Ozone exposure affects corneal epithelial fate by promoting mtDNA leakage and cGAS/STING activation.

Ozone is a common air pollutant associated with various human diseases. The human ocular surface is frequently exposed to ozone in the troposphere, but the mechanisms by which ozone affects the ocular surface health remain unclear. This study aimed to establish a mouse model to investigate the effects of ozone exposure on the ocular surface and the corneal epithelium. The findings revealed that ozone exposure disrupted corneal epithelial homeostasis and differentiation, resulting in corneal squamous metaplasia. Further, ozone exposure induced oxidative damage and cytoplasmic leakage of mitochondrial DNA (mtDNA), thereby activating the cGAS/STING signaling pathway. The activation of the cGAS/STING signaling pathway triggered the activation of downstream NF-κB and TRAF6 signaling pathways, causing corneal inflammation, thereby promoting corneal inflammation and squamous metaplasia. Finally, C-176, a selective STING inhibitor, effectively prevented and treated corneal inflammation and squamous metaplasia caused by ozone exposure. This study revealed the role of mtDNA leakage-mediated cGAS/STING activation in corneal squamous epithelial metaplasia caused by ozone exposure. It also depicted the abnormal expression pattern of corneal epithelial keratin using three-dimensional images, providing new targets and strategies for preventing and treating corneal squamous metaplasia and other ocular surface diseases.

A cocktail of small molecules maintains the stemness and differentiation potential of conjunctival epithelial cells.

PURPOSE: The conjunctival epithelial cells cultured with bovine serum or feeder cells were not suitable for clinical application. Therefore, we developed a novel serum-free and feeder cell-free culture system containing only a cocktail of three chemicals (3C) to expand the conjunctival epithelial cells. METHODS: The cell proliferative ability was evaluated by counting, crystal violet staining and Ki67 immunostaining. Co-staining of K7 and MUC5AC was performed to identify goblet cells. PAS staining was used to assess the ability of cells to synthesis and secrete glycoproteins. In vivo, eye drops containing 3C was administered to verify the role of 3C in the mouse conjunctival injury model. PAS, HE and immunofluorescence staining were performed to show conjunctival epithelial repair. RESULTS: Compared with other small molecule groups and the serum group, the cells in 3C group showed superior morphology and proliferative ability. Meanwhile, 3C maintained the well-proliferative capacity of cells even after fifth passage. The 3C group also exhibited more K7 and MUC5AC double positive cells, and the PAS staining positive areas were present in both the cytoplasm and extracellular matrix. The cell sheets treated with 3C in air-lifted culture were obviously stratified. In vivo, more goblet cells in the conjunctival epithelium were observed in the 3C group. CONCLUSION: Overall, our culture system can expand the conjunctival epithelial cells and retain their potential to differentiate into mature goblet cells, which provided a promising source of seed cells for conjunctival reconstruction. Furthermore, this system provides new insights for the clinical treatment of ocular surface diseases.

The Ocular Surface Characteristics in Prostate Cancer Patients Treated with Androgen Deprivation Therapy.

BACKGROUND: To investigate the association of long-term androgen deprivation therapy (ADT) with ocular surface characteristics in prostate cancer patients. METHODS: A total of 30 male prostate cancer patients who received ADT were selected. All candidates were scored using the Ocular Surface Disease Index (OSDI) and subsequently divided into two groups containing 9 symptomatic patients (scores >12) and 21 asymptomatic patients (scores ≤ 12). Another 20 healthy age-matched males were selected as the control group. Each candidate was assessed with respect to eyelid margin abnormality, tear film break-up time (NI-BUT), tear meniscus height (TMH), meiboscore, meibum expressibility, and demodex infection. RESULTS: The NI-BUT in the ADT group was significantly shorter than that in the control group. The scores for OSDI, eyelid margin abnormality, meibum expressibility, and meiboscores were significantly higher in the ADT group (P < 0.05). Moreover, the NI-BUT in the symptomatic ADT group was significantly shorter than that in the asymptomatic ADT group (P < 0.05). The meiboscores and meibum expressibility score in the symptomatic ADT group were significantly higher than those in the asymptomatic ADT group (P < 0.05). The presence of demodex in the symptomatic ADT group was also higher than that in the asymptomatic ADT group (P < 0.05).The length of time that patients had been taking ADT was positively correlated with meiboscores and negatively correlated with NI-BUT. CONCLUSION: Androgen levels were associated with significant changes in relative meibomian gland function. Subjective symptoms, such as dryness and foreign body sensation, were more obvious in prostate cancer patients receiving ADT, which may be caused by MGD and demodex infection. It's recommended that more attention be paid to the ocular surface in prostate cancer patients taking ADT by performing examination of NI-BUT and meibomian gland morphology and function with a view to providing more comprehensive prevention and treatment protocols.

Small Leucine-Rich Proteoglycans in Skin Wound Healing.

Healing of cutaneous wounds is a complex and well-coordinated process requiring cooperation among multiple cells from different lineages and delicately orchestrated signaling transduction of a diversity of growth factors, cytokines, and extracellular matrix (ECM) at the wound site. Most skin wound healing in adults is imperfect, characterized by scar formation which results in significant functional and psychological sequelae. Thus, the reconstruction of the damaged skin to its original state is of concern to doctors and scientists. Beyond the traditional treatments such as corticosteroid injection and radiation therapy, several growth factors or cytokines-based anti-scarring products are being or have been tested in clinical trials to optimize skin wound healing. Unfortunately, all have been unsatisfactory to date. Currently, accumulating evidence suggests that the ECM not only functions as the structural component of the tissue but also actively modulates signal transduction and regulates cellular behaviors, and thus, ECM should be considered as an alternative target for wound management pharmacotherapy. Of particular interest are small leucine-rich proteoglycans (SLRPs), a group of the ECM, which exist in a wide range of connecting tissues, including the skin. This manuscript summarizes the most current knowledge of SLRPs regarding their spatial-temporal expression in the skin, as well as lessons learned from the genetically modified animal models simulating human skin pathologies. In this review, particular focus is given on the diverse roles of SLRP in skin wound healing, such as anti-inflammation, pro-angiogenesis, pro-migration, pro-contraction, and orchestrate transforming growth factor (TGF)ß signal transduction, since cumulative investigations have indicated their therapeutic potential on reducing scar formation in cutaneous wounds. By conducting this review, we intend to gain insight into the potential application of SLRPs in cutaneous wound healing management which may pