG neurons led to decreased VEC migration and tube formation. More importantly, subconjunctival injection of a CGRP antagonist CGRP8-37 reduced suture-induced corneal hemangiogenesis and lymphangiogenesis in vivo. Taken together, our data suggest that TG sensory neuron and corneal nerve-derived CGRP promotes corneal angiogenesis.

Efficacy of Voriconazole Corneal Intrastromal Injection for the Treatment of Fungal Keratitis.

PURPOSE: To evaluate efficacy and safety of novel tricyclic corneal stroma injection (TCSI) voriconazole for the treatment of fungal keratitis. METHODS: This retrospective cohort study included data of 57 patients (57 eyes) with fungal keratitis. The TCSI group consisted of 27 patients (27 eyes) who were injected voriconazole once via TCSI procedure within one week after enrollment, in addition to conventional antifungal treatment. The control group consisted of 30 patients (30 eyes) who were treated using conventional antifungal treatment modalities. The outcome measures consist of the 3-week and 3-month best-corrected visual acuity (BCVA) values and size of infiltrate or scar, time to re-epithelialization, corneal perforation rate and/or therapeutic penetrating keratoplasty (TPK) requirement, the preoperative and post-TCSI corneal endothelial cell density (ECD), and the intraocular pressure (IOP) of the treated eye and the respective contralateral eye. RESULTS: There were no significant differences in the baseline demographic and clinical characteristics between the two groups. 3 weeks and 3 months after enrollment, the TCSI group exhibited an increase in visual acuity (P < 0.05), and there was no significant difference in the size of infiltrate or scar between two groups (P > 0.05). Time to re-epithelialization was shorter in the TCSI group than in the control group (P < 0.05). There was no statistically significant difference between corneal ECD on the day before and 7 days after TCSI and the IOP of treated and contralateral healthy eyes on the day before and 1 day, 3 days, 7 days, and 1 month after TCSI (P > 0.05). The difference in the risk of perforation and/or TPK requirement was not statistically significant between two groups (P > 0.05). CONCLUSION: Localized injection of voriconazole using TCSI may be a minimally invasive, safe, and effective adjuvant treatment modality for fungal keratitis.

Case Report: Associated Ocular Adverse Reactions With Inactivated COVID-19 Vaccine in China.

The vaccine is still the best clinical measure for effective prevention and control of coronavirus disease 2019 (COVID-19). The vaccine-associated ocular adverse reactions should be noted in detail among the medical community. We reported twelve eyes of 9 patients presented at the Department of Ophthalmology, Qilu Hospital of Shandong University from March to August 2021 with ocular complaints following COVID-19 vaccination. The main inclusion criterion was the development of ocular symptoms within 14 days after receiving a dose of an inactivated COVID-19 vaccine. The mean (SD) age was 44.7 ± 16.5 years (range, 19-78 years), among which seven (77.8%) cases were women. The mean time of ocular adverse events was 7.1 days (range, 1-14 days) after receiving the inactivated COVID-19 vaccine. One patient was diagnosed with choroiditis, 1 with uveitis, 4 with keratitis, 1 with scleritis, 1 with acute retinal necrosis, and 1 with iridocyclitis. Although the causal relationship between vaccines and ocular adverse events cannot be established from this case series report, physicians should pay attention to the ocular adverse reactions following the COVID-19 vaccine administration.

Hypoxia adaptation in the cornea: Current animal models and underlying mechanisms.

The cornea is an avascular, transparent tissue that is essential for visual function. Any disturbance to the corneal transparency will result in a severe vision loss. Due to the avascular nature, the cornea acquires most of the oxygen supply directly or indirectly from the atmosphere. Corneal tissue hypoxia has been noticed to influence the structure and function of the cornea for decades. The etiology of hypoxia of the cornea is distinct from the rest of the body, mainly due to the separation of cornea from the atmosphere, such as prolonged contact lens wearing or closed eyes. Corneal hypoxia can also be found in corneal inflammation and injury when a higher oxygen requirement exceeds the oxygen supply. Systemic hypoxic state during lung diseases or high altitude also leads to corneal hypoxia when a second oxygen consumption route from aqueous humor gets blocked. Hypoxia affects the cornea in multiple aspects, including disturbance of the epithelium barrier function, corneal edema due to endothelial dysfunction and metabolism changes in the stroma, and thinning of corneal stroma. Cornea has also evolved mechanisms to adapt to the hypoxic state initiated by the activation of hypoxia inducible factor (HIF). The aim of this review is to introduce the pathology of cornea under hypoxia and the mechanism of hypoxia adaptation, to discuss the current animal models used in this field, and future research directions.

