Human Cornea Research Articles

Hyperopic SMILE Versus FS-LASIK: A Biomechanical Comparison in Human Fellow Corneas

PURPOSE: To investigate the biomechanical properties of ex vivo human paired corneas after hyperopic correction via cap-based versus flap-based laser-assisted refractive surgery. METHODS: In this prospective experimental study, 13 pairs of human corneas unsuitable for transplantation were equally divided into two groups. The pachymetry was performed in each eye just before the laser procedure. Corneas from the right eye were treated with small incision lenticule extraction (SMILE), whereas corneas from the left eye of the same donor were treated with femtosecond laser–assisted laser in situ keratomileusis (FS-LASIK). All corneas were subjected to a refractive correction of +6.00 diopters (D) sphere with a 6.5-mm zone under a 120-µm cap (SMILE) or a 7-mm zone under a 110-µm flap (FS-LASIK). For two-dimensional biomechanical measurements, the corneoscleral buttons underwent two testing cycles (preconditioning stress-strain curve from 0.03 to 9.0 N and stress-relaxation at 9.0 N during 120 seconds) to analyze the elastic and viscoelastic material properties. The effective elastic modulus was calculated. Statistical analysis was performed with a confidence interval of 95%. RESULTS: In stress-strain measurements, the effective elastic modulus was not significantly different ( P > .311) between SMILE (13.5 ± 12.8 MPa) and FS-LASIK (7.56 ± 17.9 MPa). In stress-relaxation measurements, the remaining stress was not significantly different ( P = .841) between SMILE (124 ± 20 kPa) and FS-LASIK (126 ± 21 kPa). CONCLUSIONS: Unlike myopic correction, after hyperopic correction the cap-based procedure (SMILE) and the flap-based technique (FS-LASIK) may be considered equivalent in terms of biomechanical stability when measured experimentally in ex vivo human fellow eye corneas. [ J Refract Surg . 2021;37(12):810–815.]

Local information pattern descriptor for corneal diseases diagnosis

<span lang="EN-US">Light penetrates the human eye through the cornea, which is the outer part of the eye, and then the cornea directs it to the pupil to determine the amount of light that reaches the lens of the eye. Accordingly, the human cornea must not be exposed to any damage or disease that may lead to human vision disturbances. Such damages can be revealed by topographic images used by ophthalmologists. Consequently, an important priority is the early and accurate diagnosis of diseases that may affect corneal integrity through the use of machine learning algorithms, particularly, use of local feature extractions for the image. Accordingly, we suggest a new algorithm called local information pattern (LIP) descriptor to overcome the lack of local binary patterns that loss of information from the image and solve the problem of image rotation. The LIP based on utilizing the sub-image center intensity for estimating neighbors' weights that can use to calculate what so-called contrast based centre (CBC). On the other hand, calculating local pattern (LP) for each block image, to distinguish between two sub-images having the same CBC. LP is the sum of transitions of neighbors' weights, from sub-image center value to one and vice versa. Finally, creating histograms for both CBC and LP, then blending them to represent a robust local feature vector. Which can use for diagnosing, detecting.</span>

Topical retinoic acid induces corneal strengthening by upregulating transglutaminase 2 in murine cornea

Transglutaminase 2 (TG2) is the most abundant crosslinking enzyme in murine and human cornea, while retinoids are well-known inducers of TG2 expression. This study aims to determine if the retinoic acid supplementation can increase corneal stiffness by crosslinking through upregulating the corneal TG2 expression. The right eyes of C57BL/6 mice were treated with 2 × 10−2M retinol palmitate (VApal) eyedrops or control eyedrops and hold for 30 min, once a day for 28 consecutive days. The WB and qPCR results showed increased expression of TG2 in murine cornea with the prolongation of VApal eyedrop application. After 28 days of VApal eyedrop treatment, the increased TG2 were found catalytically active and distributed in corneal epithelium and stroma as detected by 5-(biotinamido) pentylamine (5-BP) incorporation method and immunofluorescence staining. The transmission electron microscope image revealed that VApal treated cornea manifested with increased collagen density in anterior and middle layer of stroma. The higher elastic module was found among VApal treated cornea by nano-indentation test. In cultured corneal epithelial cells and keratocytes, all-trans retinoid acid (ATRA) treatment increased the content of TG2 in cell lysis and in culture medium. These results indicate that retinoic acid induce the reinforcement of the cornea by TG2 mediated crosslinking via increasing the TG2 expression in corneal epithelium and keratocyte. As TG2 was found to be less in the cornea of keratoconus patients in several RNA-sequencing studies, retinoic acid could serve as a non-invasive prevention method for keratoconus progression.

