Normal Rat Cornea Research Articles

Prophylactic Instillation of Hydrogen-Rich Water Decreases Corneal Inflammation and Promotes Wound Healing by Activating Antioxidant Activity in a Rat Alkali Burn Model.

Many studies have demonstrated the therapeutic effects of hydrogen in pathological conditions such as inflammation; however, little is known about its prophylactic effects. The purpose of this study is to investigate the prophylactic effects of hydrogen-rich water instillation in a rat corneal alkali burn model. Hydrogen-rich water (hydrogen group) or physiological saline (vehicle group) was instilled continuously to the normal rat cornea for 5 min. At 6 h after instillation, the cornea was exposed to alkali. The area of corneal epithelial defect (CED) was measured every 6 h until 24 h after alkali exposure. In addition, at 6 and 24 h after injury, histological and immunohistochemical observations were made and real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to investigate superoxide dismutase enzyme (SOD)1, SOD2, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA expression. CED at 12 h and the number of inflammatory infiltrating cells at 6 h after injury were significantly smaller in the hydrogen group than the vehicle group. Furthermore, SOD1 expression was significantly higher in the hydrogen group than the vehicle group at both 6 and 24 h, and the number of PGC-1α-positive cells was significantly larger in the hydrogen group than the vehicle group at 6 h after injury. In this model, prophylactic instillation of hydrogen-rich water suppressed alkali burn-induced inflammation, likely by upregulating expression of antioxidants such as SOD1 and PGC-1α. Hydrogen has not only therapeutic potential but also prophylactic effects that may suppress corneal scarring following injury and promote wound healing.

Advantages of Cubosomal Formulation for Gatifloxacin Delivery in the Treatment of Bacterial Keratitis: In Vitro and In Vivo Approach Using Clinical Isolate of Methicillin-Resistant Staphylococcus aureus.

The objective of this study was to enhance the corneal permeation of gatifloxacin (GTX) using cubosomal nanoparticle as a delivery system. Cubosomal nanoparticle loaded with GTX was prepared and subjected for in vitro and in vivo investigations. The prepared GTX-loaded cubosomal particles exhibited nanoparticle size of 197.46 ± 9.40 nm and entrapment efficiency of 52.8% ± 2.93. The results of ex vivo corneal permeation of GTX-loaded cubosomal dispersion show approximately 1.3-fold increase compared to GTX aqueous dispersion. The incorporation of GTX into cubosomal particles resulted in a fourfold reduction in the minimum inhibitory concentration (MIC) value for the GTX cubosomal particles relative to GTX aqueous dispersion. Furthermore, the enhanced corneal penetration of GTX-loaded cubosomal dispersion compared was evident by a significant decrease in the area % of corneal opacity in MRSA infected rats. Moreover, these results were confirmed by photomicrographs of histological structures of corneal tissues from rats treated with GTX-cubosomal dispersion which did not present any change compared to that of the normal rat corneas. In conclusion, treatment of ocular bacterial infections and reduction in the probability of development of new resistant strains of MRSA could be accomplished with GTX-loaded cubosomal nanoparticles.

Effect of Ultraviolet-A and Riboflavin treatment on the architecture of the center and periphery of normal rat cornea: 7 days post treatment

