Development of graphitic carbon nitride quantum dots-based oxygen self-sufficient platforms for enhanced corneal crosslinking.

Keratoconus, a disorder characterized by corneal thinning and weakening, results in vision loss. Corneal crosslinking (CXL) can halt the progression of keratoconus. The development of accelerated corneal crosslinking (A-CXL) protocols to shorten the treatment time has been hampered by the rapid depletion of stromal oxygen when higher UVA intensities are used, resulting in a reduced cross-linking effect. It is therefore imperative to develop better methods to increase the oxygen concentration within the corneal stroma during the A-CXL process. Photocatalytic oxygen-generating nanomaterials are promising candidates to solve the hypoxia problem during A-CXL. Biocompatible graphitic carbon nitride (g-C3N4) quantum dots (QDs)-based oxygen self-sufficient platforms including g-C3N4 QDs and riboflavin/g-C3N4 QDs composites (RF@g-C3N4 QDs) have been developed in this study. Both display excellent photocatalytic oxygen generation ability, high reactive oxygen species (ROS) yield, and excellent biosafety. More importantly, the A-CXL effect of the g-C3N4 QDs or RF@g-C3N4 QDs composite on male New Zealand white rabbits is better than that of the riboflavin 5'-phosphate sodium (RF) A-CXL protocol under the same conditions, indicating excellent strengthening of the cornea after A-CXL treatments. These lead us to suggest the potential application of g-C3N4 QDs in A-CXL for corneal ectasias and other corneal diseases.

Effect of Corneal Allogenic Intrastromal Ring Segment (CAIRS) Implantation Surgery in Patients With Keratoconus According to Prior Corneal Cross-linking Status.

PURPOSE: To compare the effects of corneal allogenic intrastromal ring segment (CAIRS) implantation on topographical measurements and visual outcomes of patients with keratoconus with and without corneal cross-linking (CXL) prior to the time of implantation. METHODS: Sixty-seven eyes with corneal allograft intrastromal ring segment implantation (KeraNatural; Lions VisionGift) due to advanced keratoconus were included in the study. Thirty-seven eyes had no CXL and 30 eyes had had CXL before being referred to the authors. The changes in spherical equivalent (SE), uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), steep keratometry (K1), flat keratometry (K2), mean keratometry (Kmean), maximum keratometry (Kmax), and thinnest pachymetry were retrospectively analyzed 6 months after the implantation. RESULTS: The median age was 29 years in the CXL group and 24.0 years in the non-CXL group (P > .05), respectively. All topographical and visual parameters before implantation were similar in both groups (P > .05 for all parameters). At 6 months, CDVA, K1, and Kmean showed higher improvement in the non-CXL group than the CXL group (P = .030, .018, and .039, respectively). CONCLUSIONS: CAIRS surgery has a flattening effect on both the corneas with and without CXL. The cornea with prior CXL treatment had less flattening effect due to the stiffening effect of prior CXL. [J Refract Surg. 2024;40(6):e392-e397.].

Impact of keratocyte differentiation on corneal opacity resolution and visual function recovery in male rats.

Intrastromal cell therapy utilizing quiescent corneal stromal keratocytes (qCSKs) from human donor corneas emerges as a promising treatment for corneal opacities, aiming to overcome limitations of traditional surgeries by reducing procedural complexity and donor dependency. This investigation demonstrates the therapeutic efficacy of qCSKs in a male rat model of corneal stromal opacity, underscoring the significance of cell-delivery quality and keratocyte differentiation in mediating corneal opacity resolution and visual function recovery. Quiescent CSKs-treated rats display improvements in escape latency and efficiency compared to wounded, non-treated rats in a Morris water maze, demonstrating improved visual acuity, while stromal fibroblasts-treated rats do not. Advanced imaging, including multiphoton microscopy, small-angle X-ray scattering, and transmission electron microscopy, revealed that qCSK therapy replicates the native cornea's collagen fibril morphometry, matrix order, and ultrastructural architecture. These findings, supported by the expression of keratan sulfate proteoglycans, validate qCSKs as a potential therapeutic solution for corneal opacities.

Combined topography-guided photorefractive keratectomy and corneal collagen crosslinking.