Killing efficacy of a new hypothermic corneal storage medium against the micro-organisms frequently found in human donor cornea intended for transplantation

ObjectiveTo study the in vitro killing efficacy of Kerasave (AL.CHI.MI.A Srl), a medium provided with amphotericin B tablet for hypothermic storage of human donor corneas, against relevant contaminants associated with...

Bowman-stromal inlay using an intraocular lens injector for management of keratoconus.

A simple approach for transplanting Bowman layer and anterior stroma Bowman-stromal inlay (BSI) in keratoconic patients as an intervention to cease progression of ectasia and increase the overall thickness is described. A femtosecond laser was used to create BSI from human eye bank donor corneas and form an intrastromal pocket in the host cornea. The inlay was placed in the intrastromal space using an intraocular lens injector. This technique was performed successfully in 10 eyes of patients with progressive corneal ectasia. Postoperatively, the increased host pachymetry was as per BSI thickness. The tomography parameters remained stable, suggesting stabilization of keratoconus over a mean 15.9 months of follow-up. The BSI may offer a technically easy and safe technique of stromal augmentation to arrest keratoconus progression. It also opens up the possibility of surface ablation in the future for visual rehabilitation.

Vitrification of Human Cornea: Assessment By Viability, Function and Human To Rabbit Xenograft

Vitrification of Human Cornea: Assessment By Viability, Function and Human To Rabbit Xenograft

Examining the Transmission of Visible Light through Electrospun Nanofibrous PCL Scaffolds for Corneal Tissue Engineering.

The transparency of nanofibrous scaffolds is of highest interest for potential applications like corneal wound dressings in corneal tissue engineering. In this study, we provide a detailed analysis of light transmission through electrospun polycaprolactone (PCL) scaffolds. PCL scaffolds were produced via electrospinning, with fiber diameters in the range from (35 ± 13) nm to (167 ± 35) nm. Light transmission measurements were conducted using UV–vis spectroscopy in the range of visible light and analyzed with respect to the influence of scaffold thickness, fiber diameter, and surrounding medium. Contour plots were compiled for a straightforward access to light transmission values for arbitrary scaffold thicknesses. Depending on the fiber diameter, transmission values between 15% and 75% were observed for scaffold thicknesses of 10 µm. With a decreasing fiber diameter, light transmission could be improved, as well as with matching refractive indices of fiber material and medium. For corneal tissue engineering, scaffolds should be designed as thin as possible and fabricated from polymers with a matching refractive index to that of the human cornea. Concerning fiber diameter, smaller fiber diameters should be favored for maximizing graft transparency. Finally, a novel, semi-empirical formulation of light transmission through nanofibrous scaffolds is presented.

Analysis of Cryopreservation Protocols and Their Harmful Effects on the Endothelial Integrity of Human Corneas.

Corneal cryopreservation can partially solve the worldwide concern regarding donor cornea shortage for keratoplasties. In this study, human corneas were cryopreserved using two standard cryopreservation protocols that are employed in the Tissue Bank of the Teresa Herrera Hospital (Spain) to store corneas for tectonic keratoplasties (TK protocol) and aortic valves (AV protocol), and two vitrification protocols, VS55 and DP6. Endothelial viability and general corneal state were evaluated to determine the protocol that provides the best results. The potential corneal cryopreservation protocol was studied in detail taking into consideration some cryopreservation-related variables and the endothelial integrity and stroma arrangement of the resulting cryopreserved corneas. TK corneas showed mostly viable endothelial cells, while the others showed few (AV) or none (DP6 and VS55). The corneal structure was well maintained in TK and AV corneas. TK corneas showed endothelial acellular areas surrounded by injured cells and a normal-like stromal fiber arrangement. Cryoprotectant solutions of the TK protocol presented an increasing osmolality and a physiological pH value. Cooling temperature rate of TK protocol was of 1 °C/min to −40 °C and 3 °C/min to −120 °C, and almost all of dimethyl sulfoxide left the tissue after washing. Future studies should be done changing cryopreservation-related variables of the TK protocol to store corneas of optical grade.

