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.

Salivary and Lacrimal Gland Alterations of the Epidermal Fatty Acid-Binding Protein (E-FABP) in Non-Obese Diabetic Mice.

The purpose of this study was to investigate the changes in E-FABP in the salivary and lacrimal glands of the Sjögren syndrome (SS) model non-obese diabetic mice (NOD). Cotton thread and ocular vital staining tests were performed on 10-week NOD male mice (n = 24) and age- and sex-matched wild-type (WT) mice (n = 25). Tear and saliva samples were collected at sacrifice for E-FABP ELISA assays. Salivary and lacrimal gland specimens underwent immunohistochemistry stainings for E-FABP. Real-time RT-PCR was also performed for the quantification of mRNA expression levels in the salivary and lacrimal glands. Corneal vital staining scores in the NOD mice were significantly higher compared with those for the wild-type mice (p = 0.0001). The mean tear E-FABP level showed a significantly lower concentration in the NOD mice (p = 0.001). The mean saliva E-FABP level also showed a significantly lower concentration in the NOD mice (p = 0.04). Immunohistochemistry revealed intense E-FABP staining in the LG acinar epithelium and less intense staining in the acinar epitheliae of the SGs in the NOD mice compared to the WT mice. Real-time RT-PCR for the mRNA expression of E-FABP showed a significantly decreased expression in the SG and a significant increase in the LG of the NOD mice compared to the WT mice. In conclusion, the E-FABP showed marked alterations in the tear film, saliva, lacrimal, and salivary glands of the NOD mouse, which may help explain the ocular surface changes in relation to the dry eye disease in this SS model mouse and keratoconjunctivitis sicca in SS patients.

The Effects of High Molecular Weight Hyaluronic Acid Eye Drop Application in Environmental Dry Eye Stress Model Mice.

Hyaluronic acid (HA) ophthalmic solution is widely used in dry eye treatment worldwide. However, there are no reports comparing the dry eye treatment effects of high molecular weight HA with low molecular weight HA. Sixty eight-week-old C57BL/6 mice were assigned to the following 6 groups and exposed to environmental dry eye stress (EDES) that mimics office work environment: 1) 0.1% low molecular weight HA (LMWHA) eye drops, 2) 0.3% LMWHA eye drops, 3) 3% diquafosol sodium (DQ) eye drops, 4) 0.15% high molecular weight HA (HMWHA) eye drops, 5) no treatment with exposure to EDES (EDES+/Treatment-), and 6) no treatment without exposure to EDES (EDES-/Treatment-). After EDES, the HMWHA group had significantly longer break-up time (BUT) than the 0.1%, 0.3% LMWHA groups and the DQ group. After EDES, the HMWHA group had significantly lower lissamine green staining scores than the LMWHA and DQ groups. Subepithelial presumed dendritic cell density in the HMWHA group was significantly lower than the EDES+/Treatment- group. After EDES exposure, Conjunctival Muc5AC mRNA expression in the HMWHA group was significantly higher than the 0.1 and 0.3% LMWHA groups. Ophthalmic HMWHA solution may have a better dry eye treatment effect than LMWHA or DQ solution, owing to its anti-inflammatory effect.

Changes in Murine Subbasal Corneal Nerves After Scopolamine-Induced Dry Eye Stress Exposure.

Purpose: To evaluate the morphologic changes in the corneal subbasal nerve (CSN) plexus in wild-type (WT) mice after exposure to scopolamine-induced dry eye stress (DES) by using in vivo confocal microscopy. Methods: Twenty right eyes of twenty (n = 20) 8-week-old WT BALB/c male mice were investigated. The mice were divided into two experimental groups; 10 eyes of 10 mice exposed to DES for 28 days and 10 eyes of 10 mice were used as a control group. All mice underwent examinations for aqueous tear secretion quantity, tear film breakup time (TBUT), corneal vital staining. and corneal sensitivity thrice (pre-experiment, 2nd week, and 4th week). CSN density, tortuosity, reflectivity, and dendritic cell (DC) densities were examined. Results: The mean aqueous tear secretion (P < 0.0001) and TBUTs (P < 0.0001) were significantly decreased after DES. The mean corneal vital staining scores were significantly higher (fluorescein, P < 0.0001; lissamine, P < 0.0001), the mean TBUTs were significantly shorter (P < 0.0001), and the corneal sensitivities (P < 0.0001) were significantly lower in the dry eye-induced mice than the control mice. The mean CSN fiber density (P < 0.0001) and the reflectivity (P < 0.001) were significantly lower; the mean tortuosity and the mean DC density were significantly higher (P < 0.0001) in the dry eye mice. Conclusions: Our data demonstrated that prolonged exposure to DES resulted in alterations of CSN density; DC intensity, reflectivity, and tortuosity as well as in tear volume; TBUT; fluorescein and lissamine green staining scores; and the corneal sensitivity in WT mice.

