A. Igarashi

Evaluation of lipid oxidative stress status in Sjögren syndrome patients

PURPOSE We evaluated the levels of lipid oxidative stress markers and inflammatory cells from tears and conjunctiva of patients with Sjögren syndrome (SS) and normal subjects. METHODS We examined 31 eyes of 16 patients (16 females) with SS and 15 eyes of 10 healthy controls (2 males and 8 females) in this prospective study. All subjects underwent a Schirmer test, measurement of tear film break-up time, vital stainings, confocal microscopy of the conjunctiva, tear collection for hexanoyl-lysine (HEL), ELISA, and conjunctival brush cytology. Brush cytology samples underwent immunohistochemistry (IHC) staining with HEL and 4-hydroxy-2-nonenal (4HNE). Hematoxylin-eosin and IHC staining with HEL and 4HNE also were performed on conjunctival samples of SS patients and controls. RESULTS The tear stability and vital staining scores were significantly worse in eyes of SS patients compared to the controls. Conjunctival inflammatory cell density was significantly higher in SS subjects compared to controls. The numbers of conjunctival cells stained positively for HEL and 4HNE were significantly higher in SS patients compared to controls. Tear HEL concentrations correlated significantly with staining scores and inflammatory cell density in confocal microscopy. Conjunctival specimens also revealed higher numbers of cells stained positively for inflammatory markers, as well as HEL and 4HNE in the IHC stainings. CONCLUSIONS Increase of the oxidative stress status in the conjunctiva of SS patients appears to have a role in the pathogenesis of dry eye disease. A close relationship may exist between reactive oxygen species (ROS) production, lipid peroxidation related membrane damage, and inflammatory processes in dry eye.

Atopic ocular surface disease : Implications on tear function and ocular surface mucins

Purpose: To describe tear function, mucin alterations, and ocular surface disorder in patients with atopic diseases. Methods: Subjects underwent corneal sensitivity measurements, Schirmer test, tear film break-up time (BUT) assay, and fluorescein and rose Bengal staining of the ocular surface. Conjunctival impression cytology and brush cytology were also conducted. Impression cytology samples underwent PAS and immunohistochemical staining for MUC5AC. Brush cytology specimens underwent evaluation for inflammatory cell expression and RT-PCR for MUC5AC mRNA expression. Differences related to tear function and ocular surface examination parameters among patients with and without corneal ulceration and healthy control subjects were studied. Results: Mean corneal sensitivity and BUT values were significantly lower in atopic patients with corneal ulcers compared with patients without ulcers and controls (P < 0.001). Brush cytology specimens from patients with corneal ulcers revealed significantly higher expression of inflammatory cells compared with patients without ulcers and controls (P < 0.001). Impression cytology samples from eyes with corneal ulcers showed significant squamous metaplasia and reduction of goblet cell density compared with eyes without ulcers and control subjects. Specimens from eyes with corneal ulcers showed PAS (+) mucin pick up and did not stain positive for MUC5AC. MUC5AC mRNA expression was significantly lower in eyes with corneal ulcers compared with in eyes without ulcers and control subjects. Conclusions: Ocular surface inflammation, tear film instability, and decreased conjunctival MUC5AC mRNA expression are important in the pathogenesis of noninfectious corneal shield ulcers in atopic ocular surface disease.

The role of oxidative stress and inflammation in conjunctivochalasis

Purpose. To investigate the status of oxidative stress and histopathologic alterations in patients with conjunctivochalasis and compare the findings with those in healthy control subjects. Methods. Eleven patients (n = 20 eyes) with Yokoi grade 3 conjunctivochalasis and 11 health control subjects (n = 22 eyes) were prospectively recruited. ELISA for tear hexanoyl-lysine (HEL) and inflammatory cytokines, tear film break-up time tests, Schirmer test measurements, and fluorescein and rose bengal vital staining were performed. Conjunctival specimens obtained during surgery for conjunctivochalasis and cataract underwent immunohistochemical staining for HEL+8-OHdG, MMP-3, and MMP-9, and positively stained cells were counted. Transmission electron microscopy was also performed, with staining for elastic fibers in the conjunctival stroma. Results. The mean tear stability and vital staining scores were significantly worse in the conjunctivochalasis patients than in the control subjects. The tear HEL and tear cytokine levels showed significantly higher values in eyes with conjunctivochalasis. IL-1beta and IL-6 levels showed a significant correlation with corneal epithelial damage. IL-1beta and TNFalpha showed a significant correlation with 8-OHdG-stained cell counts. Specimens from patients with conjunctivochalasis revealed a significantly higher number of cells positively stained for HEL, 8-OHdG, MMP-3, and MMP-9 than did specimens from age- and sex-matched control subjects. Transmission electron microscopy showed decreased intercellular cohesiveness, with the conjunctival stroma showing an accumulation of elastic fibers. Conclusions. Lipid and DNA oxidative stress were present in the conjunctiva. Increased tear inflammation seemed to coexist with loss of conjunctival epithelial cohesiveness and increased collagenolytic activity, which may explain the conjunctival laxity observed in patients with conjunctivochalasis.

Oxidative stress induced age dependent meibomian gland dysfunction in Cu, Zn-superoxide dismutase-1 (Sod1) knockout mice.

