Use of white light in vivo confocal microscopy for the detection of spatial changes in the corneal nerves in cases of early-stage Acanthamoeba keratitis with radial keratoneuritis.

Purpose: Radial keratoneuritis (RK) is a common feature of Acanthamoeba keratitis (AK). In vivo confocal microscopy (IVCM) is noninvasive and provides real-time images for the diagnosis of corneal diseases by allowing the visualization of corneal structures and morphologies of living organisms at the cellular level. Images of AK with RK obtained using commercial white light IVCM devices have not been frequently evaluated. In the present study, a white light IVCM device was used to evaluate the corneal findings and describe spatial changes in the corneal nerves at different depths in cases of early-stage AK with RK. Methods: In this retrospective, observational study, white light IVCM images focused on RK were evaluated for Acanthamoeba cysts/trophozoites, corneal deposits, and altered corneal nerves, with special emphasis on three-dimensional spatial changes in the corneal nerves at different depths. Results: Seventeen eyes of 17 patients exhibiting early-stage AK with RK were included in the study. Acanthamoeba cysts/trophozoites were observed in the corneal epithelium of 13 eyes and stroma of 7 eyes. Alterations in the corneal nerve morphology and density were observed from the basal epithelial layer to the stromal layer in 12 eyes. Acanthamoeba trophozoites were attached to the corneal stromal nerves in five eyes. Conclusion: These findings suggest that white light IVCM can identify consistent corneal findings, particularly spatial changes in the corneal nerves, in cases of early-stage AK with RK.

Analysis of SmartPlug Insertion-Related Complications.

PURPOSE: To evaluate the complications and the treatment results of SmartPlug-related complications. METHODS: Retrospective review of all patients from a single medical hospital who received SmartPlug (Medennium, Inc., Irvine, CA) insertions from October 2007 to February 2014. All patients who developed SmartPlug-related canaliculitis and pyogenic granuloma were analyzed. Lacrimal irrigation with antibiotics was performed in most patients. RESULTS: Six hundred thirty-one eyes received SmartPlug insertion and 18 eyes developed SmartPlug-related complications (14 canaliculitis and 4 pyogenic granuloma). The mean time interval from insertion to development of the complications is 3.0 (0.5-6.9) years. Sixteen eyes received lacrimal irrigation with antibiotics, and all the eyes showed improvement without recurrence. The other two eyes had recurrence of granuloma pyogenica only after surgical excision. CONCLUSIONS: SmartPlug-related complications, including canaliculitis and granuloma pyogenica, required long-term follow-up. Most of the complications can be cured by lacrimal irrigation of antibiotics.

The corneal epitheliotrophic abilities of lyophilized powder form human platelet lysates.

PURPOSE: To evaluate whether lyophilized human platelet lysate (HPL) powder can preserve the growth factor concentrations and epitheliotrophic properties of liquid HPL, and potentially be used as a clinically-friendly treatment option. METHODS: Two commercialized liquid HPLs, UltraGRO TM (Helios, Atlanta, GA) and PLTMax (Mill Creek, Rochester, MI), were obtained and converted to lyophilized powder. After redissolution, lyophilized powder HPLs were compared with liquid HPLs, as well as human peripheral serum (HPS) and fetal bovine serum (FBS) in liquid or redissolved lyophilized powder forms. Concentrations of epidermal growth factor (EGF), transforming growth factor-ß1 (TGF-ß1), platelet-derived growth factor-AB (PDGF-AB) and platelet-derived growth factor-BB (PDGF-BB) were evaluated by enzyme-linked immunosorbent assay (ELISA). Human corneal epithelial cell line was incubated with the blood derivatives and evaluated for cell migration with scratch-induced directional wounding and proliferation with MTS assays. Cell differentiation was examined by transepithelial electrical resistance (TEER). Fluorescein staining and in vivo confocal microscopy were used to evaluate in vivo corneal epithelial wound healing in Sprague-Dawley rats that underwent corneal debridement and topical application of liquid and redissolved powder HPLs. RESULTS: Liquid form and redissolved lyophilized powder form HPLs had similar concentrations of EGF, TGF-ß1, PDGF-AB and PDGF-BB. In vitro experiments on cell migration, proliferation and differentiation and rat models on wound healing demonstrated no significant difference between the liquid and redissolved lyophilized powder forms for HPLs, HPS and FBS. In vivo confocal microscopy revealed similar wound healing process at different layers of cornea after corneal epithelial debridement between liquid form and redissolved lyophilized power form of HPLs. CONCLUSIONS: The redissolved lyophilized powder form of both commercialized HPLs showed similar growth factor concentrations and corneal epitheliotrophic abilities compared to the liquid form. Results suggest that the properties of liquid HPLs can be retained despite lyophilization and that lyophilized HPLs can be a treatment option for corneal epithelial disorders.

Comparison of corneal epitheliotrophic capacities among human platelet lysates and other blood derivatives.