Analytical optimization of the laser induced refractive index change (LIRIC) process: maximizing LIRIC without reaching the damage threshold

Abstract A method to determine the optimum laser parameters for maximizing laser induced refractive index change (LIRIC) while avoiding exceeding the damage threshold for different materials with high water content (in particular, polymers such as hydrogels or the human cornea) is proposed. The model is based upon two previous independent models for LIRIC and for laser induced optical breakdown (LIOB) threshold combined in a simple manner. This work provides qualitative and quantitative estimates for the parameters leading to a maximum LIRIC effect below the threshold of LIOB.

Gram stain and addition of amphotericin B to improve the microbial safety of human donor corneas

To determine the effectiveness of two methods to improve the microbial safety of human corneas preserved in organ culture. We compared the number of positive preservation solutions of corneas in organ culture in which the initial short-term hypothermic corneal maintenance solution was supplemented with amphotericin B 2.5 µg/mL and the historical data of microbial test results (2015–2019). In addition, we appraised the efficacy of Gram stain to detect bacterial or fungal contamination in the organ culture solutions of corneas from at-risk donors compared to the culture tests of corneas from not-at-risk donors. Statistical analysis was performed using STATA and statistical significance set at p < 0.05. The number of positive culture tests after preservation was 15 (0.5%) in 2020 compared to a mean of 37 (1.2%) in the period 2015–2019 (p < 0.01), with 10 (1.0%) positive samples in the cohort of 998 corneas from at-risk donors and 5 (0.2%) in the 2046 corneas from not-at-risk donors (p < 0.01). All corneas from at-risk donors tested positive at Gram stain and the results were available 1–3 days before those of the conventional culture tests. Amphotericin B supplementation in the short-term maintenance solution markedly reduced the number of positive microbial tests after organ culture and the early detection of contaminants, including slow-growing microorganisms, by Gram stain before the standard culture results. This meant fewer corneas being discarded and a greater likelihood of preventing post-graft infections.

Exploring the Biomechanical Properties of the Human Cornea In Vivo Based on Corvis ST.

Purpose: The aim of this study was to provide a method to determine corneal nonlinear viscoelastic properties based on the output data of corneal visualization Scheimpflug technology (Corvis ST). Methods: The Corvis ST data from 18 eyes of 12 healthy humans were collected. Based on the air-puff pressure and the corneal displacement from the Corvis ST test of normal human eyes, the work done by the air-puff attaining the whole corneal displacement was obtained. By applying a visco-hyperelastic strain energy density function of the cornea, in which the first-order Prony relaxation function and the first-order Ogden strain energy were employed, the corneal strain energy during the Corvis ST test was calculated. Then the work done by the air-puff attaining the whole corneal displacement was completely regarded as the strain energy of the cornea. The identification of the nonlinear viscoelastic parameters was carried out by optimizing the sum of difference squares of the work and the strain energy using the genetic algorithm. Results: The visco-hyperelastic model gave a good fit to the data of corneal strain energy with time during the Corvis ST test (R 2 > 0.95). The determined Ogden model parameter μ ranged from 0.42 to 0.74 MPa, and α ranged from 32.76 to 55.63. The parameters A and τ in the first-order Prony function were 0.09–0.36 and 1.21–1.95 ms, respectively. Conclusion: It is feasible to determine the corneal nonlinear viscoelastic properties based on the corneal contour information and air-puff pressure of the Corvis ST test.