The Effect of Rebamipide Ophthalmic Solution on Cytokine and Mucin Secretion in Culture of Conjunctival Epithelial Cells From the Cu, Zn-Superoxide Dismutase-1 (SOD-1) Knock-Down Mouse.

OBJECTIVES: To evaluate the in vitro effects of 1-mM rebamipide ophthalmic solution on the expression of inflammatory cytokines and MUC5AC in Cu, Zn-superoxide dismutase-1 (SOD-1) knock-down conjunctival epithelium. METHODS: Conjunctival epithelium from C57BL/6 wild-type mice was cultured and treated with rebamipide ophthalmic solution. Using cytometric bead array, we examined the levels of interleukin-(IL)-6, IL-10, IL-17, monocyte chemoattractant protein-1, interferon-γ (INF-γ), tumor necrosis factor, and IL-12p70 in the culture supernatants. The culture supernatants were obtained from the culture medium of nontreated or SOD-1 knock-down conjunctival epithelium using small interfering RNA (siRNA). In addition, ELISA was performed to ascertain the MUC5AC concentration in the culture medium. RESULTS: After rebamipide ophthalmic solution was applied, IL-6 concentration in the supernatants of conjunctival epithelial cells treated with and without siRNA showed a significant timewise decrease from 0 to 24 hr (963±42 to 0.07±0.05 pg/mL and 932±168 to 2.2±0.05 pg/mL, respectively) (P<0.001). Compared with baseline values, MUC5AC concentrations significantly increased 24 hr after rebamipide application to the conjunctival cultures-both with and without SOD-1 siRNA treatment (P<0.05 in both cases). CONCLUSIONS: Rebamipide seems to increase MUC5AC levels and suppress inflammation by decreasing IL-6 levels in mouse conjunctival epithelial cell cultures. SOD-1 siRNA-treated mouse conjunctival epithelial cell culture is a practical method for investigating changes in mucosa-associated mucins and proinflammatory cytokines in response to therapeutic interventions.

The effect of Nrf2 knockout on ocular surface protection from acute tobacco smoke exposure: evidence from Nrf2 knockout mice.

Ocular surface mucosa is the first-line ocular tissue to be exposed to environmental stress. We evaluated tear functions and keratoconjunctival epithelial alterations after sidestream cigarette smoke (SCS) exposure and tried to clarify the role of the transcription factor nuclear factor erythroid 2-related factor 2 (Nfe2l2, also known as Nrf2), on the ocular surface. In wild-type and Nrf2(-/-) mice, tear volume did not change after SCS exposure. Tear film breakup time (tear stability) in Nrf2(-/-) mice was significantly shorter than that in wild-type mice after SCS exposure. Vital staining scores, including fluorescein and Rose Bengal staining, showed significantly higher values in Nrf2(-/-) mice than in wild-type mice after SCS exposure. Excessive oxidative stress accumulation was detected in Nrf2(-/-) mice after SCS exposure using immunohistochemical analysis. Immunohistochemical analysis also revealed decreased mucin 1 (Muc1) and Muc5ac staining in Nrf2(-/-) mice after SCS exposure. mRNA expression levels of Muc1, Muc4, and Muc5ac and of SAM-pointed domain epithelial-specific transcription factor in Nrf2(-/-) mice were lower than those in wild-type mice after SCS exposure. Mean tear IL-6 concentrations increased significantly in Nrf2(-/-) mice after SCS exposure. In conclusion, SCS exposure induced decreased tear stability, ocular surface damage, and altered conjunctival phenotype in Nrf2(-/-) mice. Nrf2 could play an important role in protection of the ocular surface against SCS exposure.

