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Aim: This study aimed to test whether human platelet lysate (HPL) has neurotrophic ability for corneal nerve regeneration., Methods: We measured the neurotrophic factors in human peripheral serum (HPS) and two commercially available HPLs, UltraGRO and PLTMax. In vitro, we compared the growth rates, neuronal differentiation and immunostaining of neuron markers in mouse neuroblastoma cell line (Neuro-2a) and primary culture of mouse trigeminal ganglion cells that were cultivated in different concentrations of fetal bovine serum, HPS and HPL. In vivo, we created corneal wounds on Sprague Dawley rats with a rotating burr and evaluated the effects of topical HPL on wound healing and corneal nerve regeneration by in vivo confocal microscopy and corneal aesthesiometry., Results: HPLs had significantly higher concentrations of various neurotrophic factors compared with HPS (p<0.05). In Neuro-2a cells, 3% HPL was better at promoting neuronal growth and differentiation compared with HPS at the same concentration. HPL was also found to have superior neurotrophic effects compared with HPS in primary cultures of mouse trigeminal ganglion cells. In vivo, HPL-treated eyes had better corneal epithelial wound healing rate, nerve regeneration length and corneal touch threshold compared with eyes treated with artificial tears (p<0.05)., Conclusion: HPL has significantly higher concentrations of neurotrophic factors compared with HPS. It showed not only in vitro but also in vivo corneal neurotrophic abilities. Our results suggest that HPL may have a potential role in the treatment of diseases related to corneal nerve damage or degeneration., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: To use in vivo confocal microscopy to compare the wound-healing process in epikeratome laser-assisted in situ keratomileusis (epi-LASIK) with different management of the epithelial flaps., Methods: This prospective study comprised 46 eyes in the on-flap group and 47 eyes in the off-flap group. Epithelial flaps were repositioned or removed in the on- and off-flap eyes, respectively. In vivo confocal microscopy was performed before surgery and at 1 and 2 weeks and 1, 3, and 6 months after surgery. Corneal epithelial thickness, basal/apical surface epithelial morphology, and stromal reactions were analyzed., Results: Complete epithelialization by slit lamp biomicroscopy was faster for mitomycin C (MMC)-treated off-flap (4.27 ± 0.70 days) than on-flap (5.84 ± 0.08 days) eyes (P = 0.01). The percentage of eyes recovering to preoperative basal epithelial cell confocal morphology in the on- and off-flap groups was 87.5% and 92.3% for MMC-treated eyes and 86.3% and 90.5% in eyes without MMC treatment at 1 month after surgery. Of the studied eyes, with or without MMC, 50% and 100% returned to their preoperative apical epithelial morphology by 3 months in the on-flap and off-flap groups, respectively. Regression analysis revealed that the repositioned flap resulted in significant delay of epithelialization and apical-basal epithelial recovery (P < 0.01). Stromal reaction did not differ significantly at any of the time points. Corneal epithelial thickness was significantly thicker in the on-flap eyes (54.2 ± 4.5 μm) than in the off-flap eyes (26.7 ± 5.8 μm) at 1 month after surgery (P < 0.01)., Conclusions: On- and off-flap epi-LASIK showed comparable clinical outcomes after surgery. Off-flap epi-LASIK had more rapid re-epithelialization and normalization of epithelial morphology than did on-flap epi-LASIK, when observed by in vivo confocal microscopy. (ClinicalTrials.gov number, NCT00491439.).