Contribution of the Transcription Factors Sp1/Sp3 and AP-1 to Clusterin Gene Expression during Corneal Wound Healing of Tissue-Engineered Human Corneas.

In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.

Corneal biomechanical properties following corneal cross-linking: Does age have an effect?

PurposeTo explore the effect of age on corneal biomechanical properties following corneal cross-linking (CXL). MethodsA total of 12 pairs of human eye-banked corneas (24 corneas, from 14 females and 10 males) were used in the study. The mean donor age was 48.5 years (ranging from 26 to 71 years). Corneas were divided into three age groups: A (26–41 years), B (42–57 years) and C (58–71 years), with four pairs in each group. For each pair, the right corneas were cross-linked using accelerated CXL with UVA (10 mW/cm2) and riboflavin, while the left corneas served as controls and were not exposed to either UVA irradiation or riboflavin. The corneal elastic modulus of the anterior, mid and posterior corneal stroma was measured using nanoindentation. ResultsThe difference in the corneal elastic modulus following CXL was significant in the anterior (p = 0.00002) and mid stroma (p = 0.001); however, the difference was not significant in the posterior stroma (p = 0.27) when compared to control corneas. The corneal elastic modulus of the anterior stroma increased by 178.44% in Group A, 119.7% in Group B and 50.73% in Group C compared to control corneas. For the mid stroma, the elastic modulus increased by 47.35% in Group A, 25% in Group B and 24.56% in Group C. No differences were observed in the posterior stroma between age groups. ConclusionsCorneal elasticity showed a greater response to CXL in the younger group compared to older groups. CXL treatment showed effectiveness in enhancing stromal strength, and the effect was concentrated in the anterior and mid stroma with minimal impact on the posterior stroma in all age groups.

Nonlinear optical properties of human cornea measured by spectral domain Z-scan method.

In the myopia correction surgery by femtosecond laser, such as Laser in Situ Keratomileusis (LASIK) and Small Incision Lenticule Extraction (SMILE), the nonlinear refractive index of the cornea may cause the deviation of cutting depth. In order to improve the cornea cutting's accuracy and reduce the possibility of undercorrection, the nonlinear refractive index coefficient n2 of the human cornea must be measured with high accuracy. The spectral domain Z-scan technique can measure n2 of the highly scattering biological tissues with much better signal to noise ratio and thus better accuracy than the conventional methods. In this paper, the n2 coefficient of one ex-vivo human corneal sample was measured by the spectral domain Z-scan technique. Experimental results show that as this corneal sample gradually dehydrates, its n2 coefficients are 1.1 ± 0.1×10-19 m2/W, 1.4 ± 0.2×10-19 m2/W and 1.6 ± 0.2 ×10-19 m2/W respectively for the corneal sample with water contents of 89%, 82%, and 78%. The increase of the water content reduces the value of n2, which is reasonable since the nonlinear refractive index coefficient of water is one order of magnitude smaller than that of the cornea.

Single cell transcriptomics reveals the heterogeneity of the human cornea to identify novel markers of the limbus and stroma

The cornea is the clear window that lets light into the eye. It is composed of five layers: epithelium, Bowman’s layer, stroma, Descemet’s membrane and endothelium. The maintenance of its structure and transparency are determined by the functions of the different cell types populating each layer. Attempts to regenerate corneal tissue and understand disease conditions requires knowledge of how cell profiles vary across this heterogeneous tissue. We performed a single cell transcriptomic profiling of 19,472 cells isolated from eight healthy donor corneas. Our analysis delineates the heterogeneity of the corneal layers by identifying cell populations and revealing cell states that contribute in preserving corneal homeostasis. We identified expression of CAV1, HOMER3 and CPVL in the corneal epithelial limbal stem cell niche, CKS2, STMN1 and UBE2C were exclusively expressed in highly proliferative transit amplifying cells, CXCL14 was expressed exclusively in the suprabasal/superficial limbus, and NNMT was exclusively expressed by stromal keratocytes. Overall, this research provides a basis to improve current primary cell expansion protocols, for future profiling of corneal disease states, to help guide pluripotent stem cells into different corneal lineages, and to understand how engineered substrates affect corneal cells to improve regenerative therapies.