SPARC promotes self-renewal of limbal epithelial stem cells and ocular surface restoration through JNK and p38-MAPK signaling pathways.

The purpose of this study was to investigate the effects of secreted protein acidic and rich in cysteine (SPARC) on the maintenance of limbal epithelial stem cell (LESC) stemness and restoration of ocular surface. To determine the suitable concentration of SPARC for LESC culture, the marker expression, mitogenic effect, and holoclone-forming capacity of LESCs treated with different concentrations of SPARC were analyzed. To investigate the mechanism of SPARC's action on the preservation of LESCs stemness, the phosphorylation of related signaling pathways was evaluated by Western blotting. A corneal wound model was established to verify the function of SPARC in ocular surface repair. Consecutive subculturing, colony-forming efficiency, immunofluorescence, and 5-ethynyl-2-deoxyuridine incorporation assays indicated that 1 µg/mL SPARC was a suitable concentration to stimulate LESC proliferation and preserve their proliferative potential. Compared with a control group, 1 µg/mL SPARC effectively increased the expression of ABCG-2, Bmi-1, and Ki67, while decreasing that of CK3/12. The mitogenic effect of SPARC on LESCs was found to be mediated by the phosphorylation of c-Jun N-terminal kinase (JNK) and p38-MAPK signaling pathways, whereas the inhibitors of JNK and p38 MAPK reduced the marker expression and mitogenic capacity of LESCs. In a corneal injury model, SPARC facilitated corneal epithelial wound healing and promoted the proliferation of p63α-positive cells both in the limbus and in the epithelial healing front. SPARC promotes proliferation while suppressing spontaneous differentiation of LESCs through JNK and p38-MAPK signaling pathways, suggesting that SPARC is a promising factor for the improvement of LESCs culture in vitro and in vivo.

Construction of a full-thickness human corneal substitute from anterior acellular porcine corneal matrix and human corneal cells.

AIM: To construct functional human full-thickness corneal replacements. METHODS: Acellular porcine corneal matrix (APCM) was developed from porcine cornea by decellulariztion. The biomechanical properties of anterior-APCM (AAPCM) and posterior-APCM (PAPCM) were checked using uniaxial tensile testing. Human corneal cells were obtained by cell culture. Suspending ring was designed by deformation of an acupuncture needle. MTT cytotoxicity assay was used to check the cytotoxicity of suspending ring soaking solutions. A new three-dimensional organ culture system was established by combination of suspending ring, 48-well plate and medium together. A human full-thickness corneal substitute was constructed from human corneal cells with AAPCM in an organ coculture system. Biochemical marker expression of the construct was measured by immunofluorescent staining and morphological structures were observed using scanning electron microscopy. Pump function and biophysical properties were examined by penetrating keratoplasty and follow-up clinical observations. RESULTS: There were no cells in the AAPCM or PAPCM, whereas collagen fibers, Bowman's membrane, and Descemet's membrane were retained. The biomechanical property of AAPCM was better than PAPCM. Human corneal cells grew better on the AAPCM than on the PAPCM. There was no cytotoxicity for the suspending ring soaking solutions. For the constructed full-depth human corneal replacements keratocytes scattered uniformly throughout the AAPCM and expressed vimentin. The epithelial layer was located on the surface of Bowman's membrane and composed of three or four layers of epithelial cells expressing cytokeratin 3. One layer of endothelial cells covered the stromal surface of AAPCM, expressed Na+/K+ATPase and formed the endothelial layer. The construct was similar to normal human corneas, with many microvilli on the epithelial cell surface, stromal cells with a long shuttle shape, and zonula occludens on the interface of endothelial cells. The construct withstood surgical procedures during penetrating keratoplasty. The corneal transparency increased gradually and was almost completely restored 7d after surgery. CONCLUSION: AAPCM is an ideal scaffold for constructing full-thickness corneal replacement, and functional human full-thickness corneal replacements are successfully constructed using AAPCM and human corneal cells.

Differentiation of human embryonic stem cells into corneal epithelial progenitor cells under defined conditions.