PURPOSE: This study aimed to report the long-term results of combined topography-guided photorefractive keratectomy (PRK) and accelerated corneal collagen cross-linking (CXL) for keratoconus using the Zeiss refractive coordinated system. METHODS: A prospective interventional study was conducted in a tertiary eye care hospital in South India. Patients with mild-to-moderate progressive keratoconus and corneal pachymetry greater than 450 µm were included. They underwent customized topography-guided PRK followed by CXL. Uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), and keratometry readings and complications were evaluated at 1, 3, 6, 12, and 24 months postoperatively. RESULTS: Thirty patients (30 eyes) were included in the study. All study parameters showed a statistically significant improvement postoperatively over baseline values. At 24 months, the mean UDVA improved from 0.8 ± 0.180 logarithm of the minimum angle of resolution (logMAR) to 0.38 ± 0.118 logMAR ( P -value <0.001) and CDVA improved from 0.467 ± 0.142 logMAR to 0.227 ± 0.078 logMAR ( P -value <0.001). The mean flat, steep, and maximum keratometry values were significantly reduced by 2.133, 3, and 4.54 diopters, respectively, at the last follow-up examination ( P -value <0.001). CONCLUSION: The combined topography-guided PRK and accelerated CXL procedure seem to be a promising treatment alternative for early keratoconus. This is the first such study on the Zeiss refractive coordinated system. However, further studies with a larger study population and longer follow-up periods are required to draw final conclusions about the benefits of this procedure in keratoconus.

Second harmonic generation microscopy of electromechanical reshaping on corneal collagen.

Refractive errors remain a global health concern, as a large proportion of the world's population is myopic. Current ablative approaches are costly, not without risks, and not all patients are candidates for these procedures. Electromechanical reshaping (EMR) has been explored as a viable cost-effective modality to directly shape tissues, including cartilage. In this study, stromal collagen structure and fibril orientation was examined before and after EMR with second-harmonic generation microscopy (SHG), a nonlinear multiphoton imaging method that has previously been used to study native corneal collagen with high spatial resolution. EMR, using a milled metal contact lens and potentiostat, was performed on the corneas of five extracted rabbit globes. SHG was performed using a confocal microscopy system and all images underwent collagen fibril orientation analysis. The collagen SHG signal in controls is uniform and is similarly seen in samples treated with pulsed potential, while continuous EMR specimens have reduced, nonhomogeneous signal. Collagen fibril orientation in native tissue demonstrates a broad distribution with suggestion of another peak evolving, while with EMR treated eyes a bimodal characteristic becomes readily evident. Pulsed EMR may be a means to correct refractive errors, as when comparing its SHG signal to negative control, preservation of collagen structures with little to no damage is observed. From collagen fiber orientation analysis, it can be inferred that simple DC application alters the structure of collagen. Future studies will involve histological assessment of these layers and multi-modal imaging analysis of dosimetry.

Update on corneal crosslinking for keratoconus and corneal ectasia.

PURPOSE OF REVIEW: To review corneal crosslinking for keratoconus and corneal ectasia, and recent developments in the field. This study will review the mechanism of crosslinking, clinical approaches, current results, and potential future innovations. RECENT FINDINGS: Corneal crosslinking for keratoconus was first approved by U.S. FDA in 2016. Recent studies have confirmed the general long-term efficacy of the procedure in decreasing progression of keratoconus and corneal ectasia. New types of crosslinking protocols, such as transepithelial treatments, are under investigation. In addition, adjunctive procedures have been developed to improve corneal contour and visual function in these patients. SUMMARY: Crosslinking has been found to be well tolerated and effective with the goal of decreasing progression of ectatic corneal diseases, keratoconus and corneal ectasia after refractive surgery. Studies have shown its long-term efficacy. New techniques of crosslinking and adjunctive procedures may further improve treatments and results.

Long-term outcomes of corneal crosslinking.