Long-term effect of contact lens wear: A citation network study

BackgroundContact lenses can cause complications in 2/3 of the total population, but in most cases, they are mild and easy to manage. However, most of the studies are short-term. The aim of the study was to analyze the publications on the long-term effects of contact lens wear to understand the current research landscape through the analysis of dating networks as well as determining the different research areas and the most cited publications. MethodsThe Web of Science database was used to perform the publication search, looking for the terms “Long-term” AND “contact lens wear” within the period between 1977 and January 2021. The CitNetExplorer and CiteSpace software were then used to conduct the publication analysis. To obtain the graphics, the VOSviewer software was used. ResultsA total of 721 publications were found with 2999 citations generated within the network. The year 2010 was singled out as a “key year”, considering the number of publications that emerged in that year and increase in adaptations of contact lens worldwide. The most widely cited publication was “Effects of long-term extended contact lens wear on the human cornea”, an article by Holden et al., which was published in 1985. By using the clustering function, we were able to establish three clusters that encompassed the different research areas within this field: morphological changes, the loss of corneal sensitivity, tear film characteristics and longitudinal changes in ocular physiology. ConclusionsThe main research topic in this field focuses on the morphological changes that take place in the corneal endothelial cell layer.

Objective Assessment of Corneal and Lens Clarity in Patients with COVID-19.

The severe acute respiratory syndrome coronavirus 2 virus, which causes the coronavirus disease 2019 (COVID-19) pandemic, can bind to epithelial cells in the human cornea and conjunctiva, which may result in changes of corneal and lenticular densitometry. We aimed to report the corneal and lenticular clarity of patients who had been diagnosed previously with confirmed infection of COVID-19. This was a prospective, cross-sectional study. Fifty-three patients who had recovered from COVID-19 and 51 healthy individuals who had not had COVID-19 (control) were included in this study. Measurement of the corneal (at a corneal diameter of 12 mm) and lens densitometry of participants was performed using a Pentacam HR Scheimpflug imaging system. Three different optimal depths as anterior, central, and posterior layers and four concentric zones, consisting of diameters of 0 to 2, 2 to 6, 6 to 10, and 10 to 12 mm, were chosen to perform the corneal densitometric measurements. The mean lenticular densitometric values were calculated in zone 1 at 2.0 mm, zone 2 at 4.0 mm, and zone 3 at 6.0 mm by taking the central part of the pupil as reference point. When compared with the individuals in the control group, the corneal densitometry measurements in the patients in the COVID-19 group were considerably higher in anterior 0 to 2 mm, 2 to 6 mm, 6 to 10 mm zones and total diameter; center 0 to 2 and 2 to 6 mm zones and total diameter; posterior 0 to 2 mm zone; and total corneal 0 to 2 and 2 to 6 mm zones and total diameter (P < .05, for each). When compared with the individuals in the control group, all of the lens densitometry measurements, except for those located in zone 1, were determined to be significantly higher among the patients in the COVID-19 group (P < .05, for each). Significant alterations were found in corneal and lenticular densitometric values in patients who had had COVID-19. The virus could adversely affect cornea and lens transparency.

Evaluation of Efficacy, Efficiency, and Cell Viability of a Novel Descemet Membrane Endothelial Keratoplasty Graft Preparation Device, DescePrep, in Nondiabetic and Diabetic Human Donor Corneas.