Purpose The purpose of our study was to investigate alterations in the meibomian gland (MG) in Cu, Zn-Superoxide Dismutase-1 knockout (Sod1 −/−) mouse. Methods Tear function tests [Break up time (BUT) and cotton thread] and ocular vital staining test were performed on Sod1 −/− male mice (n = 24) aged 10 and 50 weeks, and age and sex matched wild–type (+/+) mice (n = 25). Tear and serum samples were collected at sacrifice for inflammatory cytokine assays. MG specimens underwent Hematoxylin and Eosin staining, Mallory staining for fibrosis, Oil Red O lipid staining, TUNEL staining, immunohistochemistry stainings for 4HNE, 8-OHdG and CD45. Transmission electron microscopic examination (TEM) was also performed. Results Corneal vital staining scores in the Sod1 −/− mice were significantly higher compared with the wild type mice throughout the follow-up. Tear and serum IL-6 and TNF-α levels also showed significant elevations in the 10 to 50 week Sod1 −/− mice. Oil Red O staining showed an accumulation of large lipid droplets in the Sod1 −/− mice at 50 weeks. Immunohistochemistry revealed both increased TUNEL and oxidative stress marker stainings of the MG acinar epithelium in the Sod1 −/− mice compared to the wild type mice. Immunohistochemistry staining for CD45 showed increasing inflammatory cell infiltrates from 10 to 50 weeks in the Sod1 −/− mice compared to the wild type mice. TEM revealed prominent mitochondrial changes in 50 week Sod1 −/− mice. Conclusions Our results suggest that reactive oxygen species might play a vital role in the pathogensis of meibomian gland dysfunction. The Sod1 −/− mouse appears to be a promising model for the study of reactive oxygen species associated MG alterations.

The usefulness of measuring tear periostin for the diagnosis and management of ocular allergic diseases

BACKGROUND Chronic ocular allergic diseases such as vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) are accompanied by serious comorbidities; however, the underlying pathogenesis remains obscure. Furthermore, diagnosing conjunctival lesions in patients with atopic dermatitis and estimating the severity in AKC are important for the treatment of ocular allergic diseases. OBJECTIVE We addressed whether periostin, a novel mediator and biomarker in allergic inflammation, is involved in the pathogenesis of ocular allergic diseases and whether periostin can be a biomarker for these diseases. METHODS We investigated tear periostin in patients with seasonal allergic conjunctivitis (SAC), VKC, and AKC and allergic patients without conjunctivitis and compared it with tear IL-13 and serum periostin. Furthermore, in patients with AKC, we measured tear periostin before and after topical treatment with tacrolimus. RESULTS Tears from patients with ocular allergic disease showed significantly high periostin levels than did tears from allergic patients without conjunctivitis and from patients with AKC, VKC, and SAC in descending order. Tear periostin was associated with serious comorbidities such as large papilla formation and corneal damage in AKC, although both tear IL-13 and serum periostin had little to no such abilities. Furthermore, after topical tacrolimus treatment, tear periostin tended to decrease in most patients with AKC along with their clinical improvement. CONCLUSIONS Periostin produced in conjunctival tissues stimulated by IL-13 may contribute to the pathogenesis of ocular allergic diseases. Furthermore, tear periostin can be potentially applied as a biomarker to diagnose conjunctivitis in allergic patients and to evaluate disease severity as well as the efficacy of treatments in AKC.

The Role of 2% Rebamipide Eye Drops Related to Conjunctival Differentiation in Superoxide Dismutase-1 (Sod1) Knockout Mice

Purpose The superoxide dismutase-1 knockout (Sod1-/-) mouse is an age-related dry eye mouse model. We evaluated the role of 2% rebamipide ophthalmic solution on the conjunctiva and ocular surface alterations in Sod1-/- mice. Methods Rebamipide eye drops (2%) were instilled in six 50-week-old male Sod1-/- mice and six C57BL/6 strain wild-type (WT) male mice four times a day for 2 weeks. Aqueous tear secretion quantity and tear film breakup time measurements as well as vital stainings were performed. Immunohistochemistry staining of the conjunctiva was performed using SAM pointed domain-containing ETS transcription factor (SPDEF), transglutaminase-1, and involucrin antibodies. Quantitative RT-PCR was carried out to study mRNA expression of the same markers. Results The mean tear quantities showed no significant changes in both mice strains after treatment (P = 0.24). The mean tear film breakup time (P = 0.003) and vital staining scores significantly improved in the Sod1-/- mice after treatment. Treatment with 2% rebamipide eye drops significantly decreased the corneal fluorescein (P = 0.0093) and Rose Bengal (P = 0.002) staining scores in the Sod1-/- mice. We showed a notable increase in SPDEF and a marked decrease in transglutaminase-1 and involucrin immunohistochemistry stainings, together with a significant increase in SPDEF (P = 0.0003) and a significant decline in transglutaminase-1 (P = 0.0072) and involucrin (P = 0.009) mRNA expression after treatment in the Sod1-/- mice. Conclusions Topical use of 2% rebamipide drops was observed to improve conjunctival epithelial differentiation and suppress keratinization in the Sod1-/- mice.