PURPOSE: To evaluate the corneal epitheliotropic abilities of two commercialized human platelet lysates (HPLs) and to compare the results with other blood derivatives, including human peripheral serum (HPS) and bovine fetal serum (FBS). METHODS: In vitro, human corneal epithelial cells were incubated in various concentrations (0%, 3%, 5% and 10%) of blood derivatives. Two commercialized HPLs, including UltraGRO TM (Helios, Atlanta, GA) and PLTMax (Mill Creek, Rochester, MI), were tested and compared with HPS and FBS. Scratch-induced directional wounding assay was performed to evaluate cellular migration. MTS assay was used to evaluate cellular proliferation. Cellular differentiation was examined by scanning electron microscopy, inverted microscopy and transepithelial electrical resistance. Sprague-Dawley rats were used to evaluate the effects of the blood derivatives on corneal epithelial wound healing in vivo. Different blood derivatives were applied topically every 2 hours for 2 days after corneal epithelial debridement. The concentrations of epidermal growth factor (EGF), transforming growth factor -ß1 (TGF-ß1), fibronectin, platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, and hyaluronic acid in different blood derivatives were evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: In vitro experiments demonstrated statistically comparable epitheliotropic characteristics in cellular proliferation, migration, and differentiation for the two commercialized HPLs compared to FBS and HPS. Cells cultured without any serum were used as control group. The epitheliotropic capacities were statistically higher in the two commercialized HPLs compared to the control group (p<0.05). Among the different concentrations of blood derivatives, the preparations with 3% yielded better outcomes compared to 5% and 10%. In rats, HPLs also caused improved but not statistically significant wound healing compared to HPS. All the blood derivatives had better wound healing ratios than the control group (p<0.05). In the quantification of epitheliotropic factors, UltraGRO and PLTMax had significantly higher levels of EGF, TGF- ß1, fibronectin than human peripheral serum (p<0.05). CONCLUSIONS: Both commercialized HPLs showed comparable corneal epitheliotropic abilities and wound healing rates compared to HPS and FBS in the in vivo and in vitro studies. Our results suggest that HPLs may have the potential to replace HPS in the treatment of corneal epithelial problems.

Effect of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of in vitro cultivated limbal epithelial cell sheets.

PURPOSE: The aim of this study is to evaluate the effects of air-lifting on the stemness, junctional protein formation, and cytokeratin expression of rabbit limbal stem cells cultivated in vitro, and to find out the proper timing of air-lifting before transplantation as limbal epithelial cell sheets for the treatment of limbal insufficiency. MATERIALS AND METHODS: Limbal epithelial cells were isolated from the limbus of New Zealand white rabbits and cultivated in vitro. After the cells became confluent, different durations of air-lifting (0, 1, 2, 4, and 7 days) were performed. At the end of cultivation, immunohistochemistry on cryosections was performed and observed by fluorescein microscopy and in vitro confocal microscopy for cytokeratins (K3, K10, K12, K13, and K14), junctional and structural proteins (ZO-1, p120, and actin) and stem cell markers (ABCG2 and P63). Scanning electron microscopy was used for observing the microstructure of superficial cells. Transepithelial electrical resistance (TEER) was used to measure the transepithelial permeability. RESULTS: The expression of K3, K10, K12, K13, K14, and ABCG2 showed no differences in pattern and location among different groups of airlifting. A time-dependent increase in corneal epithelial thickness was found after air-lifting. In vitro confocal microscopy demonstrated that K3, p120, and ZO-1 were expressed on the apical cell layer, whereas P63 and ABCG2 were expressed more on the basal epithelial layer. Scanning electron microscopy of the superficial layer demonstrated that airlifting induced time-dependent increase in the size of surface epithelial cells and triggered cellular differentiation. TEER results demonstrated a time-dependent increase of transepithelial electric resistance. CONCLUSIONS: During limbal epithelial cell expansion in vitro, air-lifting can increase cellular stratification, enlarge surface cells, trigger cellular differentiation, and increase the transepithelial barrier. However, the expression of cellular junctional, stem cell and cytokeratin markers seems to have no obvious differences in pattern and localization.

Serum components and clinical efficacies of autologous serum eye drops in dry eye patients with active and inactive Sjogren syndrome.

PURPOSE: Autologous serum eye drops are considered safe and efficient for the treatment of various ocular surface disorders, including dry eye diseases (DED) caused by the primary and secondary Sjogren syndrome (SS). However, the serum components in patients of SS may be different from those of normal patients and can thus lead to unpredictable therapeutic effects. This study divided the SS patients into active and inactive types based on the erythrocyte sedimentation rate and the presence or absence of active rheumatoid arthritis. METHODS: We compared the serum components of these two groups with standard and multiplex enzyme linked immunosorbent assay arrays and predicted the therapeutic effects of topical autologous serum for the treatment of DED with ocular surface disease index (OSDI) and Oxford Schema scale (OSS). RESULTS: Hyaluronic acid and transforming growth factor b1 levels were significantly higher in the active SS group compared to the inactive SS group (P < 0.01), whereas epidermal growth factors, insulin growth factor 1, and fibroblast growth factor b had no significant differences between these two groups. Active SS group had significantly higher expressions of interleukin (IL) 1 beta, IL 6, and tumor necrosis factor alpha compared to inactive SS patients (P < 0.05). There were no statistical differences in therapeutic effects between these two groups, as measured with the OSDI or OSS. CONCLUSION: Dividing the Sjogren dry eye patients into active and inactive groups may appear as a reasonable method to predict the quality of autologous serum eye drops, but there seems to be no significant predictability to the therapeutic effects.