Corneal alternations induced by topical application of benzalkonium chloride in rabbit

Benzalkonium chloride (BAC) is the most common preservative in ophthalmic preparations. Here, we investigated the corneal alternations in rabbits following exposure to BAC. Twenty-four adult male New Zealand albino rabbits were randomly divided into three groups. BAC at 0.01%, 0.05%, or 0.1% was applied twice daily to one eye each of rabbits for 4 days. The contralateral untreated eyes were used as control. Aqueous tear production and fluorescein staining scores of BAC-treated eyes were compared with those of controls. The structure of the central cornea was examined by in vivo confocal microscopy. Expression of mucin-5 subtype AC (MUC5AC) in conjunctiva was detected by immunostainig on cryosections. Corneal barrier function was assessed in terms of permeability to carboxy fluorescein (CF). The distribution and expression of ZO-1, a known marker of tight junction, and reorganization of the perijunctional actomyosin ring (PAMR) were examined by immunofluorescence analysis. Although there were no significant differences between control and BAC-treated eyes in Schirmer scores, corneal fluorescein scores and the number of conjunctival MUC5AC staining cells, in vivo confocal microscopy revealed significant epithelial and stromal defects in all BAC-treated corneas. Moreover, BAC at 0.1% resulted in significant increases in central corneal thickness and endothelial CF permeability, compared with those in control eyes, and endothelial cell damage with dislocation of ZO-1 and disruption of PAMR. Topical application of BAC can quickly impair the whole cornea without occurrence of dry eye. A high concentration of BAC breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR and remodeling of apical junctional complex in vivo.     

Meibomian Gland Dysfunction Determines the Severity of the Dry Eye Conditions in Visual Display Terminal Workers

Objective

To explore meibomian gland dysfunction (MGD) may determine the severity of dry eye conditions in visual display terminal (VDT) workers.

Methodology

Prospective, case-control study carried out in China.106 eyes of 53 patients (VDT work time >4 hour per day) were recruited as the Long time VDT group; 80 eyes of 40 control subjects (VDT work time ≤4 hour per day) served as the Short time VDT group. A questionnaire of Ocular Surface Disease Index (OSDI) and multiple tests were performed. Three dry eye tests: tear film breakup time (BUT), corneal fluorescein staining, Schirmer I test; and three MGD parameters: lid margin abnormality score, meibum expression assessment (meibum score), and meibomian gland dropout degree (meiboscore) using Keratograph 5 M.

Principal Findings

OSDI and corneal fluorescein score were significantly higher while BUT was dramatically shorter in the long time VDT group than the short time VDT group. However, the average of Schirmer tear volumes was in normal ranges in both groups. Interestingly, the three MGD parameters were significantly higher in the long time VDT group than the short time one (P<0.0001). When 52 eyes with Schirmer <10 mm and 54 eyes with Schirmer ≥10 mm were separated from the long time VDT workers, no significant differences were found between the two subgroups in OSDI, fluorescein staining and BUT, as well as the three MGD parameters. All three MGD parameters were positively correlated with VDT working time (P<0.0001) and fluorescein scores (P<0.0001), inversely correlated with BUT (P<0.05), but not correlated with Schirmer tear volumes in the VDT workers.

Conclusions

Our findings suggest that a malfunction of meibomian glands is associated with dry eye patients in long term VDT workers with higher OSDI scores whereas some of those patients presenting a normal tear volume.     

Effect of lacrimal plugs combined with deproteinized calf blood extract eye gel for filamentary keratitis

This study aims to investigate the efficacy of lacrimal plugs combined with deproteinized calf blood extract eye gel on the treatment of dry-eye-associated filamentary keratitis. Lacrimal plugs were inserted into both the upper and lower puncta of 15 patients (28 eyes). Deproteinized calf blood cxtract eye gel was applied in two patients who were not cured after this operation. All patients were asked to complete three questionnaires 1 day before the surgery and at 1 week and 1 month after the surgery. Ophthalmologic examinations were carried out and repeated at 1 day and at 1 week and 1 month after plug insertion, which include fluorescein staining, tear break-up time (BUT), Schirmer test I (STI), corneal confocal microscope (HRT-III), and impression cytology (IC). Symptoms were relieved in all patients 1 month after the application of lacrimal plugs. Deproteinized calf blood extract eye gel was applied to two patients whose symptoms remained after lacrimal plug implantation for 1 week. At 3 weeks later, the symptoms disappeared and cornea fluorescein stain was hardly identified. The lacrimal river in all patients became broader after the surgery, 11 of whom reached 0.3 mm. Cornea filamentary disappeared in all patients. The average of BUT and ST were increased to 7.50 ± 1.897 s (p < 0.001) and 8.12 ± 1.996 mm at 1 week after the operation and 7.94 s and 9.00 ± 1.897 mm at 1 month later, respectively. Photography from HRT-III suggested that the state of corneal epithelium was markedly improved, including the squamous metaplasia of the epithelial layer of the cornea and the bend of nerve fibers under the corneal epithelium. IC also suggested that the squamous metaplasia of epithelial layer of conjunctiva in these patients was improved. The symptoms of patients suffering from severe filamentary keratitis were remarkably relieved by using lacrimal plugs, which could increase the tear volume of the ocular surface, improve the condition of tear film, and promote the recovery of corneal diseases. For patients with more severe symptoms, additional usage of deproteinized calf blood extract eye gel could assist the treatment. Therefore, lacrimal plugs combined with deproteinized calf blood extract eye gel are suggested to be an effective method to treat severe filamentary keratitis.     

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 Notch1^flox/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.     

Changes of Ocular Surface and the Inflammatory Response in a Rabbit Model of Short-Term Exposure Keratopathy

Purpose

To evaluate the ocular surface change and the inflammatory response in a rabbit model of short-term exposure keratopathy.

Methods

Short term exposure keratopathy by continuous eyelid opening was induced in New Zealand white rabbits for up to 4 hours. Ultrasound pachymetry was used to detect central total corneal thickness. In vivo confocal microscopy and impression cytology were performed to evaluate the morphology of ocular surface epithelium and the infiltration of inflammatory cells. Immunohistochemistry for macrophage,neutrophil, CD4(+) T cells, and CD8(+) T cells were performed to classify the inflammatory cells. Scanning electron microscopy(SEM) was performed to detect ocular surface change.The concentrations of IL-8, IL-17, Line and TNF-αwere analyzed by multiplex immunobead assay. TUNEL staining was performed to detect cellular apoptosis.

Results

Significant decrease ofcentral total cornealthickness were found within the first 5 minutes and remained stable thereafter, while there were no changes of corneal epithelial thickness.No significant change of corneal, limbal and conjunctival epithelial morphology was found by in vivo confocal microscopy except the time dependent increase of superficial cellular defects in the central cornea. Impression cytology also demonstrated time dependent increase of sloughing superficial cells of the central cornea. Aggregations ofinflammatory cells were found at 1 hour in the limbal epithelium, 2 hours in the perilimbal conjunctival epithelium, and 3 hours in the peripheral corneal epithelium.In eyes receiving exposure for 4 hours, the infiltration of the inflammatory cells can still be detected at 8 hours after closing eyes.Immunohistochemical study demonstrated the cells to be macrophages, neutrophils, CD4-T cells and CD-8 T cells.SEM demonstrated time-depending increase of intercellular border and sloughing of superficial epithelial cells in corneal surface. Time dependent increase of IL-8, IL-17 and TNF-α in tear was found.TUNEL staining revealed some apoptotic cells in the corneal epithelium and superficial stroma at 3 hours after exposure.

