Human Cornea Research Articles

Time- and Stimulus-Dependent Characteristics of Innate Immune Cells in Organ-Cultured Human Corneal Tissue

Purpose: The pattern of immune cells infiltrating the corneal stroma has been extensively studied in mice, but data on human tissue have been far less elaborate. To further characterize the number and differentiation state of resident immune cells in organ-cultured human corneal tissue, we employed a comprehensive bioinformatic deconvolution (xCell) of bulk RNA-sequencing (RNA-seq) data, immunohistochemistry (IHC), and flow cytometry (FC). Methods: A transcriptome-based analysis of immune cell types in human corneal samples was performed. The results were validated by IHC, focusing on the identification of pro-inflammatory (M1) and regulatory (M2) macrophages. A protocol was established to identify these 2 different macrophage populations in human corneal tissue by means of FC. Subsequently, corneal samples in organ culture were differentially stimulated by IL-10, IL-4 & IL-13, or LPS and macrophage populations were evaluated regarding their response to these stimuli. Furthermore, cell survival was analyzed in correlation with time in organ culture. Results: xCell-based mathematical deconvolution of bulk RNA-seq data revealed the presence of CD8 T cells, Th17 cells, dendritic cells, and macrophages as the predominant immune cell types in organ-cultured human corneal tissue. Furthermore, RNA-seq allowed the detection of different macrophage marker genes in corneal samples, including PTPRC (CD45), ITGAM (CD11b), CD14, and CD74. Our RNA-seq data showed no evidence of a relevant presence of monocytes in human corneal tissue. The presence of different macrophage subtypes was confirmed by IHC. The disintegration and subsequent FC analysis of human corneal samples showed the presence of both M1 (HLA-DR⁺, CD282⁺, CD86⁺, and CD284⁺) and M2 (CD163⁺ and CD206⁺) macrophage subtypes. Furthermore, we found that the total number of macrophages in corneal samples decreased more than the total cell count with increasing tissue culture time. Treatment with IL-10 led to higher total cell counts per cornea and to an increased expression of the M2 marker CD163 (p < 0.05) while expression levels of various M1 macrophage markers were not significantly reduced by interleukin treatment. Conclusions: Regarding different macrophage populations, untreated human corneas showed more M1 than M2 macrophages. With increasing organ culture time, these macrophages decreased. In terms of cell dynamics, adding interleukins to the organ culture medium influenced the phenotype of macrophages within the cornea as detected by FC. Modifying the immunomodulatory properties of human grafts appears a promising approach to further reduce the risk of graft rejection in patients. In this context, treatment with interleukins was more effective in upregulating M2 macrophages than in suppressing M1 macrophages in corneal tissue.

Detection of free-living amoebae in domestic cats with and without naturally-acquired keratitis

Pathogenic free-living amoebae, most notably Acanthamoeba spp., are important pathogens of the human cornea. The importance of infection with free-living amoebae in cats with keratitis is currently unclear. The aim of this study was to determine the frequency of amoeba detection in corneas of cats with naturally-acquired keratitis and in the ocular surface microflora of cats without ocular disease. Clinical ophthalmic and in vivo corneal confocal microscopic examinations were performed on 60 cats with keratitis. Corneal scrapings were analyzed by amoeba culture; cytological evaluation; and Acanthamoeba, Hartmannella, and Vahlkampfia PCR assays. Following ophthalmic examination, conjunctival specimens collected from 60 cats without clinically apparent ocular disease were analyzed similarly.In one cat with ulcerative keratitis, amoeba cysts and trophozoites were detected by in vivo corneal confocal microscopy; an Acanthamoeba sp. was isolated from corneal specimens and detected by Acanthamoeba PCR assay; and suppurative corneal inflammation was present cytologically. An Acanthamoeba sp. was isolated from conjunctival specimens from one cat without clinically apparent ocular disease, but with suppurative inflammation demonstrated cytologically. Both Acanthamoeba isolates belonged to the T4 genotype. Naegleria-like amoebae were isolated in samples from two cats with keratitis and seven cats without clinical ocular disease, but amoebae were not detected by the other assays in these samples. Amoeba detection by culture was significantly (P = 0.01) associated with cytologically diagnosed corneoconjunctival inflammation. This study identified naturally-acquired Acanthamoeba keratitis in cats. Detection of Naegleria-like amoebae in samples from cats with and without keratitis is of uncertain pathological significance.