PURPOSE OF REVIEW: This manuscript summarizes contemporary research from 2018 to 2023 evaluating long-term (≥2 years) outcomes of corneal crosslinking (CXL) for progressive keratoconus (KCN). RECENT FINDINGS: The standard Dresden protocol (SDP) has been utilized clinically since the early 2000 s to treat ectatic disorders, primarily progressive KCN and postrefractive ectasia. Various modifications have since been introduced including accelerated and transepithelial protocols, which are aimed at improving outcomes or reducing complications. This review summarizes data demonstrating that the SDP halts disease progression and improves various visual and topographic indices (UDVA, CDVA, Kmax, K1, K2) up to 13 years postoperatively. Accelerated and transepithelial protocols have been found to be well tolerated alternatives to SDP with similar efficacy profiles. Studies focusing on pediatric populations identified overall higher progression rates after CXL. All protocols reviewed had excellent safety outcomes in adults and children. SUMMARY: Recent studies revealed that SDP successfully stabilizes KCN long term, and a variety of newer protocols are also effective. Pediatric patients may exhibit higher progression rates after CXL. Further research is required to enhance the efficacy and ease of these protocols.

Comparative evaluation of biomechanical changes and aberration profile following accelerated collagen cross-linking using hypo-osmolar and iso-osmolar riboflavin: A prospective study.

PURPOSE: To compare the changes encountered in corneal biomechanics and aberration profile following accelerated corneal collagen cross-linking (CXL) using hypo-osmolar and iso-osmolar riboflavin in corneal thicknesses of <400 and >400 microns, respectively. METHODS: This is a prospective, interventional, comparative study involving 100 eyes of 75 patients with progressive keratoconus. Eyes were divided into two groups based on corneal thickness: group 1 included eyes with a corneal thickness of <400 microns who underwent hypo-osmolar CXL, and group 2 included eyes with a corneal thickness of >400 microns who underwent iso-osmolar CXL. Corneal biomechanical and aberration profiles were evaluated and compared between groups. RESULTS: In group 1, all higher-order aberrations (HOA) except secondary astigmatism significantly decreased from baseline; however, in group 2, only coma and trefoil decreased. The corneal resistance factor and corneal hysteresis significantly improved in both groups, which was significantly greater in group 2 than in group 1. The change in inverse radius, deformation amplitude, and tomographic biomechanical index was significantly improved in group 2 as compared to group 1. CONCLUSION: Improvement in corrected distance visual acuity and decrease in HOA were significantly better in the hypo-osmolar CXL group; however, the improvement in biomechanical strength of the cornea was significantly better in the iso-osmolar group.

Two-phase mechanism in the treatment of corneal stromal fibrosis with topical losartan.

Recent studies in rabbits and case reports in humans have demonstrated the efficacy of topical losartan in the treatment of corneal scarring fibrosis after a wide range of injuries, including chemical burns, infections, surgical complications, and some diseases. It is hypothesized that the effect of losartan on the fibrotic corneal stroma occurs through a two-phase process in which losartan first triggers the elimination of myofibroblasts by directing their apoptosis via inhibition of extracellular signal-regulated kinase (ERK)-mediated signal transduction, and possibly through signaling effects on the viability and development of corneal fibroblast and fibrocyte myofibroblast precursor cells. This first step likely occurs within a week or two in most corneas with fibrosis treated with topical losartan, but the medication must be continued for much longer until the epithelial basement membrane (EBM) is fully regenerated or new myofibroblasts will develop from precursor cells. Once the myofibroblasts are eliminated from the fibrotic stroma, corneal fibroblasts can migrate into the fibrotic tissue and reabsorb/reorganize the disordered extracellular matrix (ECM) previously produced by the myofibroblasts. This second stage is longer and more variable in different eyes of rabbits and humans, and accounts for most of the variability in the time it takes for the stromal opacity to be markedly reduced by topical losartan treatment. Eventually, keratocytes reemerge in the previously fibrotic stromal tissue to fine-tune the collagens and other ECM components and maintain the normal structure of the corneal stroma. The efficacy of losartan in the prevention and treatment of corneal fibrosis suggests that it acts as a surrogate for the EBM, by suppressing TGF beta-directed scarring of the wounded corneal stroma, until control over TGF beta action is re-established by a healed EBM, while also supporting regeneration of the EBM by allowing corneal fibroblasts to occupy the subepithelial stroma in the place of myofibroblasts.

Evaluation of the results of contact lens assisted corneal cross-linking treatment in keratoconus patients with thin corneas.