Purpose: To analyze factors influencing corneal graft re-epithelialization after penetrating keratoplasty (PK) and evaluate the effect of topical autologous serum in promoting graft re-epithelialization., Design: Prospective interventional study., Methods: We analyzed 165 eyes of 165 patients who underwent PK between January 1, 2005 and December 31, 2007. Patients were divided into 2 groups according to routine use or non-use of postoperative 20% topical autologous serum. Postoperative slit-lamp examination after fluorescein staining was performed, and graft re-epithelialization time was recorded. Recipient/donor characteristics, surgical variables, and topical use of autologous serum were analyzed for their effects on post-PK graft re-epithelialization. Statistical analysis was performed by univariate and multivariate regression analysis using the ordinal logistic fit model to assess the potential risk factors influencing graft re-epithelialization after PK., Results: In univariate analysis, diabetes mellitus (DM), longer death-to-storage time and death-to-surgery time of the donor, and larger recipient size significantly delayed graft re-epithelialization (P < .05). Use of autologous serum significantly expedited graft re-epithelialization (P = .004). In multiple regression analysis, only DM in the recipient (odds ratio [OR] = 5.10, P < .001), postoperative use of autologous serum (OR = 0.54, P = .046), and larger graft size (OR = 4.44, P < .001) influenced graft re-epithelialization. The beneficial and healing effect of autologous serum is particularly significant in diabetic recipients and larger grafts., Conclusions: Several factors may influence graft re-epithelialization after PK. Graft re-epithelialization time was longer in diabetic recipients and larger grafts. Use of autologous serum may be a beneficial strategy in these patients with potentially delayed epithelial healing., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Purpose: To study healing of corneal wounds using in vivo confocal microscopy in patients who received corneal epithelial debridement during pars plana vitrectomy for proliferative diabetic retinopathy and to investigate risk factors for delayed healing., Design: Prospective, observational case series., Participants: Forty-four eyes of 40 patients were enrolled., Methods: In vivo confocal microscopy was used to evaluate selected images of the corneal basal and apical surface epithelial cells and subbasal nerves before surgery, weekly for the first month, and at 3 and 6 months after surgery. Slit-lamp biomicroscopy was carried out at the same time. Multiple linear regression analysis of selected potential risk factors was performed to investigate the main determinants of delayed corneal healing., Main Outcome Measures: Healing rate of corneal epithelial cells and subbasal nerves and factors influencing the healing., Results: By slit-lamp biomicroscopy, corneal epithelial defects were found in 22.8% of eyes at 2 weeks and in 5.4% at 1 month after surgery. In vivo confocal microscopy demonstrated incomplete healing of basal epithelial cells in 72.1%, 15.2%, and 0% of eyes and incomplete healing of surface apical epithelial cells in 81.1%, 9.1%, and 0% of eyes at 1, 3, and 6 months after surgery. The percentage of subbasal nerves regaining preoperative appearance was 0%, 6.8%, and 89.3% at 1, 3, and 6 months after surgery. Regression analysis revealed infusion of silicone oil (P = 0.020) and C(3)F(8) (P = 0.017) resulted in delayed healing by slit-lamp biomicroscopy; age (P = 0.028), diabetic treatment regimen (P = 0.014), and scleral buckling (P = 0.001) correlated with delayed recovery of basal cells by in vivo confocal microscopy. The latter 2 factors also were related to delayed reconformation of apical cells (P = 0.011 and 0.004, respectively). Neither healing of apical and basal cells showed a significant correlation to findings by slit-lamp biomicroscopy (r = 0.19 and 0.09)., Conclusions: Healing of corneal epithelial wounds in diabetic eyes is slow. Both the basal and apical epithelial layers were involved in the slow healing process. Age, diabetic treatment regimen, and several intraoperative factors may alter healing rates., Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Purpose: To evaluate the activation of ERK, a MAP kinase, in the regulation of proliferation and migration of rabbit corneal endothelial cells during the healing process after scrape wound injury., Methods: Cultured rabbit corneal endothelial cells were used in this study. Immunocytochemistry and Western blot analysis were performed to demonstrate the phosphorylation of ERK after scrape injury. The injured cell sheets were treated with different concentrations of ERK inhibitor, PD98059. The wound closure at 24, 48, and 72 hr following injury was evaluated. In order to evaluate the role of proliferation during wound healing, immunocytochemistry with Ki67 and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay were performed. The role of cell migration in wound healing was evaluated by quantification with the time lapse microscopic video imaging system., Results: Immunocytochemical and Western blot analysis revealed phosphorylation of ERK in the rabbit corneal endothelial cells after wounding. Inhibition of ERK phosphorylation by PD58059 resulted in a dose-dependent delay of wound closure. The inhibition of wound healing became significant from 48 to 72 hr in cells treated with 10 and 50 micro M of PD58059 (p < 0.05). ERK inhibition suppressed cell proliferation as indicated by Ki67 immunostaining and MTS assay. The time lapse microscopic video imaging analysis indicated delayed cell migration caused by PD98059., Conclusions: ERK activation regulates wound repair of rabbit corneal endothelial cells. Both cell proliferation and migration are involved in this process.

