A Comprehensive Assessment of Tear-Film-Oriented Diagnosis (TFOD) in a Dacryoadenectomy Dry Eye Model.

Tear film instability is a major cause of dry eye disease. In order to treat patients with short tear film breakup time (TBUT)-type dry eye, the development of tear film stabilizing agents is essential. However, the lack of an appropriate animal model of tear film instability has made drug development difficult. Although rabbit dry eye models have been reported in the past, there are only a few reports that focus on tear film instability. Herein, we assessed the tear film stability of a rabbit dry eye model induced by dacryoadenectomy. A clinical evaluation of the ocular surface, interferometry, and histological assessments of the cornea and conjunctiva were performed. Following the removal of the lacrimal glands, TBUT was shortened significantly, with dimple and random breakup patterns prominently observed. Furthermore, the blink rate in this model increased after dacryoadenectomy, suggesting that this model partially captured the phenotypes of human short TBUT-type dry eye and may be useful as an animal model for investigating potential drug candidates.

Senescence-associated secretory phenotype promotes chronic ocular graft-vs-host disease in mice and humans.

Chronic graft-vs-host disease (cGVHD) is a multifactorial inflammatory disease that affects patients undergoing hematopoietic stem cell transplantation. Multiple organs, including the lacrimal glands (LGs), are negatively affected by cGVHD and lose function due to the resultant fibrosis. An abnormal immune response is thought to be a major factor in the development of chronic ocular GVHD, which is currently treated primarily with immunosuppressive therapies. However, all the treatments yield unsatisfactory outcomes, and additional treatment strategies are needed. To meet this unmet medical need, we aimed to elucidate an additional pathway of chronic ocular GVHD. Our findings suggest a potential association between chronic ocular GVHD pathogenesis and stress-induced cellular senescence through the senescence-associated secretory phenotype (SASP). Senescent cells produce cytokines and chemokines, such as IL-6 and CXCL9. Indeed, senescent cell accumulation was presumably associated with cGVHD development in LGs, as evidenced by the improvement in LGs after the selective elimination of senescent cells (senolysis) with ABT-263. Results in the sclerodermatous cGVHD mouse model suggest that inhibiting the major components of the SASP, including IL-6 and CXCL9, with senolytics is a potential novel strategy for treating cGVHD-affected LGs. Taken together, our results indicate a potential association between the SASP and cGVHD development in LGs and suggest that targeted senolytic treatment may be a new therapeutic option for this disease.

MSCs Become Collagen-Type I Producing Cells with Different Phenotype in Allogeneic and Syngeneic Bone Marrow Transplantation.

Mesenchymal stem cells (MSCs) have been widely used in therapeutic applications for many decades. However, more and more evidence suggests that factors such as the site of origin and pre-implantation treatment have a crucial impact on the result. This study investigates the role of freshly isolated MSCs in the lacrimal gland after allogeneic transplantation. For this purpose, MSCs from transgenic GFP mice were isolated and transplanted into allogeneic and syngeneic recipients. While the syngeneic MSCs maintained a spherical shape, allogeneic MSCs engrafted into the tissue as spindle-shaped cells in the interstitial stroma. Furthermore, the MSCs produced collagen type I in more than 85% to 95% of the detected GFP+ MSCs in the recipients of both models, supposedly contributing to pathogenic fibrosis in allogeneic recipients compared to syngeneic models. These findings indicate that allogeneic MSCs act completely differently from syngeneic MSCs, highlighting the importance of understanding the exact mechanisms behind MSCs.

Accelerated Epithelial Proliferation in a Rabbit Model of Bullous Keratopathy.

Purpose: Long standing bullous keratopathy (BK) is associated with peripheral conjunctivalization and limbal deficiency. We hypothesized that cases of limbal deficiency may be induced due to accelerated proliferation of corneal epithelial cells. To investigate this hypothesis, we examined the influence of BK on the corneal epithelium in a rabbit model. Methods: Continuous curvilinear descemetorhexis was performed to remove a circular section of the corneal endothelium and Descemet's membrane (DM) using inverted Shinskey hook. Corneal tissue sections of BK eyes were observed by histochemical analysis using BrdU pulse chase methods for evaluation of corneal epithelial cell proliferation. Results: Rabbit corneas immediately became stromal edematous after DM stripping surgery, and a week later their thickness was five times that of control cornea. Vascularization of the peripheral cornea was observed in BK eyes, however, conjunctivalization was rarely observed at 6 weeks. The number of BrdU positive cells tended to be lower in the BK cornea epithelium compared to the control cornea epithelium. The number of Ki67 positive cells also showed a tendency to increase in the BK corneal epithelium. Telomere intensity in BK was similar to control corneal epithelium. Conclusion: BK may accelerate the proliferation of corneal epithelial cells in a rabbit model.