Corneal collagen cross-linking (CXL) is a treatment that is widely applied to halt the progression of ectatic diseases such as keratoconus by creating biomechanical strength in the cornea. Most of the studies assessed the effect of the CXL on the cornea without any differentiation of its effect between periphery and the center of the untreated control cornea especially after the 7 days of CXL application. We investigate the ultrastructural changes in the architecture of the center and periphery of rat corneas, 7 days after standard CXL application. Five Wistar rats (10 corneas) were used in the present study. The left eye corneas (5 mm area) were de-epithelialized and irradiated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The right eye corneas were used as a control. The sclera-cornea button was removed and processed for electron microscopy. Digital images were captured with a bottom mounted Quemesa camera and analyzed using the iTEM software. The ultrastructure of epithelium, hemi-desmosomes, Bowman's layer and stroma were organized in both untreated control and CXL rat cornea in both untreated control and CXL rat cornea. Within the same CXL cornea, both the collagen fibril (CF) diameter and interfibrillar spacing at the center were significantly smaller compared to the peripheral diameter and spacing of the cornea. When comparing the untreated control and CXL cornea, the central interfibrillar spacing of the CXL cornea was significantly smaller than the central spacing the untreated control cornea. In the CXL cornea the peripheral spacing was significantly higher compared to the peripheral interfibrillar spacing of the untreated control cornea. Within the CXL cornea, the proteoglycans (PGs) area and density of the periphery was significantly higher compared to the area and density of the center of the cornea. It suggests that CXL was more effective at the periphery of the cornea. This could be due to the higher amount of leucine rich PG lumican and higher diffusion of oxygen and riboflavin at the periphery cornea.

Effect of lentivirus-mediated gene silencing, targeting toll-like receptor 2, on corneal allograft transplantation in rats

AimThe present work aims to assess the effectiveness of lentiviral vector (LV) mediated Toll-like receptor 2 (TLR2) gene silencing in the survival of transplanted corneal allografts, against immune rejection, in rats. MethodsLV mediated TLR2 small interference RNA (SiRNA) with enhanced green fluorescent protein (eGFP) [LV-TLR2-siRNA-eGFP] was realised and transfected to both rat corneal epithelial (EC) and stromal cells (SC). Multiplicity of infection (MOI) was optimized for transfection efficiency using flow cytometric (FCM) analysis. Viability of transfected cells and the success rate of TLR2 gene silencing were respectively determined by CCK-8 assay and western blot assay. The in-vivo experiments were subjected to a penetrating keratoplasty (PK) performed between host Sprague Dawley (SD) and donor Wistar/SD rats, randomly dividing them into 4 groups including the allograft, isograft, allograft treated with LV-eGFP (LV blank control) and allograft treated with LV-TLR2-siRNA-eGFP (LV treated group). The rejection index (RI) was then recorded under a slit lamp, every day following surgery. Expression of the TLR2 and Myeloid differentiation primary response gene 88 (MyD88) were detected by using immunohistochemistry on day 9 post-surgery, whereas grafts’s TLR2 and MyD88 mRNA were determined on day 5, 9, and 14 post-surgery performing RT-PCR and, normal rat corneas were included as additional controls. ResultsTransfected cells showed the strongest eGFP expression when MOI was 200 with an efficiency of 77.5% for EC and 76.3% for SC. CCK-8 assay, as measured at 72 and 96h post transfection, showed no significant changes in the cell viability (both EC and SC) between the transfected and the control group (p>0.05, p>0.05). Western blot results demonstrated a successful down regulation of TLR2 expression by LV-TLR2-SiRNA-eGFP, in both EC and SC. In vivo, compared to allograft group, LV treated group demonstrated less edema, opacity and neovascularization. Immunohistochemical analysis revealed a lower expression of TLR2 and MyD88 in isograft and LV treated group as compared to allograft group. TLR2 and MyD88 mRNA were detected in all grafts, and increased over time. With its highest expression in allograft group (peak on day 9), the mRNA expression for TLR2 and MyD88 showed a significant difference amongst the groups, on both day 9 and 14 (p<0.001). ConclusionsLV mediated TLR2 siRNA could effectively down regulate the TLR2 expression via RNA interference and prolong the survival of corneal grafts, although not necessarily able to prevent the rejection.