Purpose: To study the healing of corneal wounds after epikeratome laser-assisted in situ keratomileusis (epi-LASIK)., Methods: Twenty-seven patients who had undergone epi-LASIK for the treatment of myopia or myopic astigmatism in 46 eyes were enrolled. A single intraoperative application of 0.02% mitomycin C (MMC) for 20 to 30 seconds was used in 24 eyes with a refraction of no less than -6.0 D (MMC group). MMC was not given to eyes with myopia less than -6.0 D (non-MMC group). The eyes were examined by in vivo confocal microscopy at 1, 3, and 7 days after surgery and then weekly during the first month and once each at 3 and 6 months. Selected images of the corneal basal-apical surface epithelia and stromal reactions quantified by z-scan profile were evaluated., Results: In vivo confocal microscopy showed that cells in most of the epithelial flaps were damaged during the first few days after surgery and were rapidly replaced by new growing cells. In the MMC and non-MMC groups, the corneal basal epithelial cells returned to their preoperative morphology in 0% and 13.6% of the eyes after 1 week, 37.5% and 36.4% after 2 weeks, and 87.5% and 86.3% after 1 month, respectively. The corneal apical surface epithelial cells in the MMC and non-MMC groups recovered their squamous morphology in 12.5% and 13.6% of the eyes at 2 weeks, 37.5% and 54.5% at 1 month, and 52.4% and 57.9% at 6 months, respectively. There was no difference in the stromal reaction between the groups at 1, 3, and 6 months after surgery., Conclusions: Damage of the epithelial flaps after epi-LASIK was observed by in vivo confocal microscopy. MMC usage may cause more damage to the epithelial flaps. There was no difference in stromal reaction between the groups with and without MMC.

The mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1) mediates activin B signals required for eyelid epithelium morphogenesis during mouse fetal development. The present study investigates the role of MEKK1 in epithelial wound healing, another activin-regulated biological process. In a skin wound model, injury markedly stimulates MEKK1 expression and activity, which are in turn required for the expression of genes involved in extracellular matrix (ECM) homeostasis. MEKK1 ablation or down-regulation by interfering RNA significantly delays skin wound closure and impairs activation of Jun NH2-terminal kinases, induction of plasminogen activator inhibitor (PAI)-1, and restoration of cell-cell junctions of the wounded epidermis. Conversely, expression of wild-type MEKK1 accelerates reepithelialization of full-thickness skin and corneal debridement wounds by mechanisms involving epithelial cell migration, a cell function that is partially abolished by neutralizing antibodies for PAI-1 and metalloproteinase III. Our data suggest that MEKK1 transmits wound signals, leading to the transcriptional activation of genes involved in ECM homeostasis, epithelial cell migration, and wound reepithelialization.

Purpose: To purify and characterize the glycoprotein lumican, isolated from human amniotic membrane (AM), and to examine its efficacy in treating corneal epithelium debridement., Methods: An affinity-purified, anti-human lumican antibody-conjugated protein A Sepharose column was used to purify soluble lumican protein from human AM. The purified AM lumican was characterized by two-dimensional and SDS gel electrophoresis, plus Western blot analysis with anti-lumican antibody. The effects of lumican on corneal epithelial wound healing were examined in an organ culture mouse eye model., Results: Lumican was found to be abundantly present in the stroma of human AM. It was extracted from the AM by isotonic, 1 M NaCl, and 4 M guanidine HCl solutions, suggesting that it is present in both the soluble and matrix-bound states. In two-dimensional gel electrophoresis, the 50-kDa human amniotic lumican purified by antibody-conjugated affinity chromatography migrated in a smear between pH 3.0 and 6.0. After endo-beta-galactosidase digestion, it existed as a single core protein at pH 6.0, suggesting that native human amniotic lumican is a glycoprotein with short sugar moiety. Addition of purified human AM lumican to cultured medium promoted re-epithelialization and enhanced cell proliferation of wild-type mouse corneal epithelial cells in an organ culture. In lumican-knockout (lum(-/-)) mice, the effect of human lumican on promoting corneal epithelial wound healing was even more dramatic than in wild-type mice., Conclusions: The diversified functions of lumican include modulation of epithelial cells in wound healing and serving as an extracellular matrix component. Administration of lumican may be beneficial for treating epithelial defects in the cornea and other tissues.

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-β1 (TGF-β1), 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-β1, 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. [ABSTRACT FROM AUTHOR]

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 -β1 (TGF-β1), 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- β1, 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. [ABSTRACT FROM AUTHOR]