Introducing an Efficient In Vitro Cornea Mimetic Model for Testing Drug Permeability

There is a growing need for novel in vitro corneal models to replace animal-based ex vivo tests in drug permeability studies. In this study, we demonstrated a corneal mimetic that models the stromal and epithelial compartments of the human cornea. Human corneal epithelial cells (HCE-T) were grown on top of a self-supporting porcine collagen-based hydrogel. Cross-sections of the multi-layers were characterized by histological staining and immunocytochemistry of zonula oc-cludens-1 protein (ZO-1) and occludin. Furthermore, water content and bssic elastic properties of the synthetized collagen type I-based hydrogels were measured. The apparent permeability coefficient (Papp) values of a representative set of ophthalmic drugs were measured and correlated to rabbit cornea Papp values found in the literature. A multilayered structure of HCE-T cells and the expression of ZO-1 and occludin in the full thickness of the multilayer were observed. The hydrogel-based corneal model exhibited an excellent correlation to rabbit corneal permeability (r = 0.96), whereas the insert-grown HCE-T multilayer was more permeable and the correlation to the rabbit corneal permeability was lower (r = 0.89). The hydrogel-based human corneal model predicts the rabbit corneal permeability more reliably in comparison to HCE-T cells grown in inserts. This in vitro human corneal model can be successfully employed for drug permeability tests whilst avoiding ethical issues and reducing costs.

Crosslinking of the Bioplast plastic material as a means of increasing its biomechanical stability and the effectiveness of keratoplasty for corneal ulcers

Purpose:to assess the effect of ultraviolet (UVA) crosslinking of Bioplast plastic material (silicon-dried human cornea) on its biomechanical stability and effectiveness for keratoplasty in corneal ulcers. Material and methods. To determine the maximum rehydration time, 12 Bioplast samples were incubated in physiological solution (NaCl 0.9 %) for 12 hours and monitored for thickness using optical coherence tomography (OCT Spectralis, Heidelberg Engineering). After the epithelium was scarified rehydrated corneas were treated withof 0.1% riboflavin solution for 30 minutes, and after it 8 rehydrated samples (experimental group) were treated with UVA for 30minutes (wavelength 370 nm, power 3 mW/cm2) using a UV-X device system (IROC AG, Switzerland). In the control group (4 rehydrated samples), no UVA crosslinking was performed. The elastic-strength indicators of the experimental and control group samples were assessed by applying uniaxial tension using a deformation machine (Autograph AGS-H, Japan). To examine the patients (presented in clinical examples)before and after keratoplasty with Bioplast subjected to UVA crosslinking, a set of standard ophthalmic tests was used. Results.Rehydration of Bioplast was the most intensive in the first three hours of incubation, during which time tissue thickness increased uniformly by 50–55 μm/hour to 700–710 μm, while further incubation showed no significant additional effect on the fluid content in the cornea. After UVA crosslinking, the strength indices and the elastic modulus of the rehydrated Bioplast samples significantly increased in comparison with the control group. Clinical examples demonstrate the effectiveness of modified Bioplast for organ-preserving penetrating keratoplasty in case of perforatedcorneal ulcer. Conclusion. UVA crosslinking of rehydrated Bioplast increases its biomechanical stability. The data of laboratory studies and the first clinical tests demonstrate that modified Bioplast may be an effective replacement of native donor cornea in urgent keratoplasty of perforated ulcers.

Quantitative structural imaging of keratoconic corneas using polarization-resolved SHG microscopy.

The human cornea is mainly composed of collagen fibrils aligned together within stacked lamellae. This lamellar structure can be affected in pathologies such as keratoconus, which is characterized by progressive corneal thinning and local steepening. In this study, we use polarization-resolved second harmonic generation (P-SHG) microscopy to characterize 8 control and 6 keratoconic human corneas. Automated processing of P-SHG images of transverse sections provides the collagen orientation in every pixel with sub-micrometer resolution. Series of P-SHG images recorded in the most anterior region of the stroma evidence sutural lamellae inclined at 22° ± 5° to the corneal surface, but show no significant difference between control and keratoconic corneas. In contrast, series of P-SHG images acquired along the full thickness of the stroma show a loss of order in the lamellar structure of keratoconic corneas, in agreement with their defective mechanical properties. This structural difference is analyzed quantitatively by computing the entropy and the orientation index of the collagen orientation distribution and significant differences are obtained along the full thickness of the stroma. This study shows that P-SHG is an effective tool for automatic quantitative analysis of structural defects of human corneas and should be applied to other collagen-rich tissues.