Conclusions

Short term exposure keratopathy can cause significant changes to the ocular surface and inflammatory response. Decrease of central total corneal thickness, aggregation of inflammatory cells, and cornea epithelial cell and superficial keratocyte apoptosis were found no less than 4 hours following the insult.     

The development of an innervated epithelial barrier model using a human corneal cell line and ND7/23 sensory neurons

The corneal epithelium is a highly innervated tissue and hence in vitro models that mimic the effects of chemicals or radiation (e.g. ultra violet) on this important barrier should include consideration of the potential role of innervation. A sensory neural cell line, ND7/23, was incorporated into a 2D and 3D model of a corneal epithelium, using a human corneal cell line, and effects on barrier integrity were neither adverse nor stimulatory. In the 3D model the nerve cell bodies were separated from the corneal epithelium, via a porous polycarbonate insert membrane. The ND7/23 cells were induced to form neurites and cease division when cultured in the keratinocyte medium employed for the corneal cells. In the absence of calcium, the epithelial barrier function was lost, shown by enhanced fluorescein leakage and relocation of ZO-1 and E-cadherin from the cell membrane. At 60 microM calcium, and above, the corneal cells formed tight junctions, with peripheral membrane location of ZO-1 and E-cadherin. The presence of the ND7/23 cells did not compromise or enhance the time taken to form these junctions, when monitored at 24-h intervals over 72 h. Both male- and female-derived human corneal cell lines showed a similar tight junction functional response to different medium calcium concentrations in the presence or absence of the ND7/23 cells. Once differentiated in keratinocyte medium, patch-clamped ND7/23 cells were capable of producing a whole-cell current when exposed to low pH (5.4), indicative of the presence of active pH-gated ion channels.     

人工泪液对患者玻璃体切除术后泪膜功能的影响

观察玻璃体切除手术后泪膜的变化和应用人工泪液后对泪膜恢复的影响。方法:60例(60眼)行玻璃体切除手术的患者随机分为两组,A组行玻手术后单用妥布霉素地塞米松眼液和眼膏,B组手术后联合应用妥布霉素地塞米松眼液、眼膏和人工泪液Dextran 70 Eye Drops。分别于术前1d,术后3d、7d、14d及30d详细询问患者是否存在干眼症状,并行泪膜破裂时间(Break-Up Time,BUT)、基础泪液分泌试验(Schirmer I test,SIt)、角膜荧光素染色(Cornea Fluorescein Staining,CFS)检查。对结果进行统计学分析。结果:(1)手术前A组和B组的干眼评分、泪膜破裂时间、角膜荧光染色评分、基础泪液分泌试验检查4个项目的检查值经统计学分析无明显差异;(2)术后各个时间点,B组的BUT检查值较A组均增加且差异显著;(3)术后7d和14d,B组的干眼评分和CFS评分较A组减少且差异显著。结论:玻璃体切除术后早期泪膜稳定性明显下降,加用人工泪液后可明显提高术后泪膜稳定性,提示玻璃体切除术后早期使用人工泪液有利于泪膜恢复。     

Solvent/Detergent Virally Inactivated Serum Eye Drops Restore Healthy Ocular Epithelium in a Rabbit Model of Dry-Eye Syndrome