Cytokeratin expression in mouse lacrimal gland germ epithelium.

PURPOSE: The lacrimal gland secretes tear fluids that protect the ocular surface epithelium, and its dysfunction leads to dry eye disease (DED). The functional restoration of the lacrimal gland by engraftment of a bioengineered lacrimal gland using lacrimal gland germ epithelial cells has been proposed to cure DED in mice. Here, we investigate the expression profile of cytokeratins in the lacrimal gland germ epithelium to clarify their unique characteristics. METHODS: We performed quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry (IHC) analysis to clarify the expression profile of cytokeratin in the lacrimal gland germ epithelium. RESULTS: The mRNA expression of keratin (KRT) 5, KRT8, KRT14, KRT15, and KRT18 in the lacrimal gland germ epithelium was increased compared with that in mouse embryonic stem cells and the lacrimal gland germ mesenchyme, as analyzed by Q-PCR. The expression level of KRT15 increased in the transition from stem cells to lacrimal gland germ epithelium, then decreased as the lacrimal gland matured. IHC revealed that the expression set of these cytokeratins in the lacrimal gland germ epithelium was different from that in the adult lacrimal gland. The expression of KRT15 was observed in the lacrimal gland germ epithelium, and it segmentalized into some of the basal cells in the intercanulated duct in mature gland. CONCLUSION: We determined the expression profile of cytokeratins in the lacrimal gland epithelium, and identified KRT15 as a candidate unique cellular marker for the lacrimal gland germ epithelium.

Successful treatment of Beauveria bassiana fungal keratitis with topical voriconazole.

We describe a 66-year-old woman who suffered from fungal keratitis after corneal transplantation. The causative organism was identified as Beauveria bassiana on the basis of morphological characteristics and the sequence of the internal transcribed spacer region of the ribosomal RNA gene. The patient was successfully treated with topical voriconazole (VRCZ) use only. We, hereby, present the first report of a case with B. bassiana fungal keratitis that responded to topical antifungal VRCZ treatment.

[A Clinical Case Report Including In Vivo Laser Confocal Microscopic Findings of Keratitis Complicated with Takayasu's Arteritis].

BACKGROUND: We present a clinical case report including in vivo laser confocal microscopic findings of keratitis complicated with Takayasu's arteritis (aortitis syndrome). CASE: A 47-year-old woman was referred to the outpatient clinic of ophthalmology with blurred vision in her both eyes at the onset of Takayasu's arteritis. Since multifocal infiltrates in the stromal corneas with injection were observed with slit-lamp biomicroscope in the both eyes, the diagnosis was keratitis. A large amount of cells infiltrating the stromal cornea and activated keratocytes were also observed with in vivo laser confocal microscope in the both eyes. Systemic and local steroidal agents were initiated, which resolved the keratitis, and the active lesions turned into mild corneal scars. In vivo laser confocal microscopy showed no infiltrating cells in the stromal cornea of both eyes. No recurrence has been observed since. CONCLUSION: A rare case of keratitis complicated with Takayasu's arteritis is reported. An immune response to the stromal cornea as the etiology of the keratitis may be indicated by in vivo laser confocal microscopy.

Wickerhamomyces anomalus fungal keratitis responds to topical treatment with antifungal micafungin.

We describe a 91-year-old woman who suffered from fungal keratitis after corneal transplantation. The causative organism was identified as Wickerhamomyces anomalus (formerly Pichia anomala or Hansenula anomala) on the basis of morphological characteristics and the sequence of the internal transcribed spacer region of the ribosomal RNA gene. The patient was successfully treated with topical micafungin (MCFG) only. We present the first report of a case of W. anomalus fungal keratitis that responded to topical treatment with the antifungal MCFG.

