Tear film instability is associated with weakened colocalization between occludin and MUC5AC in scopolamine-induced dry eye disease (DED) rats.

PURPOSE: Dry eye disease (DED) is a disease with tear film instability because of multiple factors. This study was conducted to explore roles of occludin and MUC5AC in tear film instability in DED rat model. METHODS: A total of 20 SD rats were divided into DED group (n = 10) and normal control (NC) group (n = 10). DED rat model was established by subcutaneously injecting with scopolamine hydrobromide. Clinical examinations, including tear breakup time (tBUT), Schirmer's test and corneal fluorescein staining, were conducted to determine corneal functions. Transmission electron microscopy was used to measure the ultrastructures of corneal epithelial cells. Western blotting assay was used to identify occludin expression in corneal tissues of DED rats. Real-time PCR (RT-PCR) was performed to verify gene transcription of occludin and MUC5AC. Colocalization between occludin and MUC5AC was identified with confocal fluorescence microscopy. RESULTS: Tear breakup time was significantly shorter, and corneal fluorescein staining score was predominantly higher in DED rats compared to those in normal rats (P < 0.05). Normal rats showed a steady tear secretion throughout the whole experiments, while DED rats showed a dramatic reduction on day 14. DED rats demonstrated ultrastructural damage of Golgi apparatus and endoplasmic reticulum in corneal epithelial cells. Occludin and MUC5AC expressions were significantly downregulated in corneal tissue of DED rats compared with those of normal rats (P < 0.05). Percentage of occludin-MUC5AC-colocalized corneal epithelial cells in DED rats was significantly less compared with those in normal rats (P < 0.01). CONCLUSIONS: Tear film stability was damaged in scopolamine-induced DED rats because of the weakened colocalization between occludin and MUC5AC molecule. This study would provide a potential clue for the pathogenesis and a promising theoretical basis for clinical work of DED.

Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment.

Corneal stroma-derived mesenchymal stem cells (CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells (LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.

A consumer-resource competition model with a state-dependent delay and stage-structured consumer species.

In this paper, a new consumer-resource competition model with a state-dependent maturity delay is developed, which incorporates one resource species and two stage-structured consumer species. The main innovation is that the model directly manifests the relationship between resources and maturity time of consumers through a correction term, $1-\tau'(x(t))x'(t)$. Firstly, the well-posedness of the solution is studied. At the same time, the existence and uniqueness of all equilibria are discussed. Then, the linearized stabilities of the equilibria are achieved. Finally, some sufficient conditions which ensure the global attractivity of the coexistence equilibrium are obtained.

Expression of visual system homeobox 1 in human keratoconus.

AIM: To investigate the expression of visual system homeobox 1 (VSX1) and myofibroblast marker alpha smooth muscle actin (α-SMA) in keratoconus (KC). METHODS: Thirty corneal tissue were collected from KC patients after corneal transplantation and 15 normal donor corneas were obtained. All corneal tissues divided into 4 parts for different detections. Scanning electron microscopy was used to observe the ultrastructure of the specimens. VSX1 and α-SMA localization in cornea tissues was detected using immunofluorescence histochemistry. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot were performed to analyze the expression level of VSX1 and α-SMA. RESULTS: Compared to normal cornea tissue, the collagen fibers in KC stroma were distortional and attenuated and keratocytes were abnormally changed. VSX1 and α-SMA located in the corneal stroma. The mRNA and protein expression level of VSX1 in KC were about 3 times as high as that of normal tissue (P<0.001). α-SMA was hardly expressed in the normal corneas, however, its expression in the KC was about 1.5 times higher than that of the normal corneas (P<0.0001). CONCLUSION: Compared with normal corneal the expression of VSX1 and α-SMA in KC both increased. VSX1 is related to the activation of keratocytes and involved in the pathogenesis of keratoconus.

Corneal collagen cross-linking and liposomal amphotericin B combination therapy for fungal keratitis in rabbits.