Application of autologous serum eye drops (SEDs) is a recognized means to treat severe dry-eye syndrome (DES). Due to the inconvenience and difficulty of preparing SEDs from some patients, producing SEDs from allogeneic blood donations is gaining popularity. A major safety concern associated with allogeneic blood is virus transmission. We therefore herein evaluated the possibility of applying a solvent/detergent (S/D) treatment to inactivate viruses and studied the impacts of such treatment of SEDs to resolve DES in a rabbit model. Sera prepared from the blood of five rabbits were pooled and divided into two sub-pools. One was untreated (SEDs), while the other was virally-inactivated with 1% Tri-n-butyl phosphate/1% Triton X-45 at 31°C for 1 h (S/D-SEDs). DES was induced in rabbits using 0.1% benzalkonium chloride (BAC). Rabbits were divided into five groups of two rabbits each. One group was untreated (control), three were treated twice daily for 3 weeks using PBS, SEDs, or S/D-SEDs, and the last received an additional 0.1% BAC (as the negative control). The DES condition was determined by measuring aqueous tear secretion (Schirmer’s test), corneal fluorescein staining, a corneal histologic examination, TUNEL stain apoptosis, and corneal inflammatory marker (tumor necrosis factor-α, interleukin (IL)-1β, IL-8, and IL-6) expressions. We first confirmed that SEDs and S/D-SEDs had similar protein profiles and transforming growth factor (TGF)-β contents. Animal experiments showed that tear secretion did not significantly differ between the SED and S/D-SED groups but was significantly higher than in the PBS group. Eye fluorescein staining revealed dramatic improvements in epithelial defects in groups treated with SEDs or S/D-SEDs, and hematoxylin/eosin staining revealed microscopic epithelial layers similar to those of the untreated controls. Inflammatory markers and TUNEL studies showed that healthy epithelium had been restored in groups treated with SEDs or S/D-SEDs. In conclusion, this preclinical study supports the possibility of using S/D virally inactivated SEDs to treat DES and restore a normal epithelium.     

Dipeptidyl peptidase IV (DPPIV) activity in the tear fluid as an indicator of the severity of corneal injury: a histochemical and biochemical study

Comparative histochemical and biochemical studies on the catalytically active protease Dipeptidyl peptidase IV (DPPIV), have been performed in the rabbit cornea and the tear fluid using a sensitive fluorogenic substrate, Gly-Pro-7-amino-4-Trifluoromethyl Coumarine (AFC). In both normal and experimentally injured corneas, DPPIV activity was detected histochemically and in the tear fluid biochemically. In contrast to the normal cornea where DPPIV activity was absent and in the tear fluid where it was low, during continuous wearing of contact lenses or repeated irradiation of the cornea with UVB rays, slight DPPIV activity appeared first in the superficial layers of the corneal epithelium, while later increased activity was present in the whole epithelium. This paralleled elevated DPPIV activity in the tear fluid. Moreover, during continuous contact lens wear, the increased DPPIV activity in the tear fluid was, in many cases, coincidental with the presence of capillaries in the limbal part of the corneal stroma. After severe alkali burns when corneal ulcers appeared, collagen fragments were active for DPPIV, which was associated with high DPPIV activity in the tear fluid. In conclusion, Gly-Pro-AFC was found to be useful for comparative histochemical and biochemical studies on DPPIV activity in the experimentally injured rabbit eye. Using the method of the tear film collection by a short touch of substrate punches to the respective site of the cornea or conjunctiva we can show that in experimental injuries (wearing of contact lenses, irradiation of the cornea with UVB rays), the damaged corneal cells were the main source for DPPIV activity in the tear fluid. It is suggested that the activity of DPPIV measured in the tear fluid might serve as an indicator of early corneal disorders, e.g. corneal vascularization related to contact lens wear.     

Benzalkonium Chloride Suppresses Rabbit Corneal Endothelium Intercellular Gap Junction Communication

Purpose

Gap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects.

Methods

Thirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco''s Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity.

Results

Topical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity.

Conclusions

The dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to avoid declines in corneal transparency resulting from losses in GJIC activity and endothelial function.     