Expression of S100B during the innate immune of corneal epithelium against fungi invasion

To explore the expression of S100B in corneal epithelial cells under Aspergillus stimulation both in vivo and in vitro. Immortalized human corneal epithelial cells (HCECs) were exposed to inactive Aspergillus fumigatus (A. fumigatus) conidia at 0, 4, 8, 12, 16, and 24h respectively. The corneas of Wistar rats were exposed to active A. fumigatus at 0, 12, 24, 48h and the normal rat corneas were used for normal control. The mRNA level of S100B was evaluated by real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). S100B protein expression in cornea epithelium was detected by immunohistochemical/immunocytochemical staining (IHC/ICC). Histopathology revealed a significant inflammatory cell infiltration in fungal keratitis human and rat cornea. Corneal epithelial cells didn't express or rarely express S100B at baseline. A. fumigatus significantly induced S100B mRNA expression in cultured corneal epithelial cells in a time depended manner in vitro, the mRNA began to rise significantly at 8h in vitro (P<0.05) and continue to rise as time prolonged (P<0.01). In vivo, S100B mRNA level was low in the normal corneas. However, it was increased in keratitis corneas from 12h after infection (P<0.05) and reached to a peak at 24h (P<0.001). Immunochemistry revealed an obvious staining in fungal keratitis corneas as well as immortalized HCECs compared to the normal ones respectively, indicating an increased expression of S100B protein. S100B exists in corneal epithelial cells and is over-expressed under A. fumigatus stimulation. S100B may play an important role in the innate immune response of the corneal epithelium during A. fumigatus infection.

Keratocytes Create Stromal Spaces to Promote Corneal Neovascularization Via MMP13 Expression

To investigate the exact mechanism by which keratocytes promote corneal neovascularization. The expression of matrix metalloproteinase 13 (MMP13), cluster of differentiation 146 (CD146), VEGFa, VEGFc, VEGF receptor (r)2, and VEGFr3 by normal and alkali-burned rat corneas was determined via quantitative (q)RT-PCR and/or Western blot analysis or in situ hybridization. Corneal neovascularization was observed under a slit lamp microscope and evaluated via immunohistochemistry. The cells that expressed MMP13 in the corneas were determined via sequential immunohistochemistry and in situ hybridization. The degradation of type I collagen was evaluated via the detection of hydroxyproline content and Western blot analysis. The effects of VEGFa and VEGFc on MMP13 expression were determined via luciferase reporter assay for the MMP13 promoter and primary keratocyte culture. Matrix metalloproteinase 13 was predominantly expressed by epithelial cells in normal rat corneas, but it was expressed by cells in corneal stromas after alkali burns. The formation of new blood vessels was consistent with MMP13 expression and attenuated by a selective MMP13 inhibitor in alkali-burned corneas. Keratocytes were the major cells expressing MMP13 in corneal stromas after alkali burns. Through MMP13 expression, keratocytes directly degraded collagen type I to create stromal spaces, which were convenient for newly formed blood vessels to grow into. Expression of MMP13 and collagen type I degradation via keratocytes were induced by VEGFc through VEGFr3 and inhibited by antibodies for VEGFc and VEGFr3. Keratocytes could directly degrade type I collagen and create stromal spaces, promoting corneal neovascularization through VEGFc/VEGFr3-induced MMP13 expression.

Effects of low level electromagnetic field exposure at 2.45 GHz on rat cornea

Purpose: To investigate the effects of low level electromagnetic field (low level-EMF) exposure, as frequently encountered in daily life, on the normal rat cornea using histological and stereological method.Methods: Twenty-two adult male Wistar rats were randomly divided into two groups: Study group (n = 11) and control group (n = 11). Rats in the study group were exposed to 2.45 GHz microwave (MW) radiation (11.96 ± 0.89 V/m), 0.25 W/kg specific absorption rate (SAR) for 2 hours each day for 21 days. The corneal thickness and the anterior epithelium corneal thickness were measured using two different methods.Results: Using the histological method, the mean corneal thicknesses in the control and study group were 278.9 ± 54.5 μm, and 272.4 ± 85.6 μm, respectively. There was no statistically significant difference between the groups (p > 0.05). The anterior corneal epithelium thickness was 28.1 ± 4.9 μm in the control group and 31.7 ± 5.5 μm in the study group. There were statistically differences between the groups with regard to the thickness of anterior epithelium (p < 0.05). In the measurement made by the stereological method, the percentage of the cornea occupied by anterior corneal epithelium was 15.94% in the control group and 17.9% in the study group. Despite the fact that there was a relation between increased anterior epithelial area (AEA) and radiation exposure, no statistically significant relationship in area fraction of each compartment was found between the control and study groups.Conclusions: Results of this preliminary study show that exposure to MW radiation might cause alterations in the rat cornea.