PURPOSE: We aimed to compare the efficacy and safety of accelerated contact lens-assisted cross-linking (CA-CXL) with Lotrafilcon B and Comfilcon A lenses in keratoconus (KC) patients with thin corneas. STUDY DESIGN: Retrospective, single-center study. MATERIALS AND METHODS: We retrospectively included 51 eyes of 39 KC patients with corneal thickness <400µm after epithelial scraping (Epi-off), who underwent accelerated CA-CXL treatment with Lotrafilcon B (n=20) and Comfilcon A (n=31). Uncorrected and corrected distance visual acuity (UDVA and CDVA), manifest refraction values, corneal topographic data and endothelial cell density were recorded at preoperative and postoperative 1st, 3rd and 6th month controls. RESULTS: CDVA in the Comfilcon A group was higher than CDVA before surgery at 6 months postoperatively (p<0.001). When the two lenses were compared, CDVA was found to be significantly higher in the Lotrafilcon B group in the preoperative, postoperative 1st month and 3rd month values, but there was no significant difference between the postoperative 6th month values (p=0.028, p=0.018, p=0.044, p=0.181, respectively). The maximum keratometry (Kmax) value at the 6th month after surgery in the Comfilcon A group was significantly lower than in the Lotrafilcon B group (p=0,009). There was no significant difference between the endothelial cell density values between the groups (p=0.623, p=0.609, p=0.794, p=0.458, respectively). There was no significant difference between the progression, regression, and stability rates of the two groups (p=0.714). CONCLUSIONS: Accelerated CA-CXL with Lotrafilcon B and Comfilcon A silicone hydrogel lenses is a safe and effective method to stop progression in patients with thin corneas.

Cornea-Specific Human Adipose Stem Cell-Derived Extracellular Matrix for Corneal Stroma Tissue Engineering.

Utilizing tissue-specific extracellular matrices (ECMs) is vital for replicating the composition of native tissues and developing biologically relevant biomaterials. Human- or animal-derived donor tissues and organs are the current gold standard for the source of these ECMs. To overcome the several limitations related to these ECM sources, including the highly limited availability of donor tissues, cell-derived ECM offers an alternative approach for engineering tissue-specific biomaterials, such as bioinks for three-dimensional (3D) bioprinting. 3D bioprinting is a state-of-the-art biofabrication technology that addresses the global need for donor tissues and organs. In fact, there is a vast global demand for human donor corneas that are used for treating corneal blindness, often resulting from damage in the corneal stromal microstructure. Human adipose tissue is one of the most abundant tissues and easy to access, and adipose tissue-derived stem cells (hASCs) are a highly advantageous cell type for tissue engineering. Furthermore, hASCs have already been studied in clinical trials for treating corneal stromal pathologies. In this study, a corneal stroma-specific ECM was engineered without the need for donor corneas by differentiating hASCs toward corneal stromal keratocytes (hASC-CSKs). Furthermore, this ECM was utilized as a component for corneal stroma-specific bioink where hASC-CSKs were printed to produce corneal stroma structures. This cost-effective approach combined with a clinically relevant cell type provides valuable information on developing more sustainable tissue-specific solutions and advances the field of corneal tissue engineering.

Randomized clinical trial comparing customized corneal crosslinking: epi-on in high oxygen and epi-off in room air for keratoconus.

PURPOSE: To compare clinical outcomes of customized transepithelial (epi-on) corneal crosslinking (CXL) in high oxygen and customized CXL with epithelial removal (epi-off) in room air for keratoconus (KC). SETTING: Umeå University Hospital, Umeå, Sweden. DESIGN: Prospective, randomized, single-masked, intraindividually comparing study. METHODS: 32 participants with bilateral progressive KC were treated with bilateral customized topography-guided CXL, 30 mW/cm 2 ; 7.2 to 15 J/cm 2 and were randomized to epi-on in one eye (32 eyes) and epi-off in the fellow eye (32 eyes). Uncorrected distance visual acuity (UDVA) and corrected distance visual acuity (CDVA), maximal keratometry (Kmax), subjective ocular discomfort, low-contrast visual acuities (LCVAs) at 10% and 2.5% contrast, ocular and anterior corneal wavefront aberrations, manifest refractive spherical equivalent, endothelial cell count (ECC), and adverse events were assessed through 24 months. RESULTS: Both treatments showed improvements at 24 months in UDVA; -0.16 ± 0.24 ( P < .001) and -0.13 ± 0.20 logMAR ( P = .006), respectively, CDVA; -0.10 ± 0.11 ( P < .001) and -0.10 ± 0.12 ( P = .001), Kmax; -1.74 ± 1.31 ( P < .001) and -1.72 ± 1.36 D ( P < .001). LCVA 10% improved for both protocols ( P < .001), but LCVA 2.5% improved for epi-on CXL only ( P = .001). ECC was unaltered, and no adverse events occurred. The epi-on eyes had significantly less discomfort symptoms during the whole first week posttreatment ( P < .05). CONCLUSIONS: High-oxygen customized epi-on CXL is a viable alternative to room air customized epi-off CXL, with faster improvements in CDVA and LCVA and less early ocular discomfort.