The development of cell-based therapies using stem cells represents a significant breakthrough in the treatment of limbal stem cell deficiency (LSCD). The aim of this study was to develop a novel protocol to differentiate human embryonic stem cells (hESCs) into corneal epithelial progenitor cells (CEPCs), with similar features to primary cultured human limbal stem cells (LSCs), using a medium composed of DMEM/F12 and defined keratinocyte serum-free medium (KSFM) (1:1) under different carbon dioxide (CO2) levels in culture. The differentiated cells exhibited a similar morphology to limbal stem cells under 5%, 7%, and 9% CO2 and expressed the LSC markers ABCG-2 and p63; however, CK14 was only expressed in the cells cultured under 7% and 9% CO2. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis indicated that the ABCG2, p63, and CK14 levels in the 7% CO2 and 9% CO2 groups were higher than those in the 5% CO2 group and in undifferentiated hESCs (p<0.05). The highest expression of ABCG2 and p63 was exhibited in the cells cultured under 7% CO2 at day 6 of differentiation. Western blotting indicated that the ABCG2 and p63 levels were higher at day 6 than the other time points in the 7% CO2 and 9% CO2 groups. The highest protein expression of ABCG2 and p63 was identified in the 7% CO2 group. The neural cell-specific marker tubulin ß3 and the epidermal marker K1/10 were also detected in the differentiated cells via immunofluorescent staining; thus, cell sorting was performed via fluorescence-activated cell sorting (FACS), and ABCG2-positive cells were isolated as CEPCs. The sorted cells formed three to four layers of epithelioid cells by airlifting culture and expressed ABCG2, p63, CK14, and CK3. In conclusion, the novel induction system conditioned by 7% CO2 in this study may be an effective and feasible method for CEPC differentiation.

Construction of tissue-engineered full-thickness cornea substitute using limbal epithelial cell-like and corneal endothelial cell-like cells derived from human embryonic stem cells.

The aim of this study was to construct a full-thickness artificial cornea substitute in vitro by coculturing limbal epithelial cell-like (LEC-like) cells and corneal endothelial cell-like (CEC-like) cells derived from human embryonic stem cells (hESCs) on APCM scaffold. A 400 µm thickness, 11 mm diameter APCM lamella containing Bowman's membrane was prepared as the scaffold using trephine and a special apparatus made by ourselves. LEC-like cells and CEC-like cells, derived from hESCs as our previously described, were cocultured on the scaffold using a special insert of 24-well plates that enabled seeding both sides of the scaffold. Three or four layers of epithelium-like cells and a uniform monolayer of CEC-like cells could be observed by H&E staining. The thickness, endothelial cell density, and mechanical properties of the construct were similar to that of native rabbit corneas. Immunofluorescence analysis showed expression of ABCG2 and CK3 in the epithelium-like cell layers and expression of N-cadherin, ZO-1 and Na+/K + ATPase in the CEC-like cells. The corneal substitutes were well integrated within the host corneas, and the transparency increased gradually in 8-week follow-up after transplantation in the rabbits. These results suggest that the strategy we developed is feasible and effective for construction of tissue-engineered full-thickness cornea substitute with critical properties of native cornea.

Three-Dimensional Construction of a Rabbit Anterior Corneal Replacement for Lamellar Keratoplasty.

The aim of this study was to construct a rabbit anterior corneal replacement for transplantation using acellular porcine corneal matrix (APCM) and rabbit epithelial or stromal cells. APCM was prepared from fresh porcine cornea treated with 0.5% (wt./vol.) sodium dodecyl sulfate (SDS) solution. The expanded stromal cells were first injected into APCM parallel to its surface and were cultured in a shaking culture system for 7 days to obtain the stromal construct. Next, corneal epithelial cells were cultured on the stromal construct surface for another 7 days to obtain rabbit anterior corneal lamella. The construct had a phenotype similar to that of normal cornea, with high expression of cytokeratin 3 in the epithelial cell layer and vimentin in the stromal cells. More importantly, the construct integrated well with the implanted host corneal tissue, and the implant cornea maintained transparency in the 6-month follow-up, although there was a slight haze in the central corneal area. The endothelium in the surgery cornea had a similar cell density and mosaic pattern with normal cornea as shown by confocal laser corneal microscopy, and the regenerated corneal epithelial cells on the implant surface showed a similar morphology to that of natural epithelial cells. These results demonstrate that the constructed anterior corneal replacement exhibits an excellent biological property for lamellar keratoplasty and might be a possible alternative to human corneal tissue in the future.

Herpes virus entry mediator in human corneal epithelial cells modulates the production of inflammatory cytokines in response to HSV type 1 challenge.