[Factors Affecting Outcomes of Repeated Penetrating Keratoplasty].

PURPOSE: To investigate prognosis for repeated penetrating keratoplasty (PKP) and factors that affect the outcome. METHODS: We retrospectively investigated graft survival rates, 1-year postoperative best-corrected visual acuity and irreversible rejection rates in 108 eyes of 106 patients that had repeated PKP. Factors that might affect the outcome were, age, number of previous PKP, original diseases, history of glaucoma and rejection and the use of postoperative immunosuppressant were also studied. RESULTS: Individual-factor analysis showed that history of rejection and postoperative immunosuppressant significantly increased the risk of postoperative rejection. Multi-factor analysis showed that graft survival rate was significantly lower among cases that had systemic immunosuppressants (steroids and cyclosporine). One year postoperative best-corrected visual acuity was significantly worse in cases that had history of glaucoma. In cases with history of rejection, systemic administration of postoperative immunosuppressants was significantly associated with postoperative irreversible rejection. CONCLUSION: History of rejection and glaucoma tend to have poor outcome, and the outcome might not improve by postoperative immunosuppressants.

Possible involvement of epithelial-mesenchymal transition in fibrosis associated with IgG4-related Mikulicz's disease.

OBJECTIVE: Immunoglobulin G4 (IgG4)-related Mikulicz's disease (MD) is a fibrosis-associated inflammatory disease, often accompanied by lacrimal gland swelling. Although much attention has been paid to the inflammatory aspects of this disease, the mechanisms of the fibrotic processes are still unclear. We focused on the fibrotic changes occurring in the lacrimal glands of IgG4-related MD patients, by examining molecules involved in the epithelial-mesenchymal transition (EMT). METHODS: Lacrimal gland tissue specimens were obtained from 3 IgG4-related MD patients and 3 control patients with Sjögren's syndrome (SS). The glands were examined by immunohistochemistry and transmission electron microscopy. RESULTS: Storiform fibrosis, a characteristic of IgG4-related MD, was observed in the lacrimal glands of IgG4-related MD, but rarely in those of SS. Reduced E-cadherin expression, increased phalloidin-stained filamentous actin, and increased α-smooth muscle actin, snail, and heat-shock protein 47 levels were observed in the lacrimal glands of IgG4-related MD compared with those of SS. Transmission electron microscopy revealed an abnormal periodicity of collagen bundles, and basal membrane thickening in the IgG4-related MD compared with that in the SS tissues. CONCLUSION: EMT-like changes were frequently observed in the lacrimal gland epithelia from patients with IgG4-related MD. Thus, EMT may be involved in the pathology of IgG4-related MD fibrosis.

The Cornea: An Ideal Tissue for Regenerative Medicine.

Regenerative medicine is a highly anticipated field with hopes to provide cures for previously uncurable diseases such as spinal cord injuries and retinal blindness. Most regenerative medical products use either autologous or allogeneic stem cells, which may or may not be genetically modified. The introduction of induced-pluripotent stem cells (iPSCs) has fueled research in the field, and several iPSC-derived cells/tissues are currently undergoing clinical trials. The cornea is one of the pioneering areas of regenerative medicine, and already four cell therapy products are approved for clinical use in Japan. There is one other government-approved cell therapy product approved in Europe, but none are approved in the USA at present. The cornea is transparent and avascular, making it unique as a target for stem cell therapy. This review discusses the unique properties of the cornea and ongoing research in the field.

Cellular senescence promotes meibomian gland dysfunction in a chronic graft-versus-host disease mouse model.