SARS‑CoV‑2 spike protein‑induced host inflammatory response signature in human corneal epithelial cells.

Coronavirus disease 2019 (COVID‑19), caused by the severe acute respiratory syndrome coronavirus‑2 (SARS‑CoV‑2), led to an outbreak of viral pneumonia in December2019. The present study aimed to investigate the host inflammatory response signature‑caused by SARS‑CoV‑2 in human corneal epithelial cells (HCECs). The expression level of angiotensin‑converting enzyme 2 (ACE2) in the human cornea was determined via immunofluorescence. Invitro experiments were performed in HCECs stimulated with the SARS‑CoV‑2 spike protein. Moreover, the expression levels of ACE2, IL‑8, TNF‑α, IL‑6, gasdermin D (GSDMD) and IL‑1β in HCECs were detected using reverse transcription‑quantitative PCR and/or western blotting. It was identified that ACE2 was expressed in normal human corneal epithelium and HCECs cultured invitro. Furthermore, the expression levels of IL‑8, TNF‑α and IL‑6 in HCECs were decreased following SARS‑CoV‑2 spike protein stimulation, while the expression levels of GSDMD and IL‑1β were increased. In conclusion, the present results demonstrated that the SARS‑CoV‑2 spike protein suppressed the host inflammatory response and induced pyroptosis in HCECs. Therefore, blocking the ACE2 receptor in HCECs may reduce the infection rate of COVID‑19.

Assessing the local temperature of human cornea exposed to surface ablation by different laser refractive-surgery devices: a numerical comparative study.

To evaluate the local temperature at corneal tissue after applying single laser pulse from six commercial devices; Medilex™, Katana laser-soft, MEL90, Technolas-Teneo317, Alcon EX500, and PulzarTMZ1. The temperature distribution is simulated using finite element solution of the Penne's bio-heat transfer equation on a 3-D model of human cornea using the manufacturer's assigning parameters. The obtained results showed that the heating effect of Katana laser soft is 40% lower than MEL90 and Pulzar™ Z1, while the broad beam Medilex™ showed the minimum temperature rise especially at 248-nm laser radiation. The change in laser parameters selected for ablation has significant effect on the corneal local temperature. The broad beam-based device produces lower local corneal temperature than other flying spot types.

Analytical optimization of the cutting efficiency for generic cavitation bubbles.

A theoretical method to determine the optimum laser parameters for maximizing the cutting efficiency for different materials (in particular human cornea) is proposed. The model is simple and reduced to laser beam characteristics and cavitation properties. The model further provides a method to convert energy fluctuations during the cutting process to equivalent deviations in the cavitation bubbles. The proposed model can be used for calibration, verification and validation purposes of laser systems used for cutting processes at relatively low cost and may improve the quality of the results.

The effect of prior long-term recellularization with keratocytes of decellularized porcine corneas implanted in a rabbit anterior lamellar keratoplasty model.

Decellularized porcine corneal scaffolds are a potential alternative to human cornea for keratoplasty. Although clinical trials have reported promising results, there can be corneal haze or scar tissue. Here, we examined if recellularizing the scaffolds with human keratocytes would result in a better outcome. Scaffolds were prepared that retained little DNA (14.89 ± 5.56 ng/mg) and demonstrated a lack of cytotoxicity by in vitro. The scaffolds were recellularized using human corneal stromal cells and cultured for between 14 in serum-supplemented media followed by a further 14 days in either serum free or serum-supplemented media. All groups showed full-depth cell penetration after 14 days. When serum was present, staining for ALDH3A1 remained weak but after serum-free culture, staining was brighter and the keratocytes adopted a native dendritic morphology with an increase (p < 0.05) of keratocan, decorin, lumican and CD34 gene expression. A rabbit anterior lamellar keratoplasty model was used to compare implanting a 250 μm thick decellularized lenticule against one that had been recellularized with human stromal cells after serum-free culture. In both groups, host rabbit epithelium covered the implants, but transparency was not restored after 3 months. Post-mortem histology showed under the epithelium, a less-compact collagen layer, which appeared to be a regenerating zone with some α-SMA staining, indicating fibrotic cells. In the posterior scaffold, ALDH1A1 staining was present in all the acellular scaffold, but in only one of the recellularized lenticules. Since there was little difference between acellular and cell-seeded scaffolds in our in vivo study, future scaffold development should use acellular controls to determine if cells are necessary.