Study on the optical property and biocompatibility of a tissue engineering cornea.

To study the optical property and biocompatibility of a tissue engineering cornea. : The cross-linker of N-(3-Dimethylaminoropyl)-N'ethylcarbodiimide hydrochloride (EDC)/N-Hydroxysuccinimide (NHS) was mixed with Type I collagen at 10% (weight/volume). The final solution was molded to the shape of a corneal contact lens. The collagen concentrations of 10%, 12.5%, 15%, 17.5% and 20% artificial corneas were tested by UV/vis-spectroscopy for their transparency compared with normal rat cornea. 10-0 sutures were knotted on the edges of substitute to measure the corneal buttons's mechanical properties. Normal rat corneal tissue primary culture on the collagen scaffold was observed in 4 weeks. Histopathologic examinations were performed after 4 weeks of in vitro culturing. The collagen scaffold appearance was similar to that of soft contact lens. With the increase of collagen concentration, the transparency of artificial corneal buttons was diminished, but the toughness of the scaffold was enhanced. The scaffold transparency in the 10% concentration collagen group resembled normal rat cornea. To knot and embed the scaffold under the microscope, 20% concentration collagen group was more effective during implantation than lower concentrations of collagen group. In the first 3 weeks, corneal cell proliferation was highly active. The shapes of cells that grew on the substitute had no significant difference when compared with the cells before they were moved to the scaffold. However, on the fortieth day, most cells detached from the scaffold and died. Histopathologic examination of the primary culture scaffold revealed well grown corneal cells tightly attached to the scaffold in the former culturing. Collagen scaffold can be molded to the shape of soft contact corneal lens with NHS/EDC. The biological stability and biocompatibility of collagen from animal species may be used as material in preparing to engineer artificial corneal scaffold.

Blockade of Toll-like Receptor 2 Expression and Membrane Translocation in Rat Corneal Epithelial Cells by Glucocorticoid (TobraDex) After Penetrating Keratoplasty

To evaluate the role of Toll-like receptor (TLR) 2 and the effect of glucocorticoid on immune rejection of penetrating keratoplasty. Allograft corneal transplantation was performed between host Sprague Dawley (SD) and Wistar donor rats. The expression of TLR2 messenger RNA (mRNA) and protein in corneas was determined by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, and immunofluorescence on days 5, 7, and 9 after operation. Three groups were included: allograft, allograft treated with TobraDex (Alcon, Rijksweg, Belgium), and isograft. Normal rat corneas were included as an additional control. Various degrees of congregation of inflammatory cells and neovascularization of grafts were confirmed by histopathology. Immunohistochemistry revealed that TLR2 was expressed in epithelial, stromal, and endothelial cells of normal tissue, and in all of the grafts. Immunofluorescence analysis of TLR2 showed membrane staining of epithelial cells in the allografts on days 7 and 9. This was absent in the isografts and the allografts treated with TobraDex. TLR2 mRNA was detected in normal corneas, and levels were increased in all of the grafts, as determined by quantitative reverse transcription-polymerase chain reaction. By day 9 after transplantation, a 3.6-fold increase in TLR2 mRNA was observed in the allografts compared with the isografts or the allografts treated with TobraDex, which was statistically significant, at P < 0.005. Expression of TLR2 in the rat cornea was significantly increased and concurred with the allograft rejection, but was effectively blocked by treatment with TobraDex.