PURPOSE: Aging is a well-established risk factor for meibomian gland dysfunction (MGD). We previously reported an accelerated cellular senescence phenomenon in the lacrimal glands of a murine model of chronic graft-versus-host disease (cGVHD). Herein, we aimed to elucidate the relationship between cellular senescence and MGD in cGVHD mice, utilizing the senolytic agent ABT-263. METHODS: A cGVHD mouse model was established through allogeneic bone marrow transplantation (BMT) from B10.D2 to BALB/c mice. Subsequently, cGVHD mice were treated with either ABT-263 or vehicle. The eyelids of recipients were analyzed at 4-week intervals post-BMT in both groups. RESULTS: Meibomian gland (MG) area was significantly smaller in cGVHD mice than in syngeneic control mice. ABT-263-treated mice retained a significantly larger MG area than their vehicle-treated counterparts. Pathological and immunohistochemical examinations revealed significant reductions in eyelid tissue inflammation and pathological fibrosis in the ABT-263 group compared to that in the vehicle-treated group. Additionally, expression of DNA damage markers, senescent cell markers, and senescence-associated secretory phenotype (SASP) factors was elevated in the eyelids of cGVHD mice compared with that in syngeneic mice. The expression of these cellular senescence-associated molecules was considerably suppressed in ABT-263-treated eyelids compared to that in vehicle-treated ones. CONCLUSIONS: Cellular senescence, along with expression of SASP factors, exhibited increased activity in the eyelids, particularly in the MGs of cGVHD mice. ABT-263 mitigated the severity of MGD. These findings highlight the potential of targeting cellular senescence as an effective approach for MGD treatment in cGVHD.

The Necroptosis Pathway Is Upregulated in the Cornea in Mice With Ocular Graft-Versus-Host Disease.

Purpose: To identify molecular signatures specific for ocular graft-versus-host disease (GVHD) by proteomic analysis of corneas from mice with GVHD. Methods: We identified differentially expressed proteins (DEPs) in corneal samples from GVHD model mice and syngeneic control mice 4 weeks after bone marrow transplantation. Data-independent acquisition analysis was performed on individual samples, and the roles of DEPs in biological pathways related to GVHD were evaluated via bioinformatics and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Results: Three important signaling pathways were upregulated in the cornea in mice with GVHD: (1) the necroptosis pathway, (2) the mitogen-activated protein kinase (MAPK) pathway, and (3) as previously reported, the neutrophil extracellular trap (NET) pathway. In those signaling pathways, we identified new upregulated molecules, including (1) receptor-interacting protein kinase 1 (RIPK1), RIPK3, interferon regulatory factor 9, the interferon-induced double-stranded RNA-activated protein kinase lipoxygenase, and high mobility group box1 (HMGB1) which are damage-associated molecular patterns (DAMPs) in the necroptosis pathway; (2) the sequentially upregulated interleukin 1 (IL-1) receptor-associated kinase (IRAK), an evolutionarily conserved signaling intermediate in the Toll pathway (ECSIT), and p38, which is downstream of the IL-1 receptor and increased CDC42/Rac (Rac2), a Rho family GTPase in the MAPK pathway; and (3) the integrin components CR3 and macrophage-1 antigen (MAC-1), which are DAMPs, and the pyroptosis-related protein gasdermin D (GSDMD) in the NET pathway. Conclusions: These novel molecules may help researchers elucidate the pathogenesis of GVHD and identify new therapeutic targets for corneal changes in patients with ocular GVHD.

Advances in corneal regenerative medicine with iPS cells.

The cornea is a pioneering area of regenerative medicine, and Japanese researchers have led the world in this field. In Japan, 3 different epithelial sheet regenerative medicine products have been approved for corneal epithelial stem cell deficiency, and the first-in-human studies of cultured corneal endothelial cell suspension transplants, induced pluripotent stem cell (iPS cell)-derived corneal epithelial sheet transplants, and iPS cell-derived corneal endothelial substitute cell transplants were all conducted and reported globally for the first time by Japanese researchers. In the field of corneal epithelial regenerative medicine, Pellegrini et al.  (Lancet 349:990-3, 1997) performed the first in-human transplant of autologous cultured corneal epithelial sheets. More than 20 years later, autologous cultivated corneal epithelium and autologous cultivated oral mucosal epithelium products were launched in Japan. In addition, clinical studies of iPS cell-derived corneal epithelial cell sheet transplant have begun, which may solve the issues with conventional autologous epithelial sheets. In corneal endothelium regenerative medicine, a clinical study of transplant of allogenic cultured corneal endothelial cell suspension for bullous keratopathy was reported, in which corneal endothelial cells derived from donor corneas were grown in culture and then injected into the anterior chamber with a ROCK inhibitor (Kinoshita et al. in N Engl J Med 378:995-1003, 2018). Our research group is also developing iPS-cell-derived corneal endothelium-like cells, termed corneal endothelial cell substitute from iPS cells (CECSi cells), and we are conducting a clinical study to treat bullous keratopathy with these cells (Hatou et al. in Stem Cell Res 55:102497, 2021). This review describes the progress and challenges of corneal epithelial and endothelial regenerative medicine and the promising future of corneal regenerative medicine with iPS cells.