Preparation of Natural Composite Microcapsules Containing Orchid Black Currant Fragrance and its Sustained-Release Properties on Hair Bundle

In this paper, we developed an environmentally friendly microcapsules with natural materials to replace plastic microbeads used in personal care products and studied its functionality. The microcapsules with sustained release properties of orchid black currant fragrance were prepared by microemulsion method and using maltodextrin and resistant starch as wall materials. Fourier transform infrared spectroscopy (FTIR) spectra and thermogravimetric analysis (TG) indicated the fragrance was encapsulated into microcapsules successfully, and the encapsulation ratio of microcapsule particles was 38%. Scanning electron microscopy (SEM) and dynamic light scattering laser particle size analyzer (DLS) results showed the microcapsule particles were spherical, and the particle size ranged from 1.2 to 2.2 μm with the average particle size of 1.7 μm, which can stably attach to the hair and have good slow-release properties. Adding the microcapsules to the hair conditioner and treating the real hair bundle through electrostatic adsorption, the application efficacy, sustained-release and irritating effects of the microcapsules have been evaluated. The aroma retention rate of microencapsulated fragrance treated hair is much higher at 5, 7 and 21 days than that of unencapsulated fragrance treated one, and the sensory evaluation of the aroma intensity of the hair bundles treated with encapsulated fragrances vs. fragrances showed similar results to those obtained by electronic nose test. Meanwhile, the microcapsules’ irritation on corneal have been assessed by the hen's egg chorioallantoic membrane (HET-CAM) test. The test results indicated that the prepared fragrance microcapsules have no irritating effect to human cornea, and the microcapsules are safe, green and of environmental friendliness.

Comparison of the rabbit and human corneal endothelial proteomes regarding proliferative capacity

The shortage of human donor corneas has raised important concerns about engineering of corneal endothelial cells (CECs) for clinical use. However, due to the limited proliferative capacity of human CECs, driving them into proliferation and regeneration may be difficult. Unlike human CECs, rabbit CECs have a marked proliferative capacity. To clarify the potential reason for this difference, we analysed the proteomes of four human corneal endothelium samples and four rabbit corneal endothelium samples with quantitative label-free proteomics and downstream analysis. We discovered that vitamin and selenocompound metabolism and some signaling pathways such as NF-kappa B signaling pathway differed between the samples. Moreover, TGFβ, PITX2 and keratocan were distinctively expressed in rabbit samples, which might be associated with active proliferation in rabbit CECs. This study illustrates the proteomic differences between human and rabbit CECs and might promote CEC engineering strategies.

Biofilm-Forming Potential of Ocular Fluid Staphylococcus aureus and Staphylococcus epidermidis on Ex Vivo Human Corneas from Attachment to Dispersal Phase.

The biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis, isolated from patients with Endophthalmitis, was monitored using glass cover slips and cadaveric corneas as substrata. Both the ocular fluid isolates exhibited biofilm-forming potential by the Congo red agar, Crystal violet and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino) carbonyl-2H-tetra-zolium hydroxide (XTT) methods. Confocal microscopy demonstrated that the thickness of the biofilm increased from 4–120 h of biofilm formation. Scanning electron microscopic studies indicated that the biofilms grown on cover slips and ex vivo corneas of both the isolates go through an adhesion phase at 4 h followed by multilayer clumping of cells with intercellular connections and copious amounts of extracellular polymeric substance. Clumps subsequently formed columns and eventually single cells were visible indicative of dispersal phase. Biofilm formation was more rapid when the cornea was used as a substratum. In the biofilms grown on corneas, clumping of cells, formation of 3D structures and final appearance of single cells indicative of dispersal phase occurred by 48 h compared to 96–120 h when biofilms were grown on cover slips. In the biofilm phase, both were several-fold more resistant to antibiotics compared to planktonic cells. This is the first study on biofilm forming potential of ocular fluid S. aureus and S. epidermidis on cadaveric cornea, from attachment to dispersal phase of biofilm formation.

