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Purpose: To investigate the safety and efficacy of cultured human corneal endothelial cell (hCEC) injection therapy with mature differentiated (mature) cell subpopulations (SPs) for corneal endothelial failure (CEF)., Design: Comparative, interventional case series., Methods: This study involved 18 eyes with CEF that underwent cultured hCEC injection therapy, categorized into 2 groups: (1) 11 eyes administered a relatively lower proportion (0.1 to 76.3%) of mature cell SPs (group 1 [Gr1]), and (2) 7 eyes administered a relatively higher proportion (>90%) of mature cell SPs (group 2 [Gr2]). From 1 week to 3 years postoperation, corneal endothelial cell (CEC) density (CECD), central corneal thickness (CCT), and best-corrected visual acuity (BCVA) were recorded, and the CEC parameter's "spring constant" was calculated. The proportion of mature SPs was evaluated by fluorescence-activated cell sorting analysis based on cell-surface markers., Results: At 3 years postoperation, corneal restoration with improved BCVA was attained in 10 of the 11 Gr1 eyes and all Gr2 eyes, the median CECD in Gr2 (3083 cells/mm 2 ; range, 2182-4417 cells/mm 2 ) was higher than that in Gr1 (1349 cells/mm 2 ; range, 746-2104 cells/mm 2 ) (P < .001), and the spring constant verified the superiority of the mature cultured hCECs. From 24 weeks through 3 years postoperation, the median percentage of CECD decrease was 3.2% in Gr2 and 23.6% in Gr1 (P < .005). CCT recovery was prompt and constant in Gr2, while diverse in Gr1. No adverse events were observed., Conclusion: Our findings showed that mature cell SPs for hCEC injection therapy provide rapid recovery of CCT, better CECD, and low CECD attrition over 3 years postsurgery., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: This study investigated the safety and efficacy of orthokeratology in myopic children in Japan., Study Design: Prospective clinical trial., Methods: This study enrolled myopic children aged 6-16 years with a spherical equivalent of -1.00 D to -4.00 D and astigmatism of -1.5 D or lower, whose parents could manage contact lens use and could provide written informed consent. The children were treated with orthokeratology lenses (BREATH-O CORRECT R, Universal View Co., Ltd.) for 3 years. Slit-lamp findings, visual acuity, intraocular pressure, subjective refraction, corneal topography, corneal endothelial cell density, corneal thickness, and axial length were regularly assessed., Results: This study included 96 eyes of 48 patients (average age, 10.7 ± 2.08 years). The average baseline spherical equivalent was -2.46 ± 0.97 D. The average baseline uncorrected visual acuity was 0.74 ± 0.32 logMAR, with significant improvement to -0.08 ± 0.18 logMAR at 4 weeks and 0.02 ± 0.21 logMAR at 3 years (both p < 0.001, Dunnett's test). The average baseline corneal endothelial cell density was 3053 ± 181 cells/mm 2 and 3028 ± 213 cells/mm 2 at postoperative 3 years (p = 0.9933, Dunnett's test). The average axial length was 24.70 ± 0.78 mm at baseline, 24.77 ± 0.80 mm at 12 weeks, and 25.32 ± 0.85 mm at 36 months. Although mild corneal epithelial disorders occurred in 18% of eyes, they were successfully treated with eye drops, enabling resumption of lens usage. No serious adverse events were observed., Conclusion: Orthokeratology lens (BREATH-O CORRECT R) use in children demonstrated good efficacy and safety during 3 years of follow-up., (© 2021. Japanese Ophthalmological Society.)

Purpose: To evaluate the diagnostic ability of the ratio of anterior and posterior corneal surface areas (As/Ps) comparing with other keratoconus screening indices in distinguishing forme fruste keratoconus (FFKC) from normal eyes., Methods: In this comparative study, 13 eyes of 13 patients with FFKC, 29 eyes of 29 patients with keratoconus (KC) and 88 eyes of 88 patients with normal subjects were involved. The As/Ps measured by the anterior segment optical coherence tomography (AS-OCT) and other indices measured by AS-OCT and rotating Scheimpflug-based corneal tomography were evaluated. The area under receiver-operating-characteristics (AU-ROC) was calculated to assess the diagnostic ability in discriminating FFKC from normal eyes., Results: The As/Ps, the Belin/Ambrosio display enhanced ectasia total derivation value (BAD-D) and posterior and anterior elevation values showed the AU-ROC 0.9 or more in differentiating FFKC from normal eyes (0.980, 0.951, 0.924 and 0.903, respectively). The sensitivity and specificity were 0.92 and 0.96 for the As/Ps, 1.00 and 0.90 for BAD-D, 0.85 and 0.86 for posterior elevation value, and 0.85 and 0.96 for anterior elevation value, respectively., Conclusions: Among the several indices for keratoconus screening which we evaluated, the As/Ps obtained by AS-OCT had the large AU-ROC with high sensitivity and specificity in detecting FFKC, which was comparable with BAD-D obtained by rotating Scheimpflug-based corneal tomography. The As/Ps may provide information for improving the diagnostic accuracy of KC, even in the initial stage of the disease., Competing Interests: The authors have declared that no competing interests exist.