Non-apoptotic regulated cell death in Fuchs endothelial corneal dystrophy.

Introduction: Fuchs endothelial corneal dystrophy (FECD) is the leading cause of corneal blindness in developed countries. Corneal endothelial cells in FECD are susceptive to oxidative stress, leading to mitochondrial dysfunction and cell death. Oxidative stress causes many forms of cell death including parthanatos, which is characterized by translocation of apoptosis-inducing factor (AIF) to the nucleus with upregulation of poly (ADP-ribose) polymerase 1 (PARP-1) and poly (ADP-ribose) (PAR). Although cell death is an important aspect of FECD, previous reports have often analyzed immortalized cell lines, making the evaluation of cell death difficult. Therefore, we established a new in vitro FECD model to evaluate the pathophysiology of FECD. Methods: Corneal endothelial cells were derived from disease-specific induced pluripotent stem cells (iPSCs). Hydrogen peroxide (H2O2) was used as a source for oxidative stress to mimic the pathophysiology of FECD. We investigated the responses to oxidative stress and the involvement of parthanatos in FECD-corneal endothelial cells. Results: Cell death ratio and oxidative stress level were upregulated in FECD with H2O2 treatment compared with non-FECD control, indicating the vulnerability of oxidative stress in FECD. We also found that intracellular PAR, as well as PARP-1 and AIF in the nucleus were upregulated in FECD. Furthermore, PARP inhibition, but not pan-caspase inhibition, rescued cell death, DNA double-strand breaks, mitochondrial membrane potential depolarization and energy depletion, suggesting that cell death was mainly due to parthanatos. Conclusions: We report that parthanatos may be involved in the pathophysiology of FECD and targeting this cell death pathway may be a potential therapeutic approach for FECD.

Eyes open on stem cells.

Recently, the murine cornea has reemerged as a robust stem cell (SC) model, allowing individual SC tracing in living animals. The cornea has pioneered seminal discoveries in SC biology and regenerative medicine, from the first corneal transplantation in 1905 to the identification of limbal SCs and their transplantation to successfully restore vision in the early 1990s. Recent experiments have exposed unexpected properties attributed to SCs and progenitors and revealed flexibility in the differentiation program and a key role for the SC niche. Here, we discuss the limbal SC model and its broader relevance to other tissues, disease, and therapy.

Characterization of cultivated murine lacrimal gland epithelial cells.

PURPOSE: To date, mouse lacrimal gland epithelial cells have been cultured successfully but only in cases involving newborn mouse lacrimal glands. In this work, we attempted to cultivate and characterize adult mouse lacrimal gland epithelial cells. METHODS: Lacrimal glands were removed from newborn mice (C57B/6) and isolated lacrimal gland epithelial cells were seeded onto tissue culture treated or low adherent culture dishes in Cnt-07 culture medium with or without cholera toxin. Cultivated cells were characterized by immunostaining with pan-cytokeratin, α-smooth muscle actin, and lactoferrin antibodies. Lacrimal gland cells from 7-week-old green fluorescent protein (GFP) and non-GFP (C57B/6) mice were mixed and seeded onto uncoated dishes to assess sphere-forming efficiency. Cells were also seeded onto 3T3 cell feeder layers to assess colony forming efficiency. RESULTS: Lacrimal gland epithelial cells were selectively cultured with cholera toxin, and cell type was verified by pan-cytokeratin and α-smooth muscle actin immunostaining. Sphere formation from single cells of adult mice was observed using specific medium and low adherent culture dishes. These cells could also undergo colony formation on 3T3 feeder cells. CONCLUSIONS: Adult mouse lacrimal gland epithelial cells were successfully cultivated in cholera toxin-containing medium, and were observed to form spheres from single cells.

Graft Size and Double Scroll Formation Rate in Descemet Membrane Endothelial Keratoplasty.