Fusarium infection complicating rheumatic keratitis that acutely progressed to endophthalmitis during regular infusion of tocilizumab: a case report

BackgroundFilamentous fungi are ubiquitous in plants, water, and soil. The predominant fungi that infect the human cornea include Fusarium and Aspergillus species. The onset of fungal endophthalmitis is indolent, and typically takes weeks to months to develop after corneal infection. We report a case of Fusarium infection complicating rheumatic keratitis that acutely progressed to endophthalmitis during intravenous tocilizumab therapy.Case presentationA 65-year-old female patient was referred to our department due to pain and decreased vision in her left eye. Slit-lamp examination showed a white focus on the upper peripheral cornea, hypopyon, anterior chamber fibrin formation, marked ciliary hyperemia, and whole corneal epithelial defects. As the corneal scraping smear was positive for filamentous fungi and Fusarium species were detected by aqueous humor polymerase chain reaction, anti-fungal therapy was started. Although the initial response to anti-fungal therapy was good, we observed corneal infiltration, worsening hypopyon, and vitreous opacity after tocilizumab infusion. Given that the infection continued to progress despite conservative therapy, we performed penetrating keratoplasty combined with vitrectomy. After removal of the white focus beneath the intraocular lens, a temporary corneal prosthesis was mounted and the dense vitreous opacity was removed. Finally, a frozen donor graft was sutured in place. The corneal infiltration, hypopyon, and vitreous opacity all disappeared after the operation.ConclusionThe rapid progression of Fusarium keratitis to endophthalmitis in a patient who was receiving a regular infusion of tocilizumab demonstrates that ocular condition should be closely monitored during systemic tocilizumab administration due to increased risk of infection.

Application of in vitro methods to evaluate the safety of baby care products

Draize rabbit eye irritation and skin irritation tests are widely used in the chemical industry as traditional methods to evaluate the safety of cosmetics. However, great differences among laboratories have caused great doubt in the industry. In addition, with vigorous development of the global animal protection movement, developed countries have launched the “3R” campaign, and various kinds of in vitro alternative methods have emerged. Hen's egg test on the chorioallantoic membrane (HET-CAM), which is similar to the structure of the human cornea and has a clear and complete vascular system, is based on the characteristics of the chorioallantoic membrane (CAM) in mid-term SPF egg embryos. The reconstructed human epidermis (EpiSkin®) is composed of normal human keratinocytes that are histologically similar to human epidermises seen in vivo, and it is cultured on a collagen matrix. Similar to EpiSkin®, Human Corneal Epithelium (SkinEthic™) is another reconstructed 3D human-corneal structure that is an alternative to the traditional eye irritation test. Three in vitro methods were conducted to evaluate the safety of 12 baby care products, which included the most common types. In addition, a consumer research study was also carried out for two weeks to evaluate the safety. The results of the reconstructed human epidermis model, human corneal epithelium model and consumer research showed that no irritation was found in any test products; however, HET-CAM tests showed positive results.

Robust and double-layer micro-patterned bioadhesive based on silk nanofibril/GelMA-alginate for stroma tissue engineering

We develop a robust micro-patterned double-layer film that can adhere firmly to the tissue and provide a sustained release of ascorbic acid (AA) for corneal regeneration. This double-layer film consists of a AA reservoir sodium alginate (SA) adhesive and an anisotropic layer made of micro-patterned silk nanofibrils (SNF) incorporated gelatin methacrylate (GelMA) (S/G). The S/G layer facilitates the adhesion and orientation of corneal stroma cells, depending on the pattern sizes (50 μm (P1) and 100 (P2) μm). Results reveal that more than 90% and 80% of the cells are located at angles close to the vertical axis (0–20°) in the sample with the smaller and larger pattern size, respectively. The mechanical robustness and 90% light transmission of this hybrid film originate from the micro-patterned S/G layer. However, the micro-pattern size does not show a significant role in the mechanical properties of hybrid films (tensile strength of S/G-SA, S/G-SA(P1), and S/G-SA(P2) is 3.4 ± 0.1 MPa, 3.6 ± 0.6 MPa and 3.3 ± 0.2 MPa, respectively). In addition, the strong adhesion to the tissue of this double-layer film is related to the alginate layer. AA can release in a controlled manner, which can significantly promote corneal stroma cells' attachment, alignment, and proliferation compared to the control (AA-free micro-patterned film). Our results reveal that this innovative multifunctional S/G-SA + AA film can be a proper candidate for use in stroma tissue engineering of the human cornea.