Substance P promotes transforming growth factor-ß-induced collagen synthesis in human corneal fibroblasts.

Corneal fibroblasts maintain homeostasis of the corneal stroma by mediating the synthesis and degradation of extracellular collagen, and these actions are promoted by transforming growth factor-ß (TGF-ß) and interleukin-1ß (IL-1ß), respectively. The cornea is densely innervated with sensory nerve fibers that are not only responsible for sensation but also required for physiological processes such as tear secretion and wound healing. Loss or dysfunction of corneal nerves thus impairs corneal epithelial wound healing and can lead to neurotrophic keratopathy. The sensory neurotransmitter substance P (SP) promotes corneal epithelial wound healing by enhancing the stimulatory effects of growth factors and fibronectin. We have now investigated the role of SP in collagen metabolism mediated by human corneal fibroblasts in culture. Although SP alone had no effect on collagen synthesis or degradation by these cells, it promoted the stimulatory effect of TGF-ß on collagen type I synthesis without affecting that of IL-1ß on the expression of matrix metalloproteinase-1. This effect of SP on TGF-ß-induced collagen synthesis was accompanied by activation of p38 mitogen-activated protein kinase (MAPK) signaling and was attenuated by pharmacological inhibition of p38 or of the neurokinin-1 receptor. Our results thus implicate SP as a modulator of TGF-ß-induced collagen type I synthesis by human corneal fibroblasts, and they suggest that loss of this function may contribute to the development of neurotrophic keratopathy.NEW & NOTEWORTHY This study investigates the role of substance P (SP) in collagen metabolism mediated by human corneal fibroblasts in culture. We found that, although SP alone had no effect on collagen synthesis or degradation by corneal fibroblasts, it promoted the stimulatory effect of transforming growth factor-ß on collagen type I synthesis without affecting that of interleukin-1ß on the expression of matrix metalloproteinase-1.

A comparison of keratoconus progression following collagen cross-linkage using standard or personalised keratometry thresholds.

OBJECTIVE: To define how estimates of keratoconus progression following collagen cross-linking (CXL) vary according to the parameter selected to measure corneal shape. MATERIALS AND METHODS: We estimated progression following CXL in 1677 eyes. We compared standard definitions of keratoconus progression based on published thresholds for Kmax, front K2, or back K2, or progression of any two of these three parameters, with the option of an increased threshold for Kmax values ≥ 55D. As corneal thickness reduces unpredictably after CXL, it was excluded from the principal analysis. We then repeated the analysis using novel adaptive estimates of progression for Kmax, front K2, or back K2, developed separately using 6463 paired readings from keratoconus eyes, with a variation of the Bland-Altman method to determine the 95% regression-based limits of agreement (LoA). We created Kaplan-Meier survival plots for both standard and adaptive thresholds. The primary outcome was progression five years after a baseline visit 9-15 months following CXL. RESULTS: Progression rates were 8% with a standard (≥ 1.5D) threshold for K2 or 6% with the static multi-parameter definition. With a ≥ 1D threshold for Kmax, the progression was significantly higher at 29%. With adaptive Kmax or K2, the progression rates were similar (20%) but less than with the adaptive multi-parameter method (22%). CONCLUSIONS: Estimates of keratoconus progression following CXL vary widely according to the reference criteria. Using adaptive thresholds (LoA) to define the repeatability of keratometry gives estimates for progression that are markedly higher than with the standard multi-parameter method.