AIM: To observe the therapeutic effect of corneal collagen cross-linking (CXL) in combination with liposomal amphotericin B in fungal corneal ulcers. METHODS: New Zealand rabbits were induced fungal corneal ulcers by scratching and randomly divided into 3 groups, i.e. control, treated with CXL, and combined therapy of CXL with 0.25% liposomal amphotericin B (n=5 each). The corneal lesions were documented with slit-lamp and confocal microscopy on 3, 7, 14, 21 and 28d after treatment. The corneas were examined with transmission electron microscopy (TEM) at 4wk. RESULTS: A rabbit corneal ulcer model of Fusarium was successfully established. The corneal epithelium defect areas in the two treatment groups were smaller than that in the control group on 3, 7, 14 and 21d (P<0.05). The corneal epithelium defect areas of the combined group was smaller than that of the CXL group (P<0.05) on 7 and 14d, but there were no statistical differences on 3, 21 and 28d. The corneal epithelium defects of the two treatment groups have been healed by day 21. The corneal epithelium defects of the control group were healed on 28d. The diameters of the corneal collagen fiber bundles (42.960±7.383 nm in the CXL group and 37.040±4.160 nm in the combined group) were thicker than that of the control group (24.900±1.868 nm), but there was no difference between the two treatment groups. Some corneal collagen fiber bundles were distorted and with irregular arrangement, a large number of fibroblasts could be seen among them but no inflammatory cells in both treatment groups. CONCLUSION: CXL combined with liposomal amphotericin B have beneficial effects on fungal corneal ulcers. The combined therapy could alleviate corneal inflammattions, accelerate corneal repair, and shorten the course of disease.

Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea.

AIM: To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS: A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy (TEM) and hematoxylin and eosin (H&E) staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype. RESULTS: The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process. In vitro, the methyl thiazolyl tetrazolium results revealed that extracts of the acellular ostrich corneas (AOCs) had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes. The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea (APC) transplantation. The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer. CONCLUSION: We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.

Establishment of a novel therapeutic vector targeting the trigeminal ganglion in rats.

BACKGROUND: In the pathogenesis of herpes simplex keratitis, herpes simplex virus type 1 (HSV-1) infection begins in corneal epithelium cells and then progresses through the sensory nerve endings and finally travels up forward to the trigeminal ganglion (TG), where it remains as latent virus. The available anti-HSV therapies do not completely suppress the recurrence of active HSV-1 infection. The aim of this study was to establish a novel replication-defective (rd) HSV-1 (rdHSV) vector (rdHSV-interferon gamma [IFNγ]) that could effectively target the TG. METHODS: Recombinant HSV-1 virus was inserted into a shuttle plasmid carrying IFNγ to establish the rdHSV-IFNγ vector. Safety was evaluated in vitro by 50% cellular cytotoxicity in transfected SH-SY5Y neuroblastoma cells and in vivo by Kaplan-Meier survival estimate and infection rate. Wistar rats were immunized with rdHSV-IFNγ to evaluate the TG targeting efficiency. Real-time polymerase chain reaction and Western blot assays were used to evaluate IFNγ mRNA and protein expression and rdHSV-IFNγ localization. RESULTS: The rdHSV-IFNγ vector was successfully constructed and showed high in vitro safety and overall survival and a corneal infection rate similar to that of control rats immunized with saline (control group; P>0.05). Real-time polymerase chain reaction and immunohistochemistry assays confirmed IFNγ expression and effective TG targeting on days 14 and 21, which increased with postimmunization time. Moreover, IFNγ was expressed sufficiently in the TG tissues. CONCLUSION: The rdHSV-IFNγ can act as an effective gene transporting vector that carries the therapeutic genes to the TG and triggers its expression.

Replication-defective HSV-1 effectively targets trigeminal ganglion and inhibits viral pathopoiesis by mediating interferon gamma expression in SH-SY5Y cells.

