Tohru Sakimoto

Background: Dry eye disease is a multifactorial ocular surface disorder characterized by tear film instability, inflammation, and neurosensory abnormalities that can lead to corneal pain and discomfort. In this study, we evaluated the effects of specific eye drops for dry eyes on neuronal pain receptors to gain insight into the mechanisms underlying corneal nerve pain in patients with dry eyes using a primary cell culture model of murine trigeminal ganglion cells. Methods: Trigeminal ganglia were obtained from wild-type postnatal day 7–10 mice. Primary cultures were prepared using the cell suspension method. After culturing for one week, the cells were stained with neuron-specific anti-neuronal nuclei, polymodal nociceptor, and transient receptor potential vanilloid 1 (TRPV1) antibodies. The calcium ion probe Fura2-AM^® was added to cultured cells after 2 weeks of incubation. The effects of capsaicin alone, in combination with the TRPV1 antagonist AMG9810, and in the presence of components of commercially available eye drops (cyclosporine, diquafosol tetrasodium, or rebamipide) were evaluated by monitoring calcium signals. Results: Neural excitation and capsaicin-induced increase in fluorescence intensity ratio were suppressed by AMG9810, cyclosporine, and diquafosol tetrasodium, but not by rebamipide. Conclusions: Inhibition of cellular excitation by cyclosporine and diquafosol tetrasodium may underlie their clinical pain suppressive effects. The primary culture model described here may serve as a useful tool for future studies on corneal perception.

Objective: We conducted a retrospective evaluation of the long-term prognosis associated with anterior blepharitis subsequent to topical antibiotic intervention. Methods: Inclusion criteria encompassed 92 eyes of 92 patients who exhibited clinical manifestations of anterior blepharitis and undergone a regimen of topical azithromycin. The follow-up duration extended to a minimum of one year within our medical facility. The diagnostic framework for anterior blepharitis, along with the evaluation of both objective and subjective manifestations, adhered to the Blepharitis Preferred Practice Pattern as promulgated by the American Academy of Ophthalmology. Relapse denoted the resurgence of blepharitis symptoms subsequent to the primary treatment, necessitating the instigation of either topical or oral therapeutic measures. Results: Of 92 cases of anterior blepharitis, 48 showed recurrence, with a recurrence rate of 52.2%. During the follow-up period, 2 patients experienced 5 relapses, 2 patients experienced 4 relapses, 5 patients experienced 3 relapses, 13 patients experienced 2 relapses, and 26 patients experienced 1 relapse. When the patients were divided into three groups: multiple recurrence group (22 patients), single recurrence group (26 patients), and no recurrence group (44 patients), there were no significant differences in the blepharitis finding score before and after the initial treatment among three groups. However, significantly fewer patients in the no recurrence group required further treatment after initial treatment, and the percentage of patients with residual blepharitis during follow-up was significantly lower in the no recurrence group. Conclusions: Our data suggest that patients with residual findings after blepharitis treatment are at a risk of recurrence.

We evaluated the clinical features of postoperative anterior blepharitis following cataract surgery and the efficacy of topical azithromycin retrospectively. Thirty eyes of 30 patients with a clinical diagnosis of anterior blepharitis by 6 months postoperatively among those who underwent cataract surgery at our institution between November 2020 and June 2022 were included. The diagnosis of anterior blepharitis and the assessment of objective and subjective findings were based on the American Academy of Ophthalmology Blepharitis Preferred Practice Pattern^®. Azithromycin eye drops were prescribed for all patients, and findings and symptoms before and after the drops were reviewed. The time of onset ranged from 2 weeks to 6 months after cataract surgery, with the most common onset at 2 to 3 months postoperatively (mean time of onset 79.4 ± 39.6 days). The type of anterior blepharitis was staphylococcal blepharitis in 26 eyes and seborrheic blepharitis in 4 eyes, while mixed type with posterior blepharitis was noted in 6 eyes. Symptoms at the time of examination included irritation (including foreign body sensation) in 24 eyes, tearing in 4 eyes, and redness in 3 eyes. The findings and symptoms of anterior blepharitis were alleviated or resolved with azithromycin eye drops in 26 of the 30 eyes, but the blepharitis recurred in 6 of these eyes, requiring azithromycin eye drops to be re-prescribed. The onset of anterior blepharitis after cataract surgery may be related to a gradual decrease in postoperative eye drops. Patients tended to complain of irritation and foreign body sensation, and azithromycin eye drops were effective in such cases.