To assess the indications, outcomes and time to recurrence of phototherapeutic keratectomy (PTK) for anterior corneal pathology.This study involved 714 eyes of 477 consecutive patients (mean age: 66.0 ± 15.2 years; range: 6-101 years) who underwent PTK as the initial surgical intervention for an anterior corneal pathology. In case of each patient, the cornea treated by PTK, followed up by slit-lamp examination and corrected distance visual acuity (CDVA) testing. Main outcome measures included Slit-lamp findings (1), CDVA (2), patients' complaints (3).The mean follow-up period was 44.0 ± 38.8 months (range: 1-156 months).We treated 376 granular corneal dystrophy (GCD) eyes, 238 band keratopathy (BK) eyes, 23 epithelium attachment disorder eyes, 16 gelatinous drop-like corneal dystrophy (GDLD) eyes, 13 lattice corneal dystrophy (LCD) eyes, and 48 eyes with other corneal diseases. The CDVA significantly improved from LogMAR 0.65 ± 0.61 pre PTK to LogMAR 0.26 ± 0.39 post PTK. A 2 or more lines increase of CDVA was observed in GCD eyes (67.8%), BK eyes (49.2%), epithelium attachment disorder eyes (57.1%), GDLD eyes (87.5%), LCD eyes (76.9%), and other corneal disease eyes (60.4%). The recurrence of BK was rare. GCD recurred slowly. Epithelium attachment disorder eyes remitted simultaneously, and recurred comparatively faster.PTK was proved to be a successful therapy for all 6 corneal disease categories. Disease recurrence after PTK differed among the diseases, and surgeons should recognize the different rates of disease recurrence after PTK surgery.

Keratoconus (KC) is an ectatic disorder with a high prevalence rate. However, the exact cause of the disease and possible underlying mechanisms of development remain unclear. In this present study, we aimed to investigate the anterior and the posterior corneal surface area in normal, forme fruste keratoconus (FFKC), and keratoconic eyes (as a reference group) using anterior segment optical coherence tomography (AS-OCT) in order to assess the pathological change of KC. The surface areas of the anterior or posterior cornea, and the anterior-posterior (As/Ps) ratio of corneal surface area, were measured at the central 5.0 mm-, 6.0 mm-, and 7.0 mm-diameter areas via AS-OCT, and a comparison between the normal eyes and FFKC eyes was then performed using the Mann-Whitney U test. The posterior surface area at the central 5.0 mm areas in the FFKC eyes (20.430 mm 2 ) and KC eyes (20.917 mm 2 ) seemed to become larger than that of normal eyes (20.389 mm 2 ) (normal vs FFKC; P = 0.06). Moreover, the As/Ps of the corneal surface area in the FFKC eyes (0.986) and the KC eyes (0.976) was significantly smaller than that of the normal eyes (0.988) (normal vs FFKC; P < 0.01). Anterior and posterior corneal surface area imbalance may reflect keratoconic eyes at the early stage of the disease.

Purpose: To evaluate the effect of corneal vesicles in patients with posterior corneal vesicles (PCV) on corneal endothelial cell (CEC) density and the acquisition of amblyopia., Methods: In this retrospective study of patients with PCV (18 eyes of 14 patients), CEC density was examined by noncontact specular microscopy during each follow-up examination. Best-corrected visual acuity and the objective refractive error were also examined., Results: Of the 14 total patients, 10 were diagnosed with PCV and 4 were diagnosed with bilateral suspected PCV or posterior polymorphous corneal dystrophy (PPCD), and in all patients, no ocular abnormality other than corneal vesicles was observed. In patients with PCV and patients with bilateral suspected PCV or PPCD, mean CEC density was 1131 ± 338 and 1095 ± 492 cells/mm, respectively. In both PCV group and the bilateral suspected PCV or PPCD-group patients who were followed for 164.2 ± 25.4 months (range: 123-186 months), CEC density tended not to decrease. In patients with PCV, the mean best-corrected visual acuity of the unaffected eyes was significantly higher than that of the affected eyes with corneal vesicles (-0.10 ± 0.06 and 0.05 ± 0.13, respectively, P = 0.012). Four of 9 affected eyes (1 eye was excluded because of retinal atrophy) exhibited amblyopia, and all 4 eyes had astigmatism higher than 2 diopters., Conclusions: Although CEC density of patients with PCV in this study was found to be stable over a long-term follow-up period, strict attention should be given to the possibility of amblyopia in eyes with PCV.