The purpose of this study was to evaluate the safety, efficacy, and efficiency of a Descemet membrane endothelial keratoplasty (DMEK) graft preparation device, DescePrep, through measurement of graft viability, yield, and preparation time in both healthy and diabetic (high-risk) donor eyes. Twenty nondiabetic and 10 diabetic donor corneas were processed using DescePrep, which standardizes the liquid bubble technique. Corneas were stained with trypan blue and then processed. Cell counts through specular microscopy, optical coherence tomography imaging, and slit-lamp analysis were used for the evaluation of graft separation and viability in 5 nondiabetic corneas. The remaining 25 corneas (15 nondiabetic and 10 diabetic) were evaluated for preparation success rate and processing time. Ten corneas (5 nondiabetic and 5 diabetic) were randomly selected for further evaluation of global cell loss through staining. Ninety-seven percent of corneas (29 of 30) were prepared successfully with DescePrep. The average preparation time was 2.83 ± 1.8 minutes. There was no significant difference in the time of preparation between the nondiabetic and diabetic groups (P = 0.077). The overall average cell death after processing was 7.9% ± 3.7% for all corneas. There was no significant difference in cell viability between diabetic and nondiabetic tissues after DescePrep processing (P = 0.769). DescePrep is a new DMEK preparation technique that can process both nondiabetic and diabetic donor corneas at high yields in minutes. High-yield preparation of diabetic corneas may offer eye banks access to a larger donor pool, which is important because the demand for DMEK grafts continues to rise worldwide.

Non-contact acoustic micro-tapping optical coherence elastography (AμT-OCE) in the cornea: Mechanical model, ex vivo and in vivo results, and direct comparison with mechanical tests

The cornea is one of the primary determinants of visual performance. Its highly pressurized anisotropic structure of collagen fibrils embedded in a hydrated proteoglycan matrix forms the clear refractive primary lens of the eye. Despite recent success in corneal topography, there are no clinical tools to predict shape changes from vision-correction therapies such as LASIK and CXL due to the absence of non-contact methods that can map corneal elasticity in vivo. Here we review our newest results in quantification of corneal elasticity. Recently, we produced a technology combining non-contact excitation of sub-mm wavelength mechanical waves in the cornea using air-coupled acoustic micro-tapping (AμT), with 1.6 MHz scan-rate phase-sensitive OCT to track their propagation three-dimensionally in real time. However, corneal moduli reconstruction from measured wavefields requires an anisotropic elastic model and cornea’s bounded, non-flat geometry must be taken into account. Thus, a nearly incompressible transversally isotropic (NITI) model of corneal elasticity was developed. We have performed multiple ex vivo and in vivo studies in rabbit, porcine and human corneas and compared AμT-OCE results directly with conventional mechanical tests (tensile extensometry and shear rheometry). These studies demonstrate that in-plane Young’s and out-of-plane shear moduli can be accurately mapped with non-contact, non-invasive AμT-OCE.

Novel Corneal Protein Biomarker Candidates Reveal Iron Metabolic Disturbance in High Myopia Eyes.

Myopia is a major public health concern with increasing global prevalence and is the leading cause of vision loss and complications. The potential role of the cornea, a substantial component of refractive power and the protective fortress of the eye, has been underestimated in the development of myopia. Our study acquired corneal stroma tissues from myopic patients undergoing femtosecond laser-assisted small incision lenticule extraction (SMILE) surgery and investigated the differential expression of circulating proteins between subjects with low and high myopia by means of high-throughput proteomic approaches—the quantitative tandem mass tag (TMT) labeling method and parallel reaction monitoring (PRM) validation. Across all corneal stroma tissue samples, a total of 2,455 proteins were identified qualitatively and quantitatively, 103 of which were differentially expressed between those with low and high myopia. The differentially abundant proteins (DAPs) between the groups of stroma samples mostly demonstrated catalytic activity and molecular function regulator and transporter activity and participated in metabolic processes, biological regulation, response to stimulus, and so forth. Pathway enrichment showed that mineral absorption, ferroptosis, and HIF-1 signaling pathways were activated in the human myopic cornea. Furthermore, TMT analysis and PRM validation revealed that the expression of ferritin light chain (FTL, P02792) and ferritin heavy chain (FTH1, P02794) was negatively associated with myopia development, while the expression of serotransferrin (TF, P02787) was positively related to myopia status. Overall, our results indicated that subjects with low and high myopia could have different proteomic profiles or signatures in the cornea. These findings revealed disturbances in iron metabolism and corneal oxidative stress in the more myopic eyes. Iron metabolic proteins could serve as an essential modulator in the pathogenesis of myopia.