Characterization of Tunable Poly-ε-Lysine-Based Hydrogels for Corneal Tissue Engineering.

A family of poly-ε-lysine hydrogels can be synthesized by crosslinking with bis-carboxylic acids using carbodiimide chemistry. In addition to creating hydrogels using a simple cast method, a fragmented method is used to introduce increased porosity within the hydrogel structure. Both methods have created tunable characteristics ranging in their mechanical properties, transparency, and water content, which is of interest to corneal tissue engineering and can be tailored to specific cellular needs and applications. With a worldwide shortage of cornea donor tissue available for transplant and limitations including rejection and potential infection, a synthetic material that can be used as a graft, or a partial thickness corneal replacement, would be an advantageous treatment method. These hydrogels can be tuned to have similar mechanical and transparency properties to the human cornea. They also support the attachment and growth of corneal epithelial cells and the integration of corneal stromal cells.

Molecular identity of human limbal heterogeneity involved in corneal homeostasis and privilege

PurposeThe corneal limbus maintains the homeostasis, immune and angiogenic privilege of cornea. This study aimed to depict the landscape of human limbal tissues by single-cell RNA sequencing (scRNA-seq). MethodsSingle cells of human limbus collected from donor corneas were subjected to 10x scRNA-seq, followed by clustering cell types through the t-distributed stochastic neighbor embedding (t-SNE) and unbiased computational informatic analysis. Immunofluorescent staining was performed using human corneas to validate the analysis results. Results47,627 cells acquired from six human limbal tissues were collected and subjected to scRNA-seq. 14 distinct clusters were identified and 8 cell types were annotated with representative markers. In-depth dissection revealed three limbal epithelial cell subtypes and refined the X-Y-Z hypothesis of corneal epithelial maintenance. We further unveiled two cell states with higher stemness (TP63+ and CCL20+ cells), and two other differentiated cell states (GPHA2+ and KRT6B + cells) in homeostatic limbal stem/progenitor cells (LSPCs) that differ in transcriptional profiles. Cell-cell communication analysis revealed the central role of LSPCs and their bidirectional regulation with various niche cells. Moreover, comparative analysis between limbus and skin deciphered the pivotal contribution of limbal immune cells, vascular and lymphatic endothelial cells to corneal immune and angiogenic privilege. ConclusionsThe human limbus atlas provided valuable resources and foundations for understanding corneal biology, disease and potential interventions.

Sinasol versus Optisol-GS for cold preservation of human cornea: a prospective ex vivo and clinical study.

To compare ex vivo results of donor corneas maintained in Sinasol with those stored in Optisol-GS and reporting clinical outcomes of grafted Sinasol-versus Optisol-GS-stored corneas. In phase I, paired donor corneas were maintained in Sinasol or Optisol-GS. Afterward, the corneas were subjected to slit-lamp biomicroscopic and specular microscopic examinations on days 1 and 7, and then to trypan blue staining on day 7. The same examinations were performed on the corneas that were kept in Sinasol or Optisol-GS for 14days. In phase II, the post-operative reports of 72 consecutive corneal transplantations were recorded using Sinasol- or Optisol-GS-preserved corneas. In phase I, 128 corneas from 64 donors and 59 corneas from 33 donors were investigated for 7 and 14days, respectively. The EC indices were comparable between the groups at the measurement periods. The EC losses over 7 and 14days were 3.7% and 19.9% in Sinasol against 4.6% and 20.8% in Optisol-GS. Although fair quality corneas were more common in Optisol-GS group after 7 (P = 0.04) and 14days (P = 0.034), changes of stromal edema, Descemet's fold, and other quality ratings during 14days were not different between the groups. In phase II, all the transplanted corneas were postoperatively clear with no adverse reactions. The overall results indicate that Sinasol is a safe, effective, and affordable intermediate cold storage medium for preservation of corneas.