Background: Corneal endothelial cell (CEC) disorders, such as Fuchs's endothelial corneal dystrophy, induce abnormal corneal hydration and result in corneal haziness and vision loss known as bullous keratopathy. We investigated whether injection of cultured human CECs supplemented with a rho-associated protein kinase (ROCK) inhibitor into the anterior chamber could increase CEC density., Methods: We performed an uncontrolled, single-group study involving 11 persons who had received a diagnosis of bullous keratopathy and had no detectable CECs. Human CECs were cultured from a donor cornea; a total of 1×10 6 passaged cells were supplemented with a ROCK inhibitor (final volume, 300 μl) and injected into the anterior chamber of the eye that was selected for treatment. After the procedure, patients were placed in a prone position for 3 hours. The primary outcome was restoration of corneal transparency, with a CEC density of more than 500 cells per square millimeter at the central cornea at 24 weeks after cell injection. Secondary outcomes were a corneal thickness of less than 630 μm and an improvement in best corrected visual acuity equivalent to two lines or more on a Landolt C eye chart at 24 weeks after cell injection., Results: At 24 weeks after cell injection, we recorded a CEC density of more than 500 cells per square millimeter (range, 947 to 2833) in 11 of the 11 treated eyes (100%; 95% confidence interval [CI], 72 to 100), of which 10 had a CEC density exceeding 1000 cells per square millimeter. A corneal thickness of less than 630 μm (range, 489 to 640) was attained in 10 of the 11 treated eyes (91%; 95% CI, 59 to 100), and an improvement in best corrected visual acuity of two lines or more was recorded in 9 of the 11 treated eyes (82%; 95% CI, 48 to 98)., Conclusions: Injection of human CECs supplemented with a ROCK inhibitor was followed by an increase in CEC density after 24 weeks in 11 persons with bullous keratopathy. (Funded by the Japan Agency for Medical Research and Development and others; UMIN number, UMIN000012534 .).

Aim: To investigate the viability of donor corneal endothelial cells (CECs) preserved in storage media by histological examination., Methods: Twenty-eight donor corneas were obtained from SightLife Eye Bank (Seattle, Washington), and redundant peripheral portions of those corneas were used for histological examination after removal of the centre corneal graft for transplantation. To assess cell viability in the corneal endothelium, biostaining experiments were performed using propidium iodide, calcein-AM, Hoechst 33 342, annexin V, anti-vimentin antibody and toluidine blue., Results: Histological analysis of the endothelium showed that the cytoplasm of dead cells had low-intensity fluorescence and that their nuclei stained red, while almost all living cells had green cytoplasm and blue-stained nuclei. The mean dead cell rate in the 28 donor corneas was 4.9%±3.3% (mean ±SD) (range: 0.6%-10.5%). The propidium iodide-positive cells stained positive for annexin V, negative for vimentin and pale for toluidine blue. After the specimens were incubated in a culture medium, the red nucleus dead cells dropped off from the level of the blue nucleus living cells., Conclusion: Our findings showed the existence of dead cells in storage-media-preserved donor corneal endothelium and that they dropped off after incubation, thus suggesting that the decrease of CECs following keratoplasty may be related to the presence of dead cells., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Ocular surface reconstruction (OSR) using tissue-engineered cultivated oral mucosal epithelial cell sheets (COMECS) is a promising newly developed treatment for patients with severe ocular surface disease. Until now, this technique has used exogenic and undefined components such as mouse-derived 3T3 feeder cells and fetal bovine serum. To minimize associated risks of zoonotic infection or transmission of unknown pathogens and so establish a safe and effective protocol for the next generation of treatment modality, we developed a novel technique for the COMECS protocol, using a feeder-free and serum-free (FFSF) culture system. Following this new protocol, COMECS exhibited 4-5 layers of well-stratified and differentiated cells, and we successfully produced functional COMECS that included holoclone-type stem cells. Immunohistochemistry confirmed the presence of markers for cell junction (ZO1, Desmoplakin), basement membrane assembly (Collagen 7, Laminin 5), differentiation (K13, K3), proliferation (Ki67) and stem/progenitor cells (p75) in the FFSF COMECS. When transplanted to the ocular surfaces of rabbits, the tissue survived for up to 2 weeks. This study represents a first step toward assessing the development of functional FFSF COMECS for safe and ideal OSR.

Purpose: To suggest an objective score for grading the acute ocular severity of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN), and to determine predictive factors for severe acute ocular involvement such as ocular surface epithelial defect and/or pseudomembrane formation., Design: Retrospective cohort study., Methods: The medical records of SJS (n = 87) and TEN (n = 48) patients between 2005 and 2007 were reviewed. An acute ocular severity score was determined on a scale from 0 to 3 (none, mild, severe, and very severe) according to the existence of hyperemia, corneal or conjunctival epithelial defect, and pseudomembrane formation. The associations between the severe acute ocular involvement and factors such as patient age, exposed drugs, systemic severity, and the prevalence of ocular sequelae were examined., Results: The number of cases with score grade 0, 1, 2, and 3 was 19 (21.8%), 31 (35.6%), 22 (25.3%), and 15 (17.2%) in 87 SJS cases and 12 (25.0%), 11 (22.9%), 17 (35.4%), and 8 (16.7%) in 48 TEN cases. Multivariate logistic regression analysis revealed that patient age (odds ratio [OR], 0.98; 95% confidence interval [CI], 0.96-0.99; P = .007) and nonsteroidal anti-inflammatory drugs NSAIDs or cold remedies (OR, 2.58; 95% CI, 1.26-5.29; P = .010) were predictive factors for severe acute ocular involvement. The prevalence of visual disturbance and eye dryness increased according to the increase of acute ocular severity (P = .001 and P = .007 in SJS; P = .007 and P = .014 in TEN, respectively)., Conclusions: At the onset of SJS/TEN, strict attention should be paid to ocular involvement in young patients and in patients exposed to NSAIDs or cold remedies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To clarify the relationship between clinical symptoms and histological status in patients with ocular cicatricial pemphigoid (OCP) and Stevens-Johnson syndrome (SJS)., Methods: Clinical symptoms of four OCP and eight SJS patients in the chronic phase were scored with our recently proposed grading system. The histological status of the pannus tissue removed from the corneal surface during surgery was investigated using immunohistological techniques., Results: All participants showed total loss of the palisades of Vogt and conjunctivalization of the entire corneal surface. All pannus tissues expressed the conjunctival epithelium marker CK4/13. The pannus tissue in clinically keratinized SJS expressed skin epidermal major cytokeratins, but the tissues of nonkeratinized SJS did not., Conclusions: Clinical observation and the use of our recently proposed grading system agreed with the immunohistological status with respect to keratinization, cell proliferation, and corneal/conjunctival cell typing. These findings facilitate our understanding of the pathogenesis of OCP and SJS, and will hopefully contribute to the development of future treatment strategies and improve predictions of the postoperative prognosis of ocular surface reconstruction in patients with OCP and SJS.