The effects of 3% diquafosol sodium application on the tear functions and ocular surface of the Cu,Zn-superoxide dismutase-1 (Sod1)-knockout mice.

PURPOSE: To investigate the role of a water and mucin secretagogue (3% diquafosol sodium eye drops) on the tear function and conjunctival ocular surface changes in Sod1(-/-) in comparison to the wild-type (WT) mice. METHODS: Fourteen eyes of 7 Sod1(-/-) male mice with C57BL/background and 14 eyes of 7 C57BL6 strain wild-type male mice were examined at 40 weeks in this study. All mice had application of 3% diquafosol ophthalmic solution six times a day for 2 weeks. Tear film stability and corneal epithelial damage was evaluated by fluorescein and Rose Bengal stainings. Anterior segment photography was performed before and after eye drop instillations. Aqueous tear quantity was measured with phenol red-impregnated cotton threads without anesthesia. Animals were sacrificed at 42 weeks after diquafosol treatment and the whole globe specimens were subjected to periodic acid Schiff staining. Goblet cell density was quantified by J Image software. Quantitative real-time PCR for conjunctival muc 5AC messenger RNA expression was also performed. RESULTS: Sod1(-/-) mice had significantly higher fluorescein staining scores compared to the WT mice before eye drop instillation. The mean tear film breakup time, Rose Bengal staining scores, and muc5 messenger RNA expression improved significantly with diquafosol treatment in both the WT and the knockout mice. The mean fluorescein staining score and aqueous tear quantity significantly improved in the Sod1(-/-) mice with treatment. A notable and consistent increase in goblet cells and decrease in inflammatory cell infiltrates could be confirmed in all specimens after 2 weeks of diquafosol eye drop application. CONCLUSIONS: Three percent diquafosol ophthalmic solution appears to be effective in the treatment of ocular surface disease in this age-related dry eye disease mouse model.

The effects of 2% rebamipide ophthalmic solution on the tear functions and ocular surface of the superoxide dismutase-1 (sod1) knockout mice.

PURPOSE: To investigate the efficacy of 2% rebamipide ophthalmic solution on the tear functions and ocular surface status of the superoxide dismutase-1(Sod1(-/-)) mice. METHODS: Two percent Rebamipide ophthalmic solution was applied to 40-week-old male Sod1(-/-) and wild-type (WT) mice four times a day for 2 weeks. We examined the cytokine concentrations in the tear fluid (by CytoBead assay), tear film break-up time, amount of tear production, and expressions of mucins 1, 4, and 5AC, by RT-PCR. We also performed vital staining of the ocular surface, PAS staining for muc5AC, and immunohistochemical stainings for 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-2'-deoxyguanosine (8-OHdG), in the conjunctiva to compare the results before and after rebamipide instillations. RESULTS: The tear functions and ocular surface epithelial damage scores were significantly worse in the Sod1(-/-) than in the WT mice. Application of 2% rebamipide for 2 weeks significantly improved the tear film break-up time, the amount of tear production, and the corneal epithelial damage scores, which also significantly increased the conjunctival goblet cell density and muc5 mRNA expression, in the Sod1(-/-) mice. The mean IL-6, IL-17, TNF-α, and IFN-γ levels in the tear fluid were reduced significantly along with a significant decrease in the density of cells positive for 4-HNE and 8-OHdG in the conjunctiva. CONCLUSIONS: Two percent Rebamipide ophthalmic solution significantly improved the tear stability and corneal epithelial damage, and enhanced the expression of muc5 mRNA on the ocular surface. We also observed anti-inflammatory effects in the tear film together with antioxidative effects in the conjunctiva, suggesting the efficacy of rebamipide in age-related dry eye disease attributable to SOD1 knockout.

Identification of the rat IgA Fc receptor encoded in the leukocyte receptor complex.

