Endoplasmic reticulum stress PERK-ATF4-CHOP pathway is involved in non-alcoholic fatty liver disease in type 1 diabetic rats: The rescue effect of treatment exercise and insulin-like growth factor I.

Endoplasmic Reticulum Stress (ERS) is a key factor in the development of Non-Alcoholic Fatty Liver Disease (NAFLD) in diabetes. The current study aimed to examine the effects of exercise and IGF-I on ERS markers in liver tissue. Rats were divided into five groups (n = 8 per group), including control (CON), diabetes (DIA), diabetes + exercise (DIA + EX), diabetes + IGF-I (DIA + IGF-I), and diabetes + exercise + IGF-I (DIA + EX + IGF-I). Type 1 diabetes was induced by an I.P. injection of streptozotocin (60 mg/kg). After 30 days of treatment with exercise or IGF-I alone or in combination, liver tissue was assessed for caspase 12, 8, and CHOP protein levels, and expression of ERS markers (ATF-6, PERK, IRE-1A) and lipid metabolism-involved genes (FAS, FXR, SREBP-1c) by western immunoblotting. In addition, for the evaluation of histopathological changes in the liver, Hematoxylin - Eosin and Masson's Trichrome staining were done. Compared to the control group, diabetes significantly caused liver fibrosis, induced ERS, increased caspase 12 and 8 levels in the liver, and changed expression levels of genes associated with lipid metabolism, including FAS, FXR, and SREBP-1c. Treatment with either exercise or IGF-I reduced fibrosis levels suppressed ER stress markers and apoptosis, and improved expression of genes associated with lipid metabolism. In addition, simultaneous treatment with exercise and IGF-I showed a synergistic effect compared to DIA + E and DIA + IGF-I. The results suggest that IGF-1 and exercise reduced liver fibrosis possibly by reducing ERS, creating adaptive ER stress status, and improving protein folding.

Corneal nerve healing after in situ laser nerve transection.

PURPOSE: We have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane. METHODS: Thy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption. RESULTS: The pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence. CONCLUSIONS: Interconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion.

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

Rapamycin Prolongs the Survival of Corneal Epithelial Cells in Culture.

Rapamycin has previously been shown to have anti-aging effects in cells and organisms. These studies were undertaken to investigate the effects of rapamycin on primary human corneal epithelial cells in vitro. Cell growth and viability were evaluated by bright field microscopy. Cell proliferation and cycle were evaluated by flow cytometry. The expression of differentiation markers was evaluated by quantitative PCR and Western blot. Senescence was evaluated by senescence-associated ß-Galactosidase staining and by Western blot analysis of p16. Apoptosis was evaluated by a TUNEL assay. The results demonstrated that primary HCEC treated with rapamycin had lower proliferation but considerably longer survival in vitro. Rapamycin-treated cells maintained a higher capacity to proliferate after removal of rapamycin and expressed more keratin 14, N-Cadherin, DeltaNp63 and ABCG2, and less keratin 12, consistent with their less differentiated state. Rapamycin treated cells demonstrated less senescence by X-ß-Gal SA staining and by lower expression of p16. Apoptosis was also lower in the rapamycin treated cells. These results indicate that rapamycin treatment of HCEC prevents the loss of corneal epithelial stem/progenitor cells to replicative senescence and apoptosis. Rapamycin may be a useful additive for ex vivo expansion of corneal epithelial cells.

Contact Lens-induced Limbal Stem Cell Deficiency.

Limbal stem cell deficiency (LSCD) is a pathologic condition caused by the dysfunction and/or destruction of stem cell precursors of the corneal epithelium, typified clinically by corneal conjunctivalization. The purpose of this review is to critically discuss a less well-known cause of limbal stem cell disease: contact lens (CL) wear. A literature search was conducted to include original articles containing patients with CL-induced LSCD. This review describes epidemiology, diagnostic strategies, pathogenesis, differential diagnosis, and treatment modalities for this condition.

Stability of limbal stem cell deficiency after mechanical and thermal injuries in mice.

We studied the reproducibility and stability of limbal stem cell deficiency (LSCD) in mice following controlled injuries to the corneal and limbal epithelia. In one method, corneal and limbal epithelia were entirely removed with a 0.5 mm metal burr. In the other, limbus to limbus epithelial removal with the burr was followed by thermal injury to the limbus. These two methods were compared with a previously published one. Unwounded corneas were used as control. The corneas were examined monthly for three months by slit lamp with fluorescein staining. Immunofluorescence staining for cytokeratin 12 and 8 on corneal wholemount and cross sections were performed to determine the phenotype of the epithelium. Mechanical shaving of the epithelium, with or without thermal injury, resulted in a reproducible state of LSCD marked by superficial neovascularization, reduce of keratin 12 expression and presence of goblet cells on the cornea. The phenotype was stable in 100% of the eyes up to at least three months. Thermal injury produced a more severe phenotype with more significant stromal opacification. These corneal injury models may be useful for studying the mechanisms leading to limbal stem cell deficiency.

Rapamycin inhibits the production of myofibroblasts and reduces corneal scarring after photorefractive keratectomy.

PURPOSE: Corneal stromal scarring partly involves the production of corneal myofibroblasts. The purpose of this study was to examine the effects of rapamycin (an inhibitor of the mammalian target of rapamycin [mTOR] pathway) on myofibroblast formation in vitro and in-vivo. METHODS: Human corneal fibroblasts were grown in culture and transformed into myofibroblasts using TGF-ß (2 ng/mL). The phosphorylation (activation) of the mTOR pathway was examined by immunoblotting. Cell proliferation with and without rapamycin was examined by thiazolyl blue tetrazolium bromide (MTT) assay and Ki67 staining. The expression of the myofibroblast differentiation marker smooth muscle actin (SMA) was examined by immunostaining and immunoblotting. The functional effects of rapamycin were measured using a gel contraction assay. For in vivo studies, 140 µm laser ablation was performed on rabbit corneas followed by subconjunctival rapamycin or vehicle. Corneal haze development was graded at 4 weeks, while the expression of myofibroblast markers was examined by immunostaining and immunoblotting. RESULTS: The TGF-ß activated the mTOR pathway with peak phosphorylation at 2 to 4 hours. Treatment of corneal fibroblasts with rapamycin reduced their proliferation by 46% compared to control. Rapamycin significantly inhibited TGF-ß-induced expression of myofibroblast markers (17.2% SMA positive cells with rapamycin compared to 69.0% in control). Rapamycin also significantly inhibited TGF-ß-induced collagen gel contraction. In the rabbit eyes treated with rapamycin, corneal haze development was significantly less compared to controls (0.75 ± 0.4 vs. 2.17 ± 0.7). CONCLUSIONS: Rapamycin appears to inhibit proliferation and differentiation of corneal myofibroblasts and, thus, may provide an effective therapeutic measure for preventing corneal scarring.

Loss of Notch1 disrupts the barrier repair in the corneal epithelium.

The corneal epithelium is the outermost layer of the cornea that directly faces the outside environment, hence it plays a critical barrier function. Previously, conditional loss of Notch1 on the ocular surface was found to cause inflammation and keratinization of the corneal epithelium. This was in part attributed to impaired vitamin A metabolism, loss of the meibomian glands and recurrent eyelid trauma. We hypothesized that Notch1 plays an essential role in the corneal epithelial barrier function and is a contributing factor in the pathologic changes in these mice. Notch1 was conditionally deleted in adult Notch1(flox/flox), K14-Cre-ERT(+/-) mice using hydroxy-tamoxifen. The results indicated that conditional deletion of Notch1 on the ocular surface leads to progressive impairment of the epithelial barrier function before the onset of corneal opacification and keratinization. Loss of the barrier was demonstrated both by an increase in in vivo corneal fluorescein staining and by enhanced penetration of a small molecule through the epithelium. Corneal epithelial wounding resulted in significant delay in recovery of the barrier function in conditional Notch1(-/-) mice compared to wild type. Mice with conditional deletion of Notch1 did not demonstrate any evidence of dry eyes based on aqueous tear production and had normal conjunctival goblet cells. In a calcium switch experiment in vitro, Notch1(-/-) cells demonstrated delayed membrane localization of the tight junction protein ZO-1 consistent with a defect in the epithelial tight junction formation. These findings highlight the role of Notch1 in epithelial differentiation and suggest that intrinsic defects in the corneal epithelial barrier recovery after wounding is an important contributing factor to the development of inflammatory keratinization in Notch1(-/-) mice.

The use of peer optic nerve photographs for teaching direct ophthalmoscopy.

OBJECTIVE: To use a novel teaching exercise to encourage students to practice ophthalmoscopy and to measure the learning effect both subjectively and objectively. DESIGN: Comparative case series. PARTICIPANTS: One hundred thirty-one fourth-year medical students on their 1-week ophthalmology rotations with 89 in the experimental group and 42 in the control group. METHODS: Those in the experimental group had 1 eye dilated and their optic nerve photographed on the first day. The next day, these students received an unlabeled optic nerve photograph belonging to 1 of their peers (typically 8-10 per group) and were given 3 days to identify the student matching the photograph. The students in the control group were simply encouraged to practice ophthalmoscopy on each other without the use of photographs. MAIN OUTCOME MEASURES: Both objective and subjective changes from the beginning to the end of the rotation were measured and compared between the 2 groups. RESULTS: In the 89 students who used peer optic nerve photographs, 75 (84.3%) showed improvement in direct ophthalmoscopy skills over the course of the week. In contrast, only 12 (28.6%) of the 42 control students demonstrated an objective improvement (P<0.001). The subjective confidence levels likewise were more improved in the students who took part in the optic nerve photograph exercise. CONCLUSIONS: These results suggest that the task of matching an unknown optic nerve photograph to the correct eye of a peer leads to increased self-confidence and more proficient use of the direct ophthalmoscope.

Notch inhibition during corneal epithelial wound healing promotes migration.

PURPOSE: To determine the role of Notch signaling in corneal epithelial migration and wound healing. METHODS: Immunolocalization of Notch1 was performed during epithelial wound healing in vivo in mouse corneal epithelial debridement wounds and in vitro in primary human corneal epithelial cells following a linear scratch wound. The effects of Notch inhibition, using the γ-secretase inhibitor N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) or following stable transfection with Notch1-short hairpin RNA (shRNA), was evaluated in a scratch assay and transwell migration assay. Likewise, in vitro adhesion, proliferation and the actin cytoskeleton was examined. The DAPT effect was also evaluated in vivo in a mouse model of corneal epithelial wound healing. RESULTS: The expression of Notch1 was reduced at the leading edge of a healing corneal epithelium both in vivo and in vitro. Notch inhibition using DAPT and using Notch1-shRNA both enhanced in vitro migration in scratch and transwell migration assays. Consistent with this increased migratory behavior, Notch inhibited cells demonstrated decreased cell-matrix adhesion and enhanced lamellipodia formation. Notch inhibition by DAPT was also found to accelerate corneal epithelial wound closure in an in vivo murine model without affecting proliferation. CONCLUSIONS: The results highlight the role of Notch in regulating corneal epithelial migration and wound healing. In particular, Notch signaling appears to decrease in the early stages of wound healing which contributes to cytoskeletal changes with subsequent augmentation of migratory behavior.