Background: Corneal staining with fluorescein resulting from a cause distinct from that of general epithelial barrier disturbance., Case: A 66-year-old woman saw her doctor complaining of blurred vision and foreign-body sensation in her left eye. Superficial punctate keratopathy (SPK) was detected on her left cornea. Following instillation of some eye drops, the patient developed corneal staining with fluorescein. Assuming that the staining was due to drug toxicity, eye-drop instillation was discontinued but the staining did not improve and her doctor then referred her to us. Although we tried glucocorticosteroid and artificial tear eye drops, the staining spread until it covered the patient's cornea almost completely. On a following day, we attempted to observe the patient's cornea by confocal microscopy, yet her corneal epithelium could only partially be seen due to what can best be described as a "black-cloud-like" formation on her cornea. However, immediately following that observation the corneal staining diminished without epithelial disturbance and the patient's symptoms were improved. Suspecting that some foreign materials may be stuck to her cornea, we performed impression cytology and detected the existence of MUC16., Conclusion: The patient's corneal staining may have resulted from her cornea being covered by materials related to MUC16.

Purpose: To evaluate the use of autologous serum (AS) from patients with severe ocular surface disease (OSD) in the development of transplantable corneal and oral epithelial tissue equivalents and to compare it with the use of conventional culture methods by using fetal bovine serum (FBS)., Methods: AS was obtained from patients with severe OSD secondary to Stevens-Johnson syndrome. Corneal and oral epithelial cells were cultivated in medium supplemented with either AS or FBS. Corneal and oral epithelial equivalents were constructed on denuded amniotic membranes. The bromodeoxyuridine (BrdU) ELISA cell proliferation assay and colony-forming efficiency (CFE) of cells cultivated in AS- or FBS-supplemented media were compared. The morphologic characteristics and the basement membrane assembly of cultivated epithelial equivalents were analyzed by light and electron microscopy, as well as by immunohistochemistry., Results: BrdU proliferation assay and CFE analysis showed that human corneal and oral epithelial cells cultivated in AS-supplemented media had comparable proliferative capacities compared with FBS-supplemented media. The corneal and oral epithelial equivalents cultivated in AS- and FBS-supplemented media were morphologically similar and demonstrated the normal expression of tissue-specific keratins and basement membrane assembly. The presence of a well-formed stratified epithelium, a basement membrane, and hemidesmosomal attachments was confirmed by electron microscopy., Conclusions: AS-supplemented cultures were effective in supporting the proliferation of human corneal and oral epithelial cells, as well as the development of transplantable epithelial equivalents. The use of AS is of clinical importance in the development of autologous xenobiotic-free bioengineered ocular surface equivalents for clinical transplantation.

Purpose: To evaluate the effects of early systemic administration of betamethasone or cyclosporin A (CsA) on inflammatory cytokine expression and corneal damage after alkali injury., Methods: Sixty-five Wistar rat corneas injured with 1N NaOH were divided into three groups: untreated, betamethasone-treated and CsA-treated. Both agents were administered systemically and daily during the first 7 days after injury. Interleukin (IL)-1alpha, -6 and -8 concentrations in the injured corneas were measured with ELISA at 2, 4 and 7 days after injury. Corneal damage was evaluated by scoring clinical findings., Results: In untreated injured corneas, IL-1alpha, -6 and -8 were markedly elevated during the 7-day period following injury. Both betamethasone and CsA significantly suppressed IL-1alpha and IL-8 at day 4. Only betamethasone significantly suppressed IL-6 at days 4 and 7. Both agents significantly reduced corneal opacity at day 4., Conclusion: Early systemic administration of betamethasone or CsA after alkali injury may be of benefit by suppressing inflammatory cytokine expression in the cornea.