Human FcalphaRI (CD89) is a myeloid-specific IgA Fc receptor encoded in the leukocyte receptor complex. Thus far, no gene coding for FcalphaRI has been identified in mice. Here, we show that, unlike mice, rats have the gene ( Fcar) coding for FcalphaRI. The rat Fcar gene has an exon-intron structure essentially identical to that of the human counterpart and is encoded in the leukocyte receptor complex on Chromosome 1. Southern blot analysis using the rat Fcar as a probe revealed hybridizing bands in Chinese and Syrian hamsters and gerbils, but not in mice, indicating that Fcar was lost in the lineage leading to mice after the divergence of rats and mice. Identification of FcalphaRI in rats should facilitate the elucidation of the in vivo role of this receptor.

A boy with mitochondrial disease: asymptomatic proteinuria without neuromyopathy.

Mitochondrial disorder is a relatively rare disease during childhood. Previous studies concluded that renal complications in this disease most often occur in patients with mitochondrial encephalomyopathies. We describe a boy with mitochondrial disease who presented with proteinuria while lacking neuromyopathy. Proteinuria was detected at the age of 6 years, including large amounts of low-molecular-weight proteins such as beta(2)- and alpha1-microglobulin. Renal functions were normal. Proximal tubular dysfunction and other renal manifestations were absent. Episodic neurologic problems such as migraine and nervous system diseases including epilepsy, depression, schizophrenia and amytrophic lateral sclerosis (ALS) were found in the boy's family members. Renal tubular basement membrane atrophy and interstitial fibrosis with mononuclear cell infiltration were observed. Ultrastructural examination showed mitochondria, mainly in the proximal tubules, which varied in size and had disoriented cristae. Mutation analysis using mitochondrial DNA (mtDNA) extracted from renal tissues demonstrated a A-->G point mutation at nucleotide position 3243 in the tRNA(Leu(UUR)) gene, while there was no mutation found in mtDNA extracted from peripheral leukocytes. Awareness among pediatricians of mitochondrial disorders, detection of low-molecular-weight proteinuria, renal ultrastructural examination and mutation analysis of mtDNA obtained from renal tissues could be important for early diagnosis of this disease.

A family of MHC class I-like genes located in the vicinity of the mouse leukocyte receptor complex.

Some members of the major histocompatibility complex (MHC) class I gene family are encoded outside the MHC. Here we describe a family of mouse class I-like genes mapping to the vicinity of the leukocyte receptor complex (LRC) on chromosome 7. This family, which we call Mill (MHC class I-like located near the LRC), has two members designated Mill1 and Mill2. Both genes are predicted to encode membrane glycoproteins with domain organization essentially similar to that of MHC class I heavy chains. The following features of Mill are noteworthy. (i) The deduced MILL proteins lack most of the residues known to be involved in the docking of peptides in classical MHC class I molecules. (ii) Among the known members of the class I gene family, MILL1 and MILL2 are related most closely to MICA/MICB encoded in the human MHC. (iii) Unlike all other known members of the class I gene family, Mill1 and Mill2 have an exon between those coding for the signal peptide and the alpha1 domain. (iv) Mill1 has a more restricted expression profile than Mill2. (v) The gene orthologous to Mill1 or Mill2 apparently is absent in the human. (vi) Mill1 and Mill2 show a limited degree of polymorphism in laboratory mice. The observation that the Mill family is related most closely to the MIC family, together with its apparent absence in the human, suggests its involvement in innate immunity.

The leukocyte common antigen (CD45) of the Pacific hagfish, Eptatretus stoutii: implications for the primordial function of CD45.

CD45, originally known as the leukocyte common antigen, is a prototypical transmembrane protein tyrosine phosphatase that plays a critical role in signal transduction through T-cell and B-cell receptors, as well as in T-cell and B-cell development. In the present study, we show that the Pacific hagfish, widely believed to lack the adaptive immune system, has CD45. The presence of CD45 in jawless fish is consistent with the recent discovery that CD45 also plays a crucial role in innate immunity via the regulation of signaling through type I and type II cytokine receptors. It is likely that CD45 was recruited to activate lymphocytes through antigen receptors encoded by rearranging genes in jawed vertebrates.