Effect of topical steroids on corneal epithelial healing after vitreoretinal surgery.Topical steroid use is usually cornneal in cases of corneal epithelial defect. We evaluated the effect of topical steroid treatment on corneal epithelial healing after epithelial debridement in vitreoretinal surgery. Our study population included 85 eyes undergoing vitreoretinal surgeries in our clinic. We prospectively compared the duration of corneal epithelial wound healing in 43 eyes in which topical healign was used with that in 42 eyes in which topical dexamethasone corticosteroids and corneal epithelial wound healing not used in the early postoperative corticosteroids and corneal epithelial wound healing after epithelial debridement. Factors that may retard corneal epithelial healing, including pre- healint intraoperative topical solutions, median operative time, steroid cycles for beginners oral presence of diabetes mellitus, prior ocular surgeries, pseudophakia, aphakia and the presence of intraocular gas or silicone oil in aphakic patients, were not significantly different between the two groups. The mean corneal epithelial defect closure time was
To investigate whether human corneal epithelial cells express the glucocorticoid receptor GR and to assess the influence of dexamethasone DEX on these cells. Cell proliferation was analyzed by 3- 4,5-dimethylthiazolyl 3-carboxy-methoxyphenyl 4-sulfophenyl -2H-tetrazolium inner salt MTS assay at 2, 4, and 6 days of culture. Apoptosis was studied by nucleus labeling using a fluorescent dye and immunostaining by APO 2. Immunocytochemical staining of the epithelial cells was performed with a monoclonal anti-human GR.
However, DEX also induced apoptosis of cultured corneal epithelial cells at any concentration used. These results indicate that human corneal epithelial cells express the GR and proliferate in response to DEX stimulation which also induces corneal epithelial cell apoptosis.
Purchase this article with an account. Materials and Methods Results Discussion. Author Affiliations Tristan Bourcier. You will receive an email whenever this article is corrected, updated, or cited in the literature. You can manage this and all other alerts in My Account. This feature is available to authenticated users only. Understanding the factors that control corneal wound healing, corneal cell proliferation, apoptosis, differentiation, and the modulation of these effects by specific drugs is critical for clinically relevant problems and successful therapeutic interventions.
Dexamethasone DEX is an anti-inflammatory glucocorticoid commonly used after cataract and penetrating keratoplasty surgeries. It is also used after refractive surgery photorefractive keratectomy or laser in situ keratomileusis in an attempt to reduce ocular surface inflammation and to delay corneal wound healing. Despite the widespread use and demonstrated clinical effectiveness of steroids, little is known regarding the specific effects of glucocorticoids on the function of corneal cells.
DEX has been shown to inhibit inflammation, through inhibition of phospholipase A2 activity and inhibition of transcription of metalloprotease genes. By preventing cellular division, glucocorticoids have been shown to decrease extracellular matrix and scar tissue formation.
Although the glucocorticoid receptor GR mRNA sequence has been detected in corneal epithelium by polymerase chain reaction and hot blot analyses, 16 little is known about the effect of glucocorticoids on corneal epithelial cells and the role of GR in the regulation of corneal wound healing. This study was initiated to determine whether human corneal epithelial cells express GR mRNA and the corresponding protein.
This study was performed according to the tenets of the Declaration of Helsinki. Human corneal epithelial cell primary cultures were obtained using human donor corneas that were discarded before transplantation because of low endothelial cell counts. Primary cultures of human corneal epithelium were started using explants, as previously described. The anterior cornea with intact epithelium was covered with 1.
One hour later, corneas were placed in culture medium and 1 to 2 mm 2 full-thickness epithelial explants were gently peeled off with forceps from the peripheral areas 1—2 mm inside the limbus. Four to six explants were removed from the peripheral cornea. Explants were placed epithelial side up on well tissue culture plates Costar, Cambridge, MA. A mm diameter sterile glass coverslip was placed on the explant in the well before the addition of culture medium.
One milliliter of medium was then added to the wells. The culture medium was supplemented hormonal epithelial medium SHEM 19 and consisted of a 1: Steroids were removed from the FCS by means of a dextran charcoal treatment to eliminate serum steroids. Corneal epithelial cells were allowed to migrate from the explants onto the surface of the wells. The cells reached confluence within 21 to 28 days. They were then enzymatically detached using 0. The suspended epithelial cells were then centrifuged at g for 10 minutes.
The supernatant was removed, and fresh medium was added again. Second-passage human corneal epithelial cells were used in all the experiments. DEX was purchased from Sigma—Aldrich. It was dissolved and serially diluted in absolute ethanol before addition to the culture medium.
DEX was added to the culture medium every day at the same concentration. In all experiments, the ethanol concentration in the culture media was maintained at 0. The control group consisted of epithelial cells cultured in SHEM with 0.