Astragalus IV ameliorates the dry eye injury in rabbit model via MUC1-ErbB1 pathway

The therapeutic effects and potential mechanisms of astragaloside IV on a rabbits dry eye model induced by benzalkonium chloride (BAC) was examined. In our study, a BAC-induced dry eye rabbit model was treated with eye drops containing astragaloside IV (5, 10 μM) or solvent four times a day. The clinical evaluations, such as tear break-up time (BUT) and Schirmer tear test (STT), were performed on days 0, 7, 14, 21, and 28. On day 28, the cornea and bulbar conjunctiva tissues (left eye and right eye) were collected with histology, and immunofluorescent staining conducted. The levels of MUC1 and ErbB1in the corneas were determined by realtime quantitative PCR (qRT-PCR) and the proteins levels of MUC1 and ErbB1 were detected by Western blot. It was demonstrated that both astragaloside IV (5, 10 μM) treatments resulted in an increased STT and BUT on days 7, 14, 21 and 28. Additionally, the astragaloside IV (5, 10 μM)-treated group showed increasing PAS-positive goblet cells than model group (0 μM). Moreover, the MUC1 in model group (0 μM) was decreased, while the expression of MUC1 in astragaloside IV (5, 10 μM) group was increased. Furthermore, astragaloside IV had a protective effect on BAC-induced rabbits’ dry eye and demonstrated clinical improvements, which indicated that astragaloside IV served as a potential protective agent in the clinical treatment of dry eye.Key words: Astragaloside IV, dry eye model, MUC1, ErbB1     

Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.     

Localization of occludin, ZO-1, and pan-cadherin in rabbit ciliary epithelium and iris vascular endothelium

Previous studies have used conventional electron microscopy and freeze fracture to identify the morphological equivalents of the blood-aqueous barrier in the mammalian eye. These equivalents are the tight junctions that form a part of the apicolateral junctional complex between adjacent non-pigmented ciliary epithelial cells and the tight junctions present between endothelial cells of the iris vasculature. Recent investigations have begun to unravel the molecular assembly of the tight junction and some variability has been found. Our goal in the present study was to probe the ciliary epithelium and iris vascular endothelium of the rabbit eye to determine if certain molecular constituents associated with tight junctions in other tissues are also present as parts of the blood-aqueous barrier. The selected constituents were occludin, ZO-1, and a representative, adherens junction-related cadherin. Immunohistochemical and immunoelectron microscopic methods were used. The results showed that occludin was distributed exclusively at known locations of tight junctions. ZO-1 was also expressed at these locations but its distribution extended beyond that of occludin, along the adjacent membranes. Pan-cadherin was expressed ubiquitously within the ciliary epithelium and negligibly in iris vascular endothelium. Our results demonstrate that occludin and ZO-1 are integral components of the blood-aqueous barrier of the normal rabbit eye.     

玻璃酸钠联合重组牛碱性成纤维细胞生长因子对糖尿病性白内障超声乳化术后泪膜稳定性的影响

目的:探讨玻璃酸钠联合重组牛碱性成纤维细胞生长因子(b FGF)对糖尿病性白内障超声乳化术后泪膜稳定性的影响。方法:选取2015年1月到2017年1月期间在我院接受治疗的150例糖尿病性白内障患者,根据随机数字表法分为对照组和研究组,各75例。两组均进行常规治疗,在常规治疗的基础上对照组采用玻璃酸钠滴眼液进行治疗,研究组在对照组的基础上加用bFGF眼用凝胶进行治疗,两组均治疗1个月。比较两组术前、术后1周、术后1个月的泪膜破裂时间(BUT)、基础泪液分泌试验(SIt)值、角膜荧光素染色(FL)评分、干眼症状评分和眼表疾病指数(OSDI)评分,并比较两组术前、术后1个月的最佳矫正视力。结果:术后1周,两组患者的BUT明显低于术前和术后1个月,且研究组的BUT高于对照组(P0.05);随时间推移两组患者的OSDI评分持续降低(P0.05),术后1周,两组患者干眼症状评分、SIt值、FL评分明显高于术前和术后1个月,且研究组的OSDI评分、干眼症状评分、SIt值、FL评分均低于对照组(P0.05)。术后1个月两组患者的最佳矫正视力均明显上升,且研究组明显高于对照组(P0.05)。结论:玻璃酸钠联合bFGF能有效提升糖尿病性白内障患者在超声乳化术后泪膜稳定性,利于患者术后视力的恢复,值得临床推广应用。     