We evaluated the role of NLR family pyrin domain containing 3 (NLRP3) inflammasome in sterile corneal inflammation caused by lipopolysaccharide (LPS) or alkali burns in C57BL6 mice or NLRP3 KO (Nlrp3^−/−) mice. Various molecules related to the NLRP3 inflammasome were upregulated in C57BL6 mice after both alkali burn injury and LPS treatment. After alkali burn injury, the corneal opacity grade was significantly reduced inNlrp3^−/−mice compared with C57BL6 mice. InNlrp3^−/−mice, Gr-1 immunoreactivity and MMP-9 mRNA expression in the corneal stroma were significantly reduced by both LPS treatment and alkali burn injury. Quantitative PCR and immunohistochemistry revealed that IL-1β and MMP-9 expression in the corneal stroma were down-regulated inNlrp3^−/−mice with both alkali burn injury and LPS treatment. These findings suggest that the NLRP3 inflammasome has a pro-inflammatory effect in the cornea by recruiting neutrophils to sites of inflammation.

Phlyctenular keratitis is a hypersensitivity reaction of the cornea, and a complication of eyelid margin disease in children and young adults. In this study, we compared the morphology of the meibomian glands in eyelids between phlyctenular keratitis patients and healthy young adults, using noncontact meibography. The study included 16 eyes of 13 patients diagnosed with phlyctenular keratitis and 17 eyes of 17 healthy volunteers. Slit-lamp observations of the cornea and eyelid were performed on all subjects. The morphology of the meibomian glands was scored using non-contact meibography (meiboscore). The meiboscore in worse eye was used in bilateral phlyctenular keratitis. All eyes with phlyctenular keratitis, but not normal controls, showed corneal nodules, neovascularization, and superficial punctate keratopathy. The mean meiboscore in phlyctenular keratitis patients (upper lid: 2.9 ± 0.3, lower lid: 2.7 ± 0.5) was significantly higher than in controls (upper lid: 0.4 ± 0.6, lower lid: 0.1 ± 0.3). Noncontact meibography enabled visualization of meibomian gland loss in phlyctenular keratitis patients, suggesting a relationship between abnormalities of the meibomian glands in young individuals and the pathogenesis of phlyctenular keratitis.

We investigated the presence of matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), proinflammatory cytokines, and soluble cytokine receptors in the tear fluid of patients with noninfectious corneal ulcers in the peripheral cornea. The subjects were 20 eyes of 17 patients with peripheral noninfectious corneal ulcers and 20 eyes of 20 volunteers. Tear samples were taken by the Schirmer test I method and the presence of MMPs (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, and MMP-13) and TIMPs (TIMP-1, TIMP-2, and TIMP-4) were investigated using an MMP antibody array system. The concentrations of proinflammatory cytokines {IL-1β, IL-6, and TNF-α (tumor necrosis factor-alpha)} and soluble cytokine receptors {soluble (s) IL-1R1, sIL-1R2, sIL-2Rα, sIL-4R, sIL-6R, sTNFR1, sTNFR2, s-vascular endothelial growth factor receptor (VEGFR) 1, sVEGFR2, sVEGFR3, and sgp130} were determined using the multiplex bead immunoassay system. The concentrations of MMP-8 and MMP-9 were significantly up-regulated in the tear fluid of the ulcer patients, whereas TIMPs concentrations did not change. The concentrations of IL-1β, IL-6, sIL-1R2, sIL-6R, sTNFR1, and sTNFR2 were up-regulated in the ulcer patients, whereas sgp130 and sVEGFR1 concentrations significantly decreased. The presence of some MMPs increased significantly in the patients with peripheral noninfectious corneal ulcers, whereas the presence of TIMPs remained unchanged. Although some proinflammatory cytokines were up-regulated, their antagonists, soluble cytokine receptors, were also up-regulated. It is thus possible that the up-regulation of MMPs disrupts the balance between the MMPs and TIMPs and that this balance may play a pivotal role in the pathophysiology of corneal ulceration.