The culture media were renewed every day. All the experiments were repeated six times. For each experiment—3- 4,5-dimethylthiazolyl 3-carboxy-methoxyphenyl 4-sulfophenyl -2H-tetrazolium inner salt MTS assays, apoptosis assays, and immunocytochemistry—all cultures were obtained from the same donor cornea.
Cultured epithelial cells were studied daily by means of phase-contrast microscopy. Total RNA extraction was performed on primary and secondary passages of human corneal epithelial cultures. RNA samples were also prepared from ex vivo corneal epithelium that was mechanically removed from donor corneas stored in preservative medium Inosol; Chauvin—Opsia, Toulouse, France for less than 1 month. RNA extraction was performed by the acidic phenol-chloroform guanidine thiocyanate method described by Chirgwin.
Total RNA recovery was measured by spectrophotometric absorbance at nm. The primers were synthesized by Gibco—Life Technology from previously published sequences.
A negative control was routinely introduced in all assays to confirm the absence of contamination. PCR amplification was performed in the same conditions as used for the samples. Human corneal keratocytes known to express the GR were used as a positive control. Primary cultures and second-passage corneal epithelial cells were studied by indirect immunoperoxidase staining.
A monoclonal mouse anti-human GR dilution 1: A monoclonal anti-human cytokeratin dilution 1: Monoclonal mouse anti-human APO 2. Seven-micrometer cryostat sections of donor corneas were also processed with anti-GR monoclonal antibody. Negative controls consisted of cryostat section incubated with no primary antibody and section incubated with anti-vimentin monoclonal antibody dilution 1: MTS is a tetrazolium salt that undergoes a color change caused by its bioreduction of MTS into a water-soluble formazan.
The conversion of MTS into the aqueous-soluble formazan is accomplished by dehydrogenase enzymes found in active mitochondria and is such that the reaction occurs only in living cells. The optical density was measured at nm by means of spectrophotometry.
Epithelial cell growth was analyzed by means of MTS assay after 2, 4, and 6 days of culture. Epithelial cell proliferation was analyzed with a hemocytometer and a cell counter Coulter, Hialeah, FL. We also looked for DEX-induced cell apoptosis by nucleus labeling using a fluorescent dye for nuclei after 6 days of culture.
Six to 12 photographs of each specimen were taken using the same instrument. Photographs were analyzed by two observers in a blind fashion. The number of apoptotic cells and the total number of cells were counted. Cells with ruptured cytoplasmic membranes were considered to be necrotic cells. All experiments were reproduced three times.
Data were analyzed by analysis of variance and Wilcoxon rank sum test. Commercial software SPSS ver. A unique PCR product of nucleotides was detected with ethidium bromide after gel electrophoresis Fig. The size of this band was consistent with the expected fragment size, determined from the human GR cDNA. Specific nuclear intracellular staining of GR was observed in cultured epithelial cells Fig. Both basal epithelial cells and keratocytes showed positive staining for GR.
Negative controls showed no staining data not shown. After 6 days of culture, DEX induced a biphasic dose-dependent effect on epithelial cell proliferation. There were no significant effects at days 2 and 4. All the results of the MTS assay were confirmed by the cell counter and hemocytometer proliferation assays data not shown.
In the nucleus labeling assay, the percentage of viable cells decreased when DEX was added to the culture medium Figs. There was no statistical difference in epithelial cell viability between the SHEM group and the ethanol group control group. There was an increased expression of the apoptotic marker APO 2.
There were differences of cell morphology among the different groups. Human corneal epithelium is a stratified squamous epithelium that forms a barrier between the external environment, tears, and the intraocular environment. In addition, it contributes to the maintenance of normal stroma transparency by transporting fluid out of the stroma. It is known to have a rapid self-renewing capacity. The epithelium is frequently injured through physical or chemical insult.
Wound closure after corneal abrasions involves a complex series of cellular changes in both the epithelium and the stroma. Pharmacologic control of epithelium proliferation appears to offer the potential to regulate corneal wound healing.
It has been hypothesized that stimulation of epithelial cell proliferation and inhibition of cell differentiation could promote epithelial hyperplasia associated with regression after photorefractive keratectomy PRK. The similar dose—response curves for the hemocytometer cell count and the MTS assay provided evidence that they measured the same proliferative effect under our experimental conditions.
The proliferative effect of DEX was observed in the presence of FCS from which steroids had been removed, indicating that it depended only on exogenous steroids. The apparent paradoxical biphasic effect of DEX on human corneal epithelial cell i. Thus, low doses of DEX could also have a mitogenic effect on cultured human epithelial cells.
It has been shown recently that AP1 components c- Fos , c- Jun , and Fra-2 are expressed in normal ocular surface epithelia and dysplastic epithelium. It has been suggested that immediate expression of nucleoprotein encoding proto-oncogenes could represent the molecular response that initiates the healing process. GR and AP1 interactions have recently been described in the glucocorticoid response elements GREs , where these two transcriptional factors are adjacent.