Dynamic changes of endothelial and stromal cilia are required for the maintenance of corneal homeostasis.

Primary cilia are distributed extensively within the corneal epithelium and endothelium. However, the presence of cilia in the corneal stroma and the dynamic changes and roles of endothelial and stromal cilia in corneal homeostasis remain largely unknown. Here, we present compelling evidence for the presence of primary cilia in the corneal stroma, both in vivo and in vitro. We also demonstrate dynamic changes of both endothelial and stromal cilia during corneal development. In addition, our data show that cryoinjury triggers dramatic cilium formation in the corneal endothelium and stroma. Furthermore, depletion of cilia in mutant mice lacking intraflagellar transport protein 88 compromises the corneal endothelial capacity to establish the effective tissue barrier, leading to an upregulation of α-smooth muscle actin within the corneal stroma in response to cryoinjury. These observations underscore the essential involvement of corneal endothelial and stromal cilia in maintaining corneal homeostasis and provide an innovative strategy for the treatment of corneal injuries and diseases.

Unilateral Granular Type 2 Corneal Dystrophy With Exacerbation After LASIK.

PURPOSE: The aim of this study was to report a case of unilateral granular corneal dystrophy type 2 (GCD2) with exacerbation after bilateral laser in situ keratomileusis (LASIK). METHODS: Clinical evaluation, Scheimpflug imaging, anterior segment optical coherence tomography (AS-OCT), cytology, and genetic testing were used to confirm the diagnosis of unilateral GCD2 with exacerbation after bilateral LASIK. Detailed literature review for possible unilateral GCD2 presentations was performed. RESULTS: A 54-year-old White woman presented with blurred vision in her left eye and a history of bilateral LASIK performed 8 years before. Examination revealed dense opacities in the left cornea only, which were confirmed to be confined to the LASIK interface and adjacent corneal stromal tissue, as determined by AS-OCT. The patient underwent flap lift, interface debris removal, and stromal bed phototherapeutic keratectomy. Cytological analysis showed eosinophilic corneal stromal deposits that stained with trichrome stain and were congophilic on Congo red stain. Genetic testing was positive for heterozygous GCD2 transforming growth factor ß-induced gene ( TGFBI ), c.371G>A, p.R124H mutation. There were no opacities identifiable in the right eye on serial slit-lamp examination, Scheimpflug imaging, or OCT imaging at 4 or 8 years after bilateral LASIK. Literature review failed to identify any previous reports of unilateral GCD2. CONCLUSIONS: This is the first known reported case of unilateral granular corneal dystrophy type 2. LASIK is contraindicated in eyes with corneal stromal dystrophies related to mutations in TGFBI as both flap creation and laser ablation can exacerbate visually significant opacity formation. Scheimpflug and AS-OCT imaging are useful to identify opacities in GCD2.

Effects of hypoxia in the diabetic corneal stroma microenvironment.

Corneal dysfunctions associated with Diabetes Mellitus (DM), termed diabetic keratopathy (DK), can cause impaired vision and/or blindness. Hypoxia affects both Type 1 (T1DM) and Type 2 (T2DM) surprisingly, the role of hypoxia in DK is unexplored. The aim of this study was to examine the impact of hypoxia in vitro on primary human corneal stromal cells derived from Healthy (HCFs), and diabetic (T1DMs and T2DMs) subjects, by exposing them to normoxic (21% O2) or hypoxic (2% O2) conditions through 2D and 3D in vitro models. Our data revealed that hypoxia affected T2DMs by slowing their wound healing capacity, leading to significant alterations in oxidative stress-related markers, mitochondrial health, cellular homeostasis, and endoplasmic reticulum health (ER) along with fibrotic development. In T1DMs, hypoxia significantly modulated markers related to membrane permeabilization, oxidative stress via apoptotic marker (BAX), and protein degradation. Hypoxic environment induced oxidative stress (NOQ1 mediated reduction of superoxide in T1DMs and Nrf2 mediated oxidative stress in T2DMs), modulation in mitochondrial health (Heat shock protein 27 (HSP27), and dysregulation of cellular homeostasis (HSP90) in both T1DMs and T2DMs. This data underscores the significant impact of hypoxia on the diabetic cornea. Further studies are warranted to delineate the complex interactions.