It has been widely believed that recurrence of herpes simplex keratitis (HSK) is due to the reactivation of herpes simplex virus type 1 (HSV-1) from latent sites in trigeminal ganglion (TG). However, there are also not effective vectors which could target TG for therapy. Replication-defective HSV-1 vector (rdHSV-IFNγ) was established by calcium phosphate co-transfection of complementing cells. We firstly infected rdHSV-IFNγ to SH-SY5Y, and detected IFNγ expression by western blot, evaluated 50 % cellular cytotoxicity (CC(50)) by ELISA. Antiviral activity of rdHSV-IFNγ was examined by immunofluorescence and antiviral concentration of 50 % effectiveness (EC(50)) assay. The rdHSV-IFNγ vector was immunized to Wistar rats to observe targeting function to TG. Kaplan-Meier survival analysis was utilized to assess security of rdHSV-IFNγ. RT-PCR and immunohistochemistry assay were employed to detect rdHSV-IFNγ localization in TG. Western blot was employed to detect IFNγ expression. rdHSV-IFNγ was successfully established, and performed an effective antiviral activity and higher security in SH-SY5Y. There were no significant differences of survival and corneal infection rate of rdHSV-IFNγ immunized rats among groups (P > 0.05). RT-PCR and immunohistochemistry indicated that expression of glycoprotein D (gD) in TG could target TG and decreased following with times post immunization. Furthermore, IFNγ was expressed effectively in TG tissues. Our findings indicated that established rdHSV-IFNγ vector effectively transported therapeutic gene into TG tissues. The administration of replication-defective vector carrying therapeutic genes may become a promising tool in inhibition or reoccurrence of HSK in clinical.

17-Alpha-estradiol ameliorating oxygen-induced retinopathy in a murine model.

PURPOSE: Our aim was to explore the effects and mechanism of 17-alpha-estradiol (17α-E2) on oxygen-induced retinopathy (OIR) in a murine model. METHODS: Newborn mice exposed to hyperoxia underwent subcutaneous injections of different doses of 17α-E2 from postnatal days (PND) 7 to 17. The retinal flat mounts were scored for avascular/total retinal area on PND 17. Vascular endothelial growth factor (VEGF), malondialdehyde (MDA) concentrations, and intensity, activity, and quality of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the retina were determined on PND 9, 13 (14), and 17. RESULTS: The avascular area, which is found in retinas of hyperoxia-exposed pups but not in retinas of normoxia-exposed ones, was significantly smaller in retinas of 17α-E2-treated pups. MDA and VEGF concentrations and intensity, activity, and quality of NADPH oxidase were stable in retinas of normoxia pups on PND 9, 13 (14), and 17, whereas in retinas of hyperoxia-exposed and 17α-E2-treated pups, they fluctuated markedly. VEGF concentrations were lower in retinas of hyperoxia-exposed pups than in those of normoxia ones on PND 9. Elevated VEGF concentrations were found in retinas of 17α-E2-treated pups on PND 9 and in hyperoxia-exposed pups on PND 14 and 17. Low VEGF concentrations were found in retinas of 17α-E2-treated pups on PND 14 and 17. MDA concentrations and NADPH oxidase concentration and activity, which were higher in retinas of hyperoxia-exposed pups, were lower in retinas of 17α-E2-treated pups on PND 9, 13, and 17. The most effective outcome in retinas of 1.0 µg 17α-E2-treated pups was markedly reversed by ICI182780. CONCLUSIONS: We found that 17α-E2 mitigates oxidative stress reactions and ameliorates OIR severity by decreasing NADPH oxidase expression and activity via the receptor and other pathways.

[Tear osmolarity and dry eye].

Dry eye is a common eye disease, and its incidence rate has been escalating. The increased tear osmolarity is one of the main reasons for complaint, damage and inflammation of dry eye patients. With the breakthrough of testing technology for tear osmolarity, more research and application of tear osmolarity was reported, and papers on tear osmolarity of normal eye and dry eye in different regions were also published. In this article, the progress of the tear osmolarity research, the range of tear osmolarity and its application in diagnosis and therapy of dry eye was introduced, and the prospect for the clinical application of hypotonic artificial tears was also discussed.