Purpose: To assess the effectiveness of switching from the concomitant use of brinzolamide 1% (BZM) and brimonidine 0.1% (BMD) to a BZM/BMD fixed-dose combination (BBFC) for the reduction of corneal epithelial damage. Study Design: Retrospective cohort study. Methods: This study involved 52 eyes of 52 glaucoma patients (26 women, 26 men; mean age: 67.0 ± 14.0 years) followed for more than 3 months after being switched from concomitant BZM and BMD to BBFC. Superficial punctate keratitis (SPK) was assessed by fluorescein staining according to the National Eye Institute classification, with the cornea divided into 5 areas: center, superior, nasal, temporal, and inferior. SPK density was graded as 0 (no SPK), 1 (separate SPK), 2 (moderately dense SPK), and 3 (high SPK with overlapping lesions). SPK scores and intraocular pressure (IOP) at pre switching to BBFC (pre-BBFC) and at 3-months post switching to BBFC (post-BBFC) were then compared using the Wilcoxon signed-rank test. Results: At pre-BBFC and post-BBFC, respectively, mean IOP was 12.4 ± 2.5 and 12.4 ± 2.7 mmHg, thus illustrating no significant difference in IOP between pre and post switch (p = 0.924), and the mean SPK score for center, superior, nasal, temporal, and inferior was 0.06 ± 0.24, 0.04 ± 0.19, 0.52 ± 0.67, 0.15 ± 0.36, and 0.92 ± 0.74, and 0.04 ± 0.19, 0.02 ± 0.14, 0.37 ± 0.56, 0.04 ± 0.19, and 0.75 ± 0.62, thus clearly showing a significant reduction in SPK scores for the nasal, temporal, and inferior areas at post-BBFC compared to those at pre-BBFC (p < 0.05). Conclusion: Our findings reveal that compared with the concomitant use of BZM and BMD, BBFC is effective in reducing corneal epithelial damage. [ABSTRACT FROM AUTHOR]

Punctal occlusion (PO) is considered to improve both tear-film instability and increased friction during blinking and may consequently affect blinks. The purpose of this study was to investigate the effect of PO on blinks. This study involved 16 eyes of 16 severe aqueous deficient dry eye (ADDE) patients (mean age: 65.7 years). In all eyes, tear meniscus radius (TMR), spread grade (SG) of the tear-film lipid layer (i.e., SG 1-5: 1 being the best), fluorescein break-up time (FBUT), corneal epithelial damage score (CED), conjunctival epithelial damage score, corneal filament (CF) grade, lid-wiper epitheliopathy (LWE) grade, and superior limbic keratoconjunctivitis (SLK) grade were evaluated at before and at more than 1-month after PO. Moreover, using a custom-made high-speed blink analyzer, palpebral aperture height, blink rate, upper-eyelid closing-phase amplitude/duration/maximum velocity, and upper-eyelid opening-phase amplitude/duration/maximum velocity were measured at the same time point. After PO, TMR, SG, FBUT, CED, and the CF, LWE, and SLK grades were significantly improved, and upper-eyelid opening/closing-phase amplitude and maximum velocity significantly increased (all p < 0.04). The findings of this study suggest that PO improves ocular surface lubrication and that blink-related parameters can reflect the friction that occurs during blinking in eyes with severe ADDE. [ABSTRACT FROM AUTHOR]

In this study, the severity of eye pain (EP) and associated objective findings were evaluated in aqueous-deficient dry eye (ADDE) patients using PainVision®, a quantitative pain-measuring device. This study involved 53 eyes of 53 ADDE patients (6 males and 47 females; mean age: 64.4 ± 13.4 [mean ± SD] years). Of those, 18 eyes of 18 patients underwent punctal occlusion, and EP and objective findings in those patients were evaluated before and after treatment. In all patients, the severity of EP as measured by PainVision® was assessed using the Pain Degree (PD). The median PD for the 53 patients was 30.6 µA/µA (interquartile range, 16.9–93.2), and the nasal and central corneal staining score and the upper lid-wiper epitheliopathy score were significantly correlated with PD (R = 0.33, 0.33, and 0.28, respectively) (all: p < 0.05). Using the least squares method, the central corneal staining score most significantly affected PD. In the 18 cases that underwent punctal occlusion, PD was significantly reduced (median PD: 24.8 to 7.1 µA/µA; p < 0.0001). Using the least squares method, the central corneal staining score and tear meniscus radius were significantly more influential as factors contributing to PD before and after treatment, and central corneal epithelial damage was the factor most associated with ADDE-related EP. [ABSTRACT FROM AUTHOR]

To identify primary cilia in human corneal endothelial cells (CECs) obtained from patients with bullous keratopathy (BK). This study involved CEC specimens obtained from 10 eyes of 10 consecutive patients (three males and seven females; mean age: 74.5 years, range: 68–90 years) with BK who underwent Descemet's stripping automated endothelial keratoplasty at Baptist Eye Institute, Kyoto, Japan between August 2019 and September 2020. Three corneal buttons obtained from 3 patients who underwent penetrating keratoplasty for keratoconus were used as 'non-BK' controls. All specimens were evaluated with immunofluorescence staining using an antibody against acetylated α-tubulin. Ciliary expression was observed in six of the 10 CEC specimens; i.e. in two specimens obtained from BK patients after glaucoma surgery (trabeculectomy), in two specimens obtained from patients with Fuchs endothelial corneal dystrophy, and in two specimens obtained from a patient with BK after laser iridotomy for primary angle closure. There was acetylated α-tubulin staining but no hair-like structures in two specimens, and ciliary expression was unknown in two specimens due to the absence of cells. The length of the primary cilia varied between all specimens. In contrast, no primary cilia were observed in the corneal buttons obtained from the three keratoconus patients. The findings in this study clearly demonstrate the expression of primary cilia in the CECs of patients afflicted with BK. [ABSTRACT FROM AUTHOR]