Effect of tetrandrine on the expression of leptin (LP) and vascular endothelial growth factor (VEGF) in corneal neovascularization of rats

To investigate the effect of tetrandrine (Tet) on the expression of leptin (LP) and vascular endothelial growth factor (VEGF) in corneal neovascularization (CNV). CNV model was made by alkaline burning. The expressions of LP and VEGF in CNV before and after using Tet were detected slit lamp biomicroscopy, photography and immunohistochemical staining. Immunohistochemistry showed LP was observed in the epithelium, stroma and endothelium of normal rat corneas. The epithelial and stromal layers of the normal rat corneas demonstrated minimal or no VEGF expression. The expression of CNV increased markedly throughout the whole cornea. In the Tet experimental group there were weak (P<0.05) expression levels for LP and VEGF in the corneal stroma. Tet can effectively inhibit the growth of CNV after alkali burns in rats, and reduce the expression of LP and VEGF in CNV. Key words: Alkali burns, corneal neovascularization, leptin, vascular endothelial growth factor, tetrandrine.

Role of epidermal growth factor receptor (EGFR) in corneal remodelling in diabetes

This study examined the role of epidermal growth factor receptor (EGFR) signalling on the organization and remodelling of collagen fibrils (CFs) and proteoglycans (PGs) in the stroma of diabetic rat cornea. Diabetes was induced in female Wistar rats (n = 5) by streptozotocin (STZ) injection (55 mg/kg). Treatment with a selective inhibitor of EGFR tyrosine kinase, AG1478, was started on the same day as the induction of diabetes and administered every other day for 4 weeks. Corneas were fixed in 4% paraformaldehyde at 4 degrees to allow for analysis of CF diameters and in 2.5% glutaraldehyde in sodium acetate buffer containing cuprolinic blue to enable the study of PG distribution. AnalySIS soft imaging software was used to analyse CFs and PGs. Epithelial thickness, and median diameter and area fraction of CF in corneal stroma were decreased in diabetic rat cornea compared with normal cornea (p < 0.001), whereas the median PG area and area fractions were significantly increased (p < 0.001). Treatment with AG1478, although it had no action on normal cornea, prevented these diameter and area fraction changes in CFs and PGs. The cornea of AG1478-treated diabetic rats showed a slight increase in CF diameter and area fraction and a decreased number density. These data show that the distribution of corneal stroma CFs and PGs was altered after 4 weeks of diabetes and that, furthermore, treatment with an EGFR signalling inhibitor normalized these abnormalities. The data suggest that EGFR plays an important role in the development of diabetes-induced corneal remodelling.

Localization of semaphorin 3A in the rat cornea

Semaphorin 3A (Sema3A) functions to guide the growth of neurons during development. We investigated the localization of Sema3A in the cornea, one of the most sensitive tissues in the body. Immunoblot analysis and reverse transcription–polymerase chain reaction analysis revealed that Sema3A protein and mRNA are expressed in the normal rat cornea. Immunofluorescence staining of frozen sections or tissue blocks prepared from the cornea revealed the presence of Sema3A in wing cells and basal cells (but not superficial cells) of the corneal epithelium, in keratocytes, and in the corneal endothelium. The expression pattern of Sema3A in the corneal epithelium differed from those of zonula occludens-1 and connexin43. These observations show that Sema3A is expressed in all cells of the rat cornea with the exception of superficial epithelial cells.

Expression of VEGF-C in rat cornea after alkali injury.

The expression of VEGF-C and molecular mechanisms of lymphangiogenesis in rat cornea after alkali injury was studied. The rat alkali injured corneal models were made. Under electron microscopy, the lymphatic vessels in the rat injured corneas were examined. The expression of VEGF-C proteins was detected by using immunohistochemical assay at day 1, 3, 5, 7, 9 after injury. The expression levels of VEGF-C mRNA were quantified with reverse transcription polymerase chain reaction (RT-PCR). The results showed that the lymphatic vessels were found in the injured rat corneas 14 days after the injury. The VEGF-C protein was detectable 3 days after injury, reached the peak 5 days after injury, and gradually decreased. In the control group, no VEGF-C proteins were detected. The VEGF-C mRNA was minimally detected in the normal rat corneas, but it was highly expressed 5 days after the injury. The difference was statistically significant. It was concluded that VEGF-C might be one of the most important relevant factors in corneal lymphangiogenesis after alkali injury.

