Diabetes-Associated Hyperglycemia Causes Rapid-Onset Ocular Surface Damage.

Purpose: The metabolic alterations due to chronic hyperglycemia are well-known to cause diabetes-associated complications. Short-term hyperglycemia has also been shown to cause many acute changes, including hemodynamic alterations and osmotic, oxidative, and inflammatory stress. The present study was designed to investigate whether diabetes-associated hyperglycemia can cause rapid-onset detrimental effects on the tear film, goblet cells, and glycocalyx and can lead to activation of an inflammatory cascade or cellular stress response in the cornea. Methods: Mouse models of type 1 and type 2 diabetes were used. Tear film volume, goblet cell number, and corneal glycocalyx area were measured on days 7, 14, and 28 after the onset of hyperglycemia. Transcriptome analysis was performed to quantify changes in 248 transcripts of genes involved in inflammatory, apoptotic, and stress response pathways. Results: Our data demonstrate that type 1 and type 2 diabetes-associated hyperglycemia caused a significant decrease in the tear film volume, goblet cell number, and corneal glycocalyx area. The decrease in tear film and goblet cell number was noted as early as 7 days after onset of hyperglycemia. The severity of ocular surface injury was significantly more in type 1 compared to type 2 diabetes. Diabetes mellitus also caused an increase in transcripts of genes involved in the inflammatory, apoptotic, and cellular stress response pathways. Conclusions: The results of the present study demonstrate that diabetes-associated hyperglycemia causes rapid-onset damage to the ocular surface. Thus, short-term hyperglycemia in patients with diabetes mellitus may also play an important role in causing ocular surface injury and dry eye.

Local Renin-Angiotensin System Activation and Myofibroblast Formation in Graft Versus Host Disease-Associated Conjunctival Fibrosis.

Purpose: The present study was designed to investigate the role of myofibroblast transdifferentiation and the conjunctival renin-angiotensin system (RAS) in the pathogenesis of graft-versus-host disease (GVHD)-associated conjunctival fibrosis. Methods: A mouse model of major histocompatibility-matched allogeneic transplantation was used to induce GVHD, with male B10.D2 mice as donors and female BALB/c mice as recipients. Male BALB/c to female BALB/c syngeneic transplantation was used as control. Y chromosome staining in the spleen cells obtained from female recipient mice was used to confirm engraftment. The phenol red thread test and fluorescein staining were used to quantify tears and corneal keratopathy. Eyes were harvested at 4 and 8 weeks after the transplant for alpha-smooth muscle actin (α-SMA), angiotensinogen, and angiotensin-converting enzyme (ACE) immunostaining. Conjunctiva was harvested for gene expression quantification of α-SMA, angiotensinogen, and ACE. Results: More than 80% of the spleen cells in the recipient mice were chromosome Y positive, thus conforming successful engraftment. A significant decrease in tear secretion and a marked increase in corneal keratopathy score after allogeneic transplantation indicated the onset of ocular GVHD in these mice. A significant increase in α-SMA gene expression and the presence of a large number of α-SMA-positive cells was noted in the bulbar orbital conjunctiva of mice after allogeneic transplantation. Allogenic transplantation also caused a significant increase in the gene expression and protein expression of angiotensinogen and ACE in the subconjunctival eyelid area. Conclusions: Results of the present study demonstrate that GVHD-associated conjunctival fibrosis is accompanied by myofibroblast formation and activation of the local conjunctival RAS.

Effect of High Glucose on Ocular Surface Epithelial Cell Barrier and Tight Junction Proteins.

Purpose: Patients with diabetes mellitus are reported to have ocular surface defects, impaired ocular surface barrier function, and a higher incidence of corneal and conjunctival infections. Tight junctions are critical for ocular surface barrier function. The present study was designed to investigate the effect of high glucose exposure on human corneal and conjunctival epithelial cell barrier function and tight junction proteins. Methods: Human corneal and conjunctival epithelial cells were exposed to 15 mM and 30 mM glucose for 24 and 72 hours. The barrier function was measured using transepithelial electrical resistance (TEER). The cell migration was quantified using scratch assay. The cells were harvested for protein extraction and mRNA isolation. Gene and protein expression of claudins, zonula occludens (ZOs), and occludin was quantified using real-time PCR and Western blot. Results: Glucose caused a significant decrease in TEER after 72 hours of exposure in both corneal and conjunctival epithelial cells. Glucose did not cause any notable change in migration of either corneal or conjunctival epithelial cells. Glucose exposure did not cause any notable change in protein expression of claudin-1, ZO-1, ZO-2, ZO-3, or occludin. On the other hand, 15 mM glucose caused an increase in gene expression of claudin-1, claudin-3, ZO-2, ZO-3, and occludin, a likely response to osmotic stress since 15 mM mannitol also caused consistently similar increase in gene expression of these proteins. Conclusions: High glucose exposure causes impairment of corneal and conjunctival epithelial cell barrier function, but this detrimental effect is not caused by a decrease in expression of tight junction proteins: claudin-1, ZO-1, ZO-2, ZO-3, and occludin.

Graft Versus Host Disease-Associated Dry Eye: Role of Ocular Surface Mucins and the Effect of Rebamipide, a Mucin Secretagogue.

Purpose: The present study was designed to investigate the role of ocular surface glycocalyx and mucins in graft versus host disease (GVHD)-associated dry eye. The ameliorative effect of topical rebamipide, a mucin secretagogue, on GVHD-associated dry eye was also tested. Methods: A mouse model of allogeneic transplantation was used to induce ocular GVHD with C57BL/6 as donors and B6D2F1 as recipient mice. Phenol red thread method and fluorescein staining was used to quantify tear secretion and corneal keratopathy. At 8 weeks after the allogeneic transplantation, corneas were harvested to perform glycocalyx staining and confocal microscopy. Goblet cell staining was performed using periodic acid Schiff's staining. Corneal and tear film levels of Mucin 1, 4, 16, 19, and 5AC were quantified using ELISA and real-time PCR. Rebamipide was applied topically twice daily to mice eyes. Results: Allogeneic transplantation resulted in ocular GVHD-associated dry eye characterized by a significant decrease in tear film volume and the onset of corneal keratopathy. Ocular GVHD caused a significant decrease in the area and thickness of corneal glycocalyx. A significant decrease in the goblet cells was also noted. A significant decrease in mucin 4 and 5AC levels was also observed. Topical treatment with rebamipide partially attenuated ocular GVHD-mediated decrease in tear film volume and significantly reduced the severity of corneal keratopathy. Conclusions: Ocular GVHD has detrimental impact on ocular surface glycocalyx and mucins. Rebamipide, a mucin secretagogue, partially prevents ocular GVHD-associated decrease in tear film and reduces the severity of corneal keratopathy.