Analysis of higher-order aberrations (HOAs) is one reported method for evaluating dry eye disease (DED)-related loss of visual function. Tear film (TF) instability and corneal epithelial damage (CED) are both reportedly responsible for HOAs in DED, although, to the best of our knowledge, there are no reported methods that allow concurrent evaluation of their effects. In this study, we used a videokeratographer (VK) to continuously measure HOAs in DED after eye opening and investigated factors of ocular surface abnormalities that determine HOAs. This study involved 96 DED cases that underwent DED symptom assessment with a questionnaire and examination of tear volume, TF abnormalities (i.e., TF lipid-layer interference grades and spreading grades, and non-invasive breakup time and fluorescein breakup time), and CED, and their correlation with HOAs evaluated via VK. The results show that HOAs at 1 or 2 s after eye opening can reflect TF instability and CED within the central 4-millimeter-diameter area of the optical zone in DED eyes concurrently. This finding may be useful for the rapid and non-invasive detection and evaluation of degraded visual function in DED cases with a variety of clinical features. [ABSTRACT FROM AUTHOR]

Toll-like receptor 3 (TLR3) and interferon-beta promoter stimulator-1 (IPS-1) are associated with antiviral responses to double-stranded RNA viruses and contribute to innate immunity. We previously reported that conjunctival epithelial cell (CEC) TLR3 and IPS-1 pathways respond to the common ligand polyinosinic:polycytidylic acid (polyI:C) to regulate different gene expression patterns as well as CD11c + cell migration in murine-model corneas. However, the differences in the functions and the roles of TLR3 and IPS-1 remain unclear. In this study, we investigated the differences of TLR3 or IPS-1-induced gene expression in corneal epithelial cells (CECs) in response to polyI:C stimulation using cultured murine primary CECs (mPCECs) derived from TLR3 and IPS-1 knockout mice via comprehensive analysis. The genes associated with viral responses were upregulated in the wild-type mice mPCECs after polyI:C stimulation. Among these genes, Neurl3, Irg1, and LIPG were dominantly regulated by TLR3, while interleukin (IL)-6 and IL-15 were dominantly regulated by IPS-1. CCL5, CXCL10, OAS2, Slfn4, TRIM30α, and Gbp9 were complementarily regulated by both TLR3 and IPS-1. Our findings suggest that CECs may contribute to immune responses and that TLR3 and IPS-1 possibly have different functions in the corneal innate immune response. [ABSTRACT FROM AUTHOR]

Spot break (SB), a tear film breakup (TFBU) subtype seen in decreased wettability dry eye (DE), is characterized by a spot-like TFBU that appears immediately after eye opening. It is sometimes difficult to detect using currently available devices for evaluating non-invasive TFBU. The purpose of this study was to investigate the effectiveness of using a newly developed videokeratography indicator for detecting SB. The study involved 44 eyes of 44 DE patients (21 eyes with SB (SB group) and 23 eyes with random break in which fluorescein breakup time was ≤ 5 s (s) (RB group)). All eyes were examined using videokeratography, with digital Meyer-ring images being obtained. By calculation of the degree of luminance blur on the cornea in the Meyer-ring images, termed 'disturbance value' (DV), DVs at 0 s (DV(0)]), 2 s (DV(2)), and 5 s (DV(5)) after eye opening, and the changes of DV between each time, were compared between the SB and RB groups. Results: No significant differences in DV(2) and DV(5) and the rate of change between DV(2) and DV(5) were found between the two groups. However, DV(0) and rate of change between DV(0) and DV(2) in the SB group were significantly greater (p < 0.001) than those in the RB group. SB characteristics were successfully detected by videokeratography using a new videokeratography DV indicator. [ABSTRACT FROM AUTHOR]

This study aims to clarify the immunogenicity in acquired and innate immune responses of cultured human corneal endothelial cells (hCECs) applied for cell injection therapy, a newly established modality for corneal endothelium failures. Thirty-four patients with corneal endothelial failure received injection of allogeneic hCEC suspension into anterior chamber. No sign of immunological rejection was observed in all 34 patients during the 5–8 years postoperative follow-up period. Cell injection therapy was successful in 2 patients treated for endothelial failure after penetrating keratoplasty and one patient with Descemet membrane stripping automated endothelial keratoplasty failure. ELISPOT assays performed in allo-mixed lymphocyte reaction to the alloantigen identical to that on the injected hCECs, elicited sparse IFN-γ-specific spots in the peripheral blood mononuclear cells of patients who received hCEC injection. The therapy generated simple and smooth graft-host junctions without wound stress. The injection of C57BL/6 CECs into the anterior chamber of BALB/c mice, which rejected C57BL/6 corneas 6 weeks ago, induced no sign of inflammatory reactions after the second challenge of alloantigen. Collectively, injection of the hCEC cell suspension in the aqueous humor induces immune tolerance that contributes to the survival of the reconstituted endothelium. [ABSTRACT FROM AUTHOR]