Glucose Transporter 1 Expression in Corneal Wound Repair under High Serum Glucose Level

Purpose: To determine glucose transporter (GLUT) 1 mRNA and protein expression during corneal epithelial wound healing in diabetic rat. Methods: Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. At 10 days after injection, unilateral 3-mm epithelial debridement was carried out in the central cornea. At 2, 4, 6, and 24 hours after wounding, whole corneal epithelium was collected and GLUT1 protein and mRNA levels were determined by Western blotting and reverse transcription-polymerase chain reaction, respectively. Sugar content in collected samples was measured by the Anthrone reaction. Normal rats were used as controls. Results: Glucose transporter 1 protein and mRNA levels in unwounded cornea were similarly low in the diabetic and control groups. Healing of corneal wounds was slower in diabetic rats than in controls. After wounding, GLUT1 mRNA and protein expression in both groups were similarly enhanced compared to unwounded epithelium. Sugar content at all time points did not show significant alteration in any group, although in diabetic rats it was significantly higher than in controls throughout the time course. Conclusion: Glucose transporter 1 expression in diabetic rat cornea showed little difference from that in normal rat cornea, suggesting minimal influence of GLUT1 on the delayed healing of diabetic corneal wounds.

Tyrosine phosphorylation in desmosomes and hemidesmosomes of the corneal epithelium.

We examined the localization of phosphotyrosine (p-Tyr)-modified proteins in the normal corneal epithelium using affinity-purified rabbit anti-p-Tyr antibody. Normal rat cornea was fixed and semi-thin and ultra-thin frozen sections were prepared. Immunofluorescence microscopy showed that p-Tyr was distributed along the cell membrane of the corneal epithelium. Immunogold electron microscopy revealed that the labeling is exclusively localized in the desmosomes and hemidesmosomes. A fraction enriched with desmosomes was extracted from the bovine corneal epithelium and examined by Western blotting. Immunoblotting for p-Tyr showed eight prominent bands (290, 200, 190, 115, 85, 62, 50, and 47 kD) in the desmosomal fraction. The enrichment of p-Tyr in desmosomes and hemidesmosomes of the corneal epithelium suggests that these cell-to-cell and cell-to-substrate junctions are involved in signal transduction.

Effect of Inhibitors of Arachidonic Acid Metabolism on Corneal Reepithelialization in the Rat

We examined the ability of inhibitors of arachidonic acid metabolism to influence the rate of healing of organ-cultured rat corneas with 3-mm diameter central epithelial abrasions. In control corneas, and in the presence of the cyclooxygenase inhibitors indomethacin (1 μM) or flurbiprofen (1 μM), the defect was completely reepithelialized by 25 hr. In contrast, corneas cultured with the lipoxygenase inhibitors quercetin (100 μM), esculetin (100 μM), or baicalein (10 μM) or the dual cyclooxygenase/lipoxygenase inhibitors BW 755C (100 μM) or BW A540C (100 μM) had significantly delayed epithelial healing rates when compared with the controls; complete healing of the epithelial defects required 32·5-40 hr. Dose-response studies with esculetin and BW 755C demonstrated that the concentrations for 50% inhibition of reepithelialization (65·3 μM for esculetin, 39·6 μM for BW 755C) were significantly greater than those for inhibition of 12-lipoxygenase activity (16·6 μM for esculetin, 21·1 μM for BW 755C), the major lipoxygenase activity in normal rat cornea. Addition of 12(S)-5,8,10,14-hydroxyeicosatetraenoic acid [12(S)-HETE, 0·01-10 μM], the main 12-lipoxygenase metabolite of arachidonic acid in normal rat cornea, to the organ cultures did not influence the rate of epithelial wound healing in the absence or presence of 100 μM esculetin. Our results suggest that lipoxygenase activity is an important factor in regulating corneal epithelial wound healing in the rat, presumably by influencing epithelial cell migration. The lipoxygenase enzyme and metabolite(s) responsible for regulating reepithelialization, and the mechanism of action, remain to be determined.