Corneal stromal localization of TGF beta isoforms in spontaneous persistent epithelial defects after PRK in rabbits.

The purpose of this study was to evaluate transforming growth factor beta (TGFß) isoform localization in rabbit corneas with spontaneous persistent epithelial defects (PEDs) after photorefractive keratectomy (PRK). Four cryofixed corneas from a previously reported series of PEDs in rabbits that had PRK were evaluated with triplex immunohistochemistry (IHC) for TGFß3, myofibroblast marker alpha-smooth muscle actin (α-SMA) and mesenchymal marker vimentin. One cornea had sufficient remaining tissue for triplex IHC for TGFß1, TGFß2, or TGFß3 (each with α-SMA and vimentin) using isoform-specific antibodies. All three TGFß isoforms were detected in the subepithelial stroma at and surrounding the PED. Some of each TGFß isoform co-localized with α-SMA of myofibroblasts, which could be TGFß isoform autocrine production by myofibroblasts or TGFß-1, -2, and -3 binding to these myofibroblasts.

How Would Nature See Our Corneal Triumphs? The LXXIX Edward Jackson Lecture.

PURPOSE: To describe discrepancies between clinical observation and current teachings in corneal endothelial disease, particularly in Fuchs endothelial dystrophy and its potential association with primary open angle glaucoma. DESIGN: Perspective. METHODS: A perspective is presented on Fuchs dystrophy, a disorder that commonly presents with a compromised endothelium but minimal stromal edema, indicating that the corneal imbibition pressure is relatively "too high." RESULTS: The discrepancy between the relative lack of stromal edema in the absence of an endothelial cell layer cannot be explained by the current theories involving a circulatory pumping mechanism over the endothelial cell layer, but may point to the following: (1) secondary involvement of the corneal endothelium in Fuchs dystrophy; (2) separate hydration systems for maintaining the imbibition pressure (vertical static hydration) and corneal nutrition (horizontal dynamic hydration); (3) the cornea as net contributor of aqueous humor; (4) a close relationship between the corneal imbibition and intraocular pressure, with potentially a shared regulatory system; and (5) a potential steroid-type hormone dependency of this regulatory system. CONCLUSIONS: Clinical observation shows that the stromal imbibition pressure is "too high" in Fuchs endothelial dystrophy, indicating that it may not primarily be an endothelial disease, but a type of "corneal glaucoma."

Mesenchymal Stem Cell-Laden In Situ-Forming Hydrogel for Preventing Corneal Stromal Opacity.

PURPOSE: The aims of this study were to construct a mesenchymal stem cell (MSC)-laden in situ-forming hydrogel and study its effects on preventing corneal stromal opacity. METHODS: The native gellan gum was modified by high temperature and pressure, and the rabbit bone marrow MSCs were encapsulated before adding Ca 2+ to initiate cross-linking. The effects of the hydrogel on 3D culture and gene expression of the rabbit bone marrow MSCs were observed in vitro. Then, the MSC-hydrogel was used to repair corneal stromal injury in New Zealand white rabbits within 28 days postoperation. RESULTS: The short-chain gellan gum solution has a very low viscosity (<0.1 Pa·s) that is ideal for encapsulating cells. Moreover, mRNA expressions of 3D-cultured MSCs coding for corneal stromal components (decorin, lumican, and keratocan) were upregulated (by 127.8, 165.5, and 25.4 times, respectively) ( P < 0.05) on day 21 in vitro and were verified by Western blotting results. For the in vivo study, the corneal densitometry of the experimental group was (20.73 ± 1.85) grayscale units which was lower than the other groups ( P < 0.05). The MSC-hydrogel downregulated mRNA expression coding for fibrosis markers (α-smooth muscle actin, vimentin, collagen type 5-α1, and collagen type 1-α1) in the rabbit corneal stroma. Furthermore, some of the 5-ethynyl-2'-deoxyuridine (EdU)-labeled MSCs integrated into the upper corneal stroma and expressed keratocyte-specific antigens on day 28 postoperation. CONCLUSIONS: The short-chain gellan gum allows MSCs to slowly release to the corneal stromal defect and prevent corneal stromal opacity. Some of the implanted MSCs can integrate into the corneal stroma and differentiate into keratocytes.