AIMS: To investigate the role of herpes virus entry mediator (HVEM) in viral entry and inflammatory cytokine production in response to herpes simplex virus (HSV) type 1 challenge in human corneal epithelial cells. METHODS: HVEM expression in human corneal epithelial cells was determined by immunofluorescence and flow cytometry. The HSV-1 virus expressing ß-galactosidase was used to challenge corneal epithelial cells and viral entry assays were performed to ascertain HSV-1 entry into cells. Levels of cytokines TNF-α, IL-6, IFN-x03B3;, IL-12, and IL-18 and chemokines MIP-1α, MIP-1ß and MIP-2 were detected in corneal epithelial cells treated with control or HVEM siRNA in response to HSV-1 challenge. RESULTS: Human corneal epithelial cells were positive for HVEM expression and showed high susceptibility to HSV-1 entry. Silencing of HVEM did not alter viral entry dramatically. However, levels of the cytokine IFN-x03B3; and chemokines MIP-1α and MIP-1ß were measured to be higher in HVEM siRNA-treated cells than control after HSV-1 challenge. CONCLUSIONS: HVEM in human corneal epithelial cells may act to dampen the production of some cytokines and chemokines and thus it may modulate the innate immune response against HSV-1. This may provide a novel mechanism for the pathogenesis of HSV-1 infection in the cornea.

Isolation and transplantation of corneal endothelial cell-like cells derived from in-vitro-differentiated human embryonic stem cells.

The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we report the derivation of CEC-like cells from human embryonic stem cells (hESCs) through the periocular mesenchymal precursor (POMP) phase. Using the transwell coculture system of hESCs with differentiated human corneal stromal cells, we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell-conditioned medium. Within 1 week, CEC-like cells that expressed the corneal endothelium (CE) differentiation marker N-cadherin and transcription factors FoxC1 and Pitx2 were detectable. Fluorescence-activated cell sorting (FACS)-based isolation of the N-cadherin/vimentin dual-positive population enriches for CEC-like cells. The isolated CEC-like cells were labeled with carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) and seeded onto posterior acellular porcine corneal matrix lamellae to construct the CEC-like cell sheets. Pump function parameters of the CEC-like cell sheets approximated those of human donor corneas. Importantly, when the CEC-like cell sheets were transplanted into the eyes of rabbit CE dysfunction models, the corneal transparency was restored gradually. In conclusion, CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CEC biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future.

A human corneal endothelium equivalent constructed with acellular porcine corneal matrix.

BACKGROUND & OBJECTIVES: Artificial corneal endothelium equivalents can not only be used as in vitro model for biomedical research including toxicological screening of drugs and investigation of pathological corneal endothelium conditions, but also as potential sources of grafts for corneal keratoplasty. This study was aimed to demonstrate the feasibility of constructing human corneal endothelium equivalents using human corneal endothelial cells and acellular porcine corneal matrix. METHODS: Porcine corneas were decellularized with sodium dodecyl sulphate (SDS) solution. Human corneal endothelial cells B4G12 were cultured with leaching liquid extracted from the acellular porcine corneal matrix, and then cell proliferative ability was evaluated by MTT assay. B4G12 cells were transplanted to a rat corneal endothelial deficiency model to analyze their in vivo bio-safety and pump function, and then seeded and cultured on acellular porcine corneal matrix for two wk. Corneal endothelium equivalents were analyzed using HE staining, trypan blue and alizarin red S co-staining, immunofluorescence and corneal swelling assay. RESULTS: The leaching liquid from acellular porcine corneal matrix had little influence on the proliferation ability of B4G12 cells. Animal transplantation of B4G12 cells showed that these cells had similar function to the native cells without causing a detectable immunological reaction and neoplasm in vivo. These formed a monolayer covering the surface of the acellular porcine corneal matrix. Trypan blue and alizarin red S co-staining showed that B4G12 cells were alive after two wk in organ culture and cell boundaries were clearly delineated. Proper localizations of ZO-1 and Na+/K+ ATPase were detected by immunofluorescence assay. Functional experiments were conducted to show that the Na+/K+ ATPase inhibitor ouabain could block the ionic-pumping function of this protein, leading to persistent swelling of 51.7 per cent as compared to the control. INTERPRETATION & CONCLUSIONS: Our findings showed that B4G12 cells served as a good model for native corneal endothelial cells in vivo. Corneal endothelium equivalents had properties similar to those of native corneal endothelium and could serve as a good model for in vitro study of human corneal endothelium.