To evaluate the toxicity of Amphotericin B (AmB) in Optisol™-GS Corneal Storage Media (Bausch & Lomb) on corneal epithelial cell (CEC) morphology and migration ability. Sclerocorneal strips were removed from male Japanese white rabbits, and then stored at 4 °C in Optisol™-GS containing 0 µg/ml of AmB (control group) and 2.5, 5, 25, and 50 µg/ml of AmB (AmB groups; four eyes per group). After 7 days of storage, CEC morphology was evaluated by hematoxylin-eosin staining, immunohistochemical staining (ZO-1), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, to evaluate CEC migration ability, three corneal blocks (6–8 × 3 mm each) from one preserved cornea were cultured for 24 h, and the area of CEC migration (2 mm at the central region) onto the stromal surface was then measured. At 5, 25, and 50 µg/ml of AmB, deformation and vacuolation of CECs were observed in all preserved corneas. ZO-1 expression was significantly reduced in corneas preserved at AmB concentrations of 25 and 50 µg/ml. TUNEL Labeling Index was significantly increased at AmB concentrations of ≥5 µg/ml. CEC migration was inhibited in a dose-dependent manner at AmB concentrations of 25 and 50 µg/ml compared to the control group. The addition of AmB to Optisol™-GS can be toxic to CECs and inhibit their migration at a concentration of ≥5 µg/ml. AmB at a concentration of 2.5 µg/ml can be considered safe for the preservation of donor corneal tissue used in corneal epithelial transplantation surgery. [ABSTRACT FROM AUTHOR]

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

This study aimed to uncover the mechanism responsible for the clinical efficacy of cell injection therapy with fully differentiated cultured cells. Analysis of polarized expression of ion transporters on cultured human corneal endothelial cells (CECs) subpopulations (SPs) was performed. The intracellular pH (pHi) between two CEC SPs, distinct in the proportion of differentiated cells, was measured, and the association with mitochondrial respiration homeostasis was investigated. The effects of the ion transporter inhibition by their selective inhibitors or siRNA transfection were also explored. Na+/K+-ATPase, Aquaporin 1, SLC4A11, NBCe1, NHE1 as transporters, and ZO-1, were all selectively expressed in differentiated SPs, but were almost null in the cell-state-transitioned SPs. We also confirmed that the pHi of CEC SPs affected their mitochondrial respiration by modulating the expression of these ion transporters via inhibitors or siRNA transfection. Ion and water transporters might participate in the maintenance of pHi and mitochondria homeostasis in differentiated SPs, which may contribute, combined with integral barrier functions, to efficient water efflux. The differences in intracellular pH between the two SPs is attributed to variations in the expression profile of specific ion transporters and mitochondrial functions, which may associate with the efficacy of the SPs in cell injection therapy. [ABSTRACT FROM AUTHOR]

Purpose: Collagen fibrils in the corneal stroma in macular corneal dystrophy, on average, are more closely spaced than in the normal cornea. This study was conducted to investigate if this occurs uniformly across the stroma or is more prevalent at certain stromal depths. Methods: Microbeam synchrotron X-ray fiber diffraction patterns were obtained in 25 μm steps across the whole thickness of a thin strip of a macular corneal dystrophy cornea obtained at keratoplasty. Data were analyzed for mean collagen interfibrillar spacing at all positions. Serum was analyzed immunochemically to determine immunophenotype, and transmission electron microscopy was carried out to visualize stromal ultrastructure. Results: Keratan sulphate was not detectable in blood serum, classifying the disease as macular corneal dystrophy type I. Collagen interfibrillar spacing dropped linearly with stromal depth from the anterior to posterior cornea, measuring 5–10% less in the posterior 100 µm of the MCD stroma compared to the anterior 100 µm ( p < 0.001). Isolated pockets of collagen fibrils with unusually large diameters were identified in the deep stroma. Conclusions: Collagen fibril spacing is reduced and large-diameter collagen fibrils are seen in macular corneal dystrophy type I, with the deep stroma affected more. We speculate that the ultrastructural abnormalities are more prevalent in the posterior stroma because the structural influence of sulphated keratan sulphate glycosaminoglycans/proteoglycans is high in this region of the cornea. [ABSTRACT FROM AUTHOR]

PURPOSE. Previously we reported the occurrence of estrogen receptor α (ERβ), estrogen receptor β (ERβ) and androgen receptor (AR) in mouse corneas. The present study was designed to investigate the occurrence of various sex steroid hormone receptors, including ERα, progesterone receptor (PR) and AR, in human corneas. METHODS. We used reverse transcription-polymerase chain reaction (RT-PCR) to look for sex hormone receptor mRNAs (ERα, PR and AR) in human corneal epithelial cells obtained at autopsy. Next, using an immunocytochemical technique, we localized these receptors in donor human corneas. RESULTS. mRNAs encoding all receptors tested for were found in corneal epithelial cells obtained from male and female donor eyes. Immunocytochemical examination revealed that the receptors were located in the nuclei of corneal epithelial, stromal, and endothelial cells. CONCLUSIONS. Since receptors for both male and female sex hormones are present in human corneas of both genders, we postulate that the receptors may influence the biological functions of corneal cells through direct interaction with specific hormones. [ABSTRACT FROM AUTHOR]