Purpose.: We evaluated an anti-inflammatory effect of TNF receptor 1 (TNFR1) ectodomain shedding in ocular surface. Methods.: Human corneal epithelial cell (HCEC) was first pretreated by TNF-α. Ectodomain shedding was stimulated by uridine triphosphate (UTP) or peptidoglycan (PGN), with or without shedding inhibition using TNF-α processing inhibitor (TAPI). The phosphorylation of the NF-κB inhibitory protein, IκB, was assessed by Western blotting and concentrations of soluble TNFR1 (sTNFR1) in culture medium were analyzed by ELISA. Tear fluid from patients with Sjögren syndrome and graft-versus-host disease (GVHD) was collected and analyzed by ELISA for sTNFR1 concentration. Five dry eye patients underwent topical treatment using diquafosol sodium eye drops, a purinergic P2Y2 receptor agonist, and the tear fluid of the patients was sampled before and 4 weeks after the treatment for sTNFR1 ELISA. Results.: Phosphorylation of IκB was diminished by adding UTP or PGN, and this down-regulation of IκB phosphorylation was reversed by adding TAPI. In HCEC medium, sTNFR1 release was increased significantly by adding UTP or PGN, and inhibited significantly by adding TAPI. In the tears of the patients with Sjögren syndrome and GVHD, sTNFR1 expression was upregulated. In the tears of the patients with short breakup time (BUT) dry eye, sTNFR1 concentrations (ng/mL) in the tears were 1.30 ± 0.58 ng/mL for the pretreatment baseline, and 1.64 ± 0.70 after treatment, statistically significantly higher than those for the pretreatment (P < 0.01). Conclusions.: Ectodomain shedding of sTNFR1 blocked TNF-α–induced intracellular signaling in corneal epithelium. The upregulation of sTNFR1 in inflamed ocular surfaces suggests an anti-inflammatory role of sTNFR1 ectodomain shedding at the ocular surface.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with the potential to degrade all types of extracellular matrix. The ADAM (a disintegrin and metalloproteinase) family of peptidases was recently identified as cleaving the extracellular domain of transmembrane proteins. This was termed ectodomain shedding. We investigated the MMP expression in patients with corneal diseases and the potential role of ADAMs in corneal pathophysiology. We detected upregulation of the active form of MMP-2 and MMP-9 in the tear fluid from patients with corneal melting or recurrent corneal erosion. Using human corneal epithelial cells, we observed ADAM17-dependent ectodomain shedding of soluble tumor necrosis factor receptor 1 and soluble interleukin-6 (IL-6) receptor (sIL-6R). The production of sIL-6R was also induced by messenger RNA splicing in the human corneal epithelial cells. IL-6/sIL-6R-induced signal transducer and activator of transcription 3 phosphorylation was observed in cultured human corneal fibroblasts, suggesting that IL-6 trans-signaling induced inflammatory cellular signaling in the human corneal fibroblasts. We demonstrated that MMPs are significantly upregulated in collagen-destructive disorders of the cornea. Additionally, we observed that ectodomain shedding by ADAMs in corneal epithelial cells mediated the production of soluble cytokine receptors. Trans-signaling of IL-6 can induce an inflammatory response in corneal stroma, indicating the significance of IL-6 trans-signaling in ocular surface inflammation. Thus, MMPs and ADAMs play an important role in the pathophysiology of corneal diseases.