PURPOSE: This study aimed to evaluate the usefulness of intentional double scroll formation of donor Descemet membrane (DM) inside a glass tube inserter (the Fogla technique) in DM endothelial keratoplasty (DMEK) for controlled insertion and unfolding of grafts. METHODS: Eleven consecutive patients who underwent DMEK were included in this study. We sought to specify graft characteristics in which double scroll configuration was successfully formed using the Fogla technique. We compared donor age, graft size, surgical time, unfolding time, and visual outcomes between patients with and without double scroll configuration. The ability to form double scroll formation of DM grafts of various diameters and unfolding time of DM grafts was evaluated using total seven eye-bank eyes in ex vivo experiments. RESULTS: A double scroll configuration inside a glass tube was successfully obtained in six DMEK grafts (54.5%). When comparing clinical features between those with and without double scroll configuration, only graft size was significantly larger in those with double scroll configuration (7.9 ± 0.2 mm) than in those without (7.4 ± 0.4, P = 0.03). There were no significant differences in other features and clinical outcomes, although unfolding-time was shorter in eyes with double scroll configuration (4.6 ± 2.0 min) compared to those without (8.6 ± 8.1, P = 0.21). Ex vivo experiments showed that unfolding time was significantly shorter in double scroll configuration (2.71 ± 0.49 min) than in single scroll (5.02 ± 0.79, P = 0.01). CONCLUSIONS: A double scroll configuration within a glass tube can be obtained more frequently in larger DMEK grafts (8.0 mm in diameter), which may result in easier and faster DMEK procedures.

The Anterior Eye Chamber as a Visible Medium for In Vivo Tumorigenicity Tests.

Pluripotent stem cell (PSC)-based cell therapies have increased steadily over the past few years, and assessing the risk of tumor formation is a high priority for clinical studies. Current in vivo tumorigenesis studies require several months and depend strongly on the site of grafting. In this study, we report that the anterior eye chamber is preferable to the subcutaneous space for in vivo tumorigenesis studies for several reasons. First, cells can easily be transplanted into the anterior chamber and monitored in real-time without sacrificing the animals due to the transparency of the cornea. Second, tumor formation is faster than with the conventional subcutaneous method. The median tumor formation time in the subcutaneous area was 18.50 weeks (95% CI 10.20-26.29), vs. 4.0 weeks (95% CI 3.34-.67) in the anterior chamber (P = .0089). When hiPSCs were spiked with fibroblasts, the log10TPD50 was 3.26, compared with 4.99 when hiPSCs were transplanted without fibroblasts. There was more than a 40-fold difference in the log10TPD50 values with fibroblasts. Furthermore, the log10TPD50 for HeLa cells was 1.45 and 100% of animals formed tumors at a concentration greater than 0.1%, indicating that the anterior chamber tumorigenesis assays can be applied for cancer cell lines as well. Thus, our method has the potential to become a powerful tool in all areas of tumorigenesis studies and cancer research.

Four cases of corneal perforation in patients with chronic graft-versus-host disease.

PURPOSE: To report the clinical features and investigate the underlying pathological processes of spontaneous corneal perforation in patients with ocular chronic graft-versus-host disease (cGVHD). METHODS: A full ophthalmological evaluation of corneal perforation in four patients with cGVHD was performed. Three of them underwent deep anterior lamellar keratoplasty and samples from two of three patients were used for histopathological analyses. RESULTS: Three patients were successfully treated by corneal transplantation. One patient was treated with a therapeutic soft contact lens, and the wound healed within 2 days. The common clinical features of these patients were (1) the presence of definite dry eye related to cGVHD in 3 of 4 patients and probable dry eye in one patient, (2) a central or paracentral site of corneal ulceration and perforation, with no sign of infection, and (3) prior use of a topical or systemic corticosteroid, and/or topical non-steroidal anti-inflammatory drugs. Immunohistochemical findings revealed an increased number of cluster of differentiation 68(+) (CD68(+)) macrophages and matrix metalloproteinase 9 (MMP-9) expression in the tissue surrounding the perforation. CONCLUSIONS: Our report extends current information on the clinical features and pathological processes of corneal perforation in cGVHD by showing increased MMP-9 expression and the accumulation of CD68(+) positive macrophages in the affected areas.