Solubilized ubiquinol for preserving corneal function

Defective cellular metabolism, impaired mitochondrial function, and increased cell death are major problems that adversely affect donor tissues during hypothermic preservation prior to transplantation. These problems are thought to arise from accumulated reactive oxygen species (ROS) inside cells. Oxidative stress acting on the cells of organs and tissues preserved in hypothermic conditions before surgery, as is the case for cornea transplantation, is thought to be a major reason behind cell death prior to surgery and decreased graft survival after transplantation. We have recently discovered that ubiquinol – the reduced and active form of coenzyme Q10 and a powerful antioxidant – significantly enhances mitochondrial function and reduces apoptosis in human donor corneal endothelial cells. However, ubiquinol is highly lipophilic, underscoring the need for an aqueous-based formulation of this molecule. Herein, we report a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products. Docking studies showed that γ-CD has the strongest binding affinity with ubiquinol compared to α- or β-CD. Complexed ubiquinol showed significantly higher stability compared to free ubiquinol in different aqueous ophthalmic products including Optisol-GS® corneal storage medium, balanced salt solution for intraocular irrigation, and topical Refresh® artificial tear eye drops. Greater ROS scavenging activity was noted in a cell model with high basal metabolism and ROS generation (A549) and in HCEC-B4G12 human corneal endothelial cells after treatment with ubiquinol/γ-CD compared to free ubiquinol. Furthermore, complexed ubiquinol was more effective at lowering ROS, and at far lower concentrations, compared to free ubiquinol. Complexed ubiquinol inhibited lipid peroxidation and protected HCEC-B4G12 cells against erastin-induced ferroptosis. No evidence of cellular toxicity was detected in HCEC-B4G12 cells after treatment with complexed ubiquinol. Using a vertical diffusion system, a topically applied inclusion complex of γ-CD and a lipophilic dye (coumarin-6) demonstrated transcorneal penetrance in porcine corneas and the capacity for the γ-CD vehicle to deliver drug to the corneal endothelium. Using the same model, topically applied ubiquinol/γ-CD complex penetrated the entire thickness of human donor corneas with markedly greater ubiquinol retention in the endothelium compared to free ubiquinol. Lastly, the penetrance of ubiquinol/γ-CD complex was assayed using human donor corneas preserved for 7 days in Optisol-GS® per standard industry practices, and demonstrated higher amounts of ubiquinol retained in the corneal endothelium compared to free ubiquinol. In summary, ubiquinol complexed with γ-CD is a highly stable composition that can be incorporated into a variety of aqueous-phase products for ophthalmic use including donor corneal storage media and topical eye drops to scavenge ROS and protect corneal endothelial cells against oxidative damage.

Three-Month Outcomes of Myopic Astigmatism Correction With Small Incision Guided Human Cornea Treatment.

To evaluate refractive and visual outcomes of small incision guided human cornea treatment (SmartSight; SCHWIND eye-tech-solutions) in the treatment of myopic astigmatism with the use of a new femtosecond laser system. This retrospective, observational case series study included 104 eyes of 56 patients who underwent SmartSight to correct myopic astigmatism and completed the 3-month follow-up. Procedures were performed with a SCHWIND ATOS femtosecond laser. Preoperatively, mean manifest spherical equivalent refraction was -6.12 ± 1.99 diopters (D) (range: -3.00 to -11.50 D) and astigmatism was 1.02 ± 0.56 D (range: 0.25 to 2.75 D). Three months postoperatively, it was +0.47 ± 0.32 D (range: -0.75 to +1.00 D) and 0.27± 0.22 D (range: 0.00 to 0.75 D), respectively (both P < .05). Spherical equivalent correction within ±0.50 D was achieved in 62 eyes (60%), and cylindrical correction in 90 eyes (87%). Preoperative corrected distance visual acuity (CDVA) was 20/20 or better in 94 eyes (90%) and postoperative uncorrected distance visual acuity was 20/20 or better in 96 eyes (92%). No eye lost two or more Snellen lines of CDVA. Myopic astigmatism correction with Smart-Sight provided good results for efficacy, safety, predictability, and visual outcomes in the first 3 months of follow-up. [J Refract Surg. 2021;37(5):304-311.].