A comparative evaluation of corneal epithelial cell cultures for assessing ocular permeability

The purpose of this study was to evaluate the potential value of different epithelial cell culture systems as in vitro models for studying corneal permeability. Transformed human corneal epithelial (HCE-T) cells and Statens Serum Institut rabbit corneal (SIRC) cells were cultured on permeable filters. SkinEthic human corneal epithelium (S-HCE) and Clonetics human corneal epithelium (C-HCE) were received as ready-to-use systems. Excised rabbit corneas (ERCs) and human corneas (EHCs) were mounted in Ussing chambers, and used as references. Barrier properties were assessed by measuring transepithelial electrical resistance, and by determining the apparent permeability of markers with different physico-chemical properties, namely, fluorescein, sodium salt; propranolol hydrochloride; moxaverine hydrochloride; timolol hydrogenmaleate; and rhodamine 123. SIRC cells and the S-HCE failed to develop epithelial barrier properties, and hence were unable to distinguish between the permeation markers. Barrier function and the power to differentiate compound permeabilities were evident with HCE-T cells, and were even more pronounced in the case of C-HCE, corresponding very well with data from ERCs and EHCs. A net secretion of rhodamine 123 was not observed with any of the models, suggesting that P-glycoprotein or similar efflux systems have no significant effects on corneal permeability. Currently available corneal epithelial cell culture systems show differences in epithelial barrier function. Systems lacking functional cell-cell contacts are of limited value for assessing corneal permeability, and should be critically evaluated for other purposes.     

Intracellular activities of chloride, potassium and sodium ions in rabbit corneal epithelium

The mechanism of ion transport in the epithelium of rabbit cornea was studied by determining the intracellular ion activity of Cl-, Na+ and K+ under various conditions. Ionic activities were measured by means of microelectrodes containing liquid ion-exchangers selective for Cl-, Na+ or K+. The Cl- activity in basal cells of the epithelium in Na+ containing bathing solutions amounts to 28 +/- 2 mM (n = 11). This value is 1.9-times greater than expected on the basis of passive distribution across the tear side membrane. This finding suggests the existence of a Cl- accumulating process. Replacement of Na+ in the aqueous bathing solution by choline or tetraethylammonium results in a reversible decrease in Cl- activity to 22 +/- 1 mM (n = 11, P less than 0.025). The ratio of observed and predicted Cl- activity decreased significantly from 1.9 to 1.4 (P less than 0.05). The decrease in Cl- activity due to Na+ replacement was rather slow. In contrast, after readmittance of Na+ to the aqueous bathing solution, Cl- activity rose to a stable level within 30 min. These results indicate involvement of Na+ in Cl- accumulation into the basal cells of the epithelium. The K+ and Na+ activities of the basal cells of rabbit corneal epithelium in control bathing solutions were 75 +/- 4 mM (n = 13) and 24 +/- 3 mM (n = 12), respectively. The results can be summarized in the following model for Cl- transport across corneal epithelium. Cl- is accumulated in the basal cells across the aqueous side membrane, energized by a favourable Na+ gradient. Cl- will subsequently leak out across the tear side membranes. Na+ is extruded again across the aqueous side membrane of the epithelium by the (Na+ + K+)-ATPase.     