PURPOSE. To investigate the expression of growth factor mRNA and the level of growth factor protein in preserved human amniotic membrane (AM). METHODS. RT-PCR was used to examine the expression of mRNA for eight growth factors (EGF, TGF-α, KGF, HGF, bFGF, TGF-β1, -β2, -β3) and two growth factor receptors (KGFR and HGFR) in human AM preserved at -80°C for one month. In addition, ELISAs were used to measure the protein concentrations of seven growth factors (EGF, TGF-α, KGF, HGF, bFGF, TGF-β1, -β2) in preserved human corneas and in AM both with and without amniotic epithelium. RESULTS. RT-PCR revealed that human AM expresses mRNA for EGF, TGF-α, KGF, HGF, bFGF, TGF-β1, -β2, -β3, KGFR and HGFR, while ELISAs showed that it contains EGF, TGF-α, KGF, HGF, bFGF, TGF-β1, -β2. AM without amniotic epithelium also contains all seven growth factors examined, however, in this tissue the protein levels of EGF, KGF, HGF and bFGF were found to be significantly lower than in native AM. CONCLUSIONS. Preserved human AM expresses mRNAs for a number of growth factors and contains several growth factor proteins that might benefit epithelialization after AM transplantation. High levels of EGF, KGF, HGF and bFGF in AM with amniotic epithelium as compared to AM without amniotic epithelium suggest an epithelial origin for these growth factors. We feel that EGF, KGF and HGF in particular might play important roles in ocular surface wound healing after AM transplantation. [ABSTRACT FROM AUTHOR]

A healthy corneal epithelium, which is essential for proper vision and protection from external pathogens, is continuously replenished throughout life by stem cells located at the limbus. In diseased or injured eyes, however, in which stem cells are deficient, severe ocular problems manifest themselves. These are notoriously difficult to manage and as a result the last 20 or so years has seen a number of therapeutic strategies emerge that aim to recover the ocular surface and restore vision in limbal stem cell deficient eyes. The dominant concept involves the generation of laboratory cultivated epithelial cell sheets expanded from small biopsies of the epithelial limbus (for patient or donors) or another non-corneal epithelial tissue such as the oral mucosa. Typically, cells are grown on sterilised human amniotic membrane as a substrate, which then forms part of the graft, or specially formulated plastic culture dishes from which cells sheets can be released by lowering the temperature, and thus the adherence of the plastic to the cells. Overall, clinical results are promising, as is discussed, with new cultivation methodologies and different cell lineages currently being investigated to augment the treatment options for visual disturbance caused by a corneal epithelial limbal stem cell deficiency. [ABSTRACT FROM AUTHOR]

PAX6, a paired box transcription factor, is necessary for eye development. However, how it regulates the cell identity of human corneal epithelial cells (CECs) is not well understood. We aimed to clarify the function of PAX6 in human CECs using gene knockout via the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated protein 9 (Cas9) system. We designed guide RNAs for different targets in PAX6. PAX6-depleted CECs maintained the epithelial morphology, but became larger. Global analyses using microarray revealed that down-regulated genes were primarily CEC-specific and included keratin 12, keratin 3, clusterin (CLU), aldehyde dehydrogenase 3 family member A1 (ALDH3A1), angiopoietin-like 7 (ANGPTL7) and transketolase (TKT), while up-regulated genes were primarily epidermis-related and included keratin 10, keratin 1, involucrin (IVL), filaggrin (FLG). These findings suggest that PAX6 maintains CEC identity by regulating differentiation. [ABSTRACT FROM AUTHOR]

To investigate the response to polyinosinic:polycytidylic acid [poly(I:C)], a double-stranded RNA Toll-like receptor 3 agonist that mimics viral infection, in the barrier function of two established human telomerase reverse transcriptase-immortalized cell lines, termed HCLE for the human corneal-limbal epithelial line and HCjE for the human conjunctival-epithelial line. In this study, HCLE and HCjE cells were used to evaluate the underlying mechanism of epithelial-cell barrier function regulation. Briefly, HCLE and HCjE cells were first cultured on 12-well Transwell® (Corning®) filter-plates, and reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical examinations were then performed to assess tight junction (TJ)-related protein expression and cellular distribution. Next, the barrier function of the cells was measured via transepithelial electrical resistance (TEER) and paracellular molecular flux. The cells were then stimulated with poly(I:C) and the TEER and TJ-related protein expressions were analyzed. Similar to that in in vivo epithelium, the expression of claudin (CLDN) subtypes CLDN-1, -4, and -7 was observed in the HCLE and HCjE cells, and the barrier function in the HCLE cells was tighter than that in the HCjE cells. Post stimulation with poly(I:C), TEER of the HCLE and HCjE cells increased in a dose- and time-dependent manner, the production of TJ-related protein mRNA and CLDN-4 protein were elevated, and the barrier function of the HCLE and HCjE cells increased, thus possibly indicating that the increased barrier function is a defense mechanism against viral infection. • Poly(I:C) is a synthetic analog of double-stranded RNA. • Poly(I:C) increases the barrier of hTERT immortalized corneal and conjunctival epithelia in a dose- and time-dependent manner. • Production of TJ-related protein mRNA and claudin-4 protein are elevated post stimulation with poly(I:C). • Most viruses synthesize dsRNA during their replication, and poly(I:C) stimulation mimics a viral infection. • Thus, we theorize that the increased barrier function must be a type of host defense reaction against viral infections. [ABSTRACT FROM AUTHOR]