Behrad Y. Milani

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-β (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-β 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-β-induced expression of myofibroblast markers (17.2% SMA positive cells with rapamycin compared to 69.0% in control). Rapamycin also significantly inhibited TGF-β-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.

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.

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.

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 Notch1flox/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.

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.

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 β-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-β-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.

In Vivo Evaluation of a Decellularized Limbal Graft for Limbal Reconstruction

Corneal and limbal epithelial function is highly dependent on its underlying matrix. In this study, we report the in vitro and in vivo effects of a decellularized limbal matrix on corneal and limbal epithelial differentiation and repair. We demonstrate that a limbal matrix helps to maintain epithelial cells in a more proliferative and less differentiated state. We introduce a novel focal injury model to the limbus using an excimer laser and further show that transplanting a decellularized limbal graft after the limbal injury helps to promote epithelialization and reduce corneal haze formation. These results suggest that a decellularized limbal graft may be therapeutically beneficial in clinical cases of focal limbal deficiency.