Punctal plugs versus artificial tears for treating dry eye: a comparative observation of their effects on contrast sensitivity

This study aimed to compare the effects of treatment with punctal plugs versus artificial tears on visual function and tear film stability for dry eye. A total of 56 consecutive eyes of 28 dry eye patients observed at our clinic from May to October in 2009 were divided into two groups. One group (32 eyes of 16 patients) was treated with artificial tears, and punctal plugs were used in the other group (24 eyes of 12 patients). A questionnaire was used in these patients before treatment and was repeated 2 weeks after treatment. Fluorescent staining for tear film break-up time (BUT), the Schirmer test I (STI), and contrast sensitivity was performed at the same time. The questionnaire indicated that all patients complained about the uncomfortable symptoms associated with dry eye. These symptoms were relieved after the application of artificial tears or punctal plugs, and there was no significant difference between these two groups. We found that the corneal fluorescent staining disappeared after treatment. The BUT was improved significantly after treatment in both groups, but the improvement was greater in patients who received punctal plugs than those that received artificial tears. There was no remarkable change in the STI in the artificial tears group, but a significant change was observed in the punctal plugs group. The contrast sensitivities were greatly improved in simulated daylight, night, and glare disability conditions after treatment with artificial tears and punctal plugs. However, the changes in contrast sensitivity did not significantly differ between groups. Both artificial tears and punctal plugs relieved dry eye symptoms, repaired corneal lesions, enhanced tear film stability, and improved contrast sensitivity. Punctal plugs could improve tear film stability and elongate the BUT better than artificial tears.     

A non-invasive method for an in vivo assessment of corneal epithelium permeability through tetrapolar impedance measurements

The permeability of the cornea epithelial layer has an important role in optimal function of the cornea. To assess this property quantitatively, methods must be based on the passive electrical properties of living tissues, as they can take advantage of the fundamental role that ionic permeability plays in such properties. For such techniques, measurement of the translayer electrical resistance (TER) has been consistently used to examine the ion transport mechanisms in the corneal epithelial cells; however, this technique has been only possible in vitro. To enhance the applications of this method, in this work we present a novel sensor to perform non-invasive in vivo TER measurements. Herein, the epithelial permeability was assessed using non-invasive tetrapolar impedance measurements that were performed with four electrodes placed on the corneal surface. The geometry of these electrodes was previously optimized to maximize the sensitivity of the corneal epithelium. To evaluate the feasibility of this sensor, the permeability of a rabbit corneal epithelium was monitored by applying a solution of benzalkonium chloride (0.05% BAC). The results validate the capability of the sensor to evaluate the cornea epithelial permeability in vivo.     

A Native-Like Corneal Construct Using Donor Corneal Stroma for Tissue Engineering

Tissue engineering holds great promise for corneal transplantation to treat blinding diseases. This study was to explore the use of natural corneal stroma as an optimal substrate to construct a native like corneal equivalent. Human corneal epithelium was cultivated from donor limbal explants on corneal stromal discs prepared by FDA approved Horizon Epikeratome system. The morphology, phenotype, regenerative capacity and transplantation potential were evaluated by hematoxylin eosin and immunofluorescent staining, a wound healing model, and the xeno-transplantation of the corneal constructs to nude mice. An optically transparent and stratified epithelium was rapidly generated on donor corneal stromal substrate and displayed native-like morphology and structure. The cells were polygonal in the basal layer and became flattened in superficial layers. The epithelium displayed a phenotype similar to human corneal epithelium in vivo. The differentiation markers, keratin 3, involucrin and connexin 43, were expressed in full or superficial layers. Interestingly, certain basal cells were immunopositive to antibodies against limbal stem/progenitor cell markers ABCG2 and p63, which are usually negative in corneal epithelium in vivo. It suggests that this bioengineered corneal epithelium shared some characteristics of human limbal epithelium in vivo. This engineered epithelium was able to regenerate in 4 days following from a 4mm-diameter wound created by a filter paper soaked with 1 N NaOH. This corneal construct survived well after xeno-transplantation to the back of a nude mouse. The transplanted epithelium remained multilayer and became thicker with a phenotype similar to human corneal epithelium. Our findings demonstrate that natural corneal stroma is an optimal substrate for tissue bioengineering, and a native-like corneal construct has been created with epithelium containing limbal stem cells. This construct may have great potential for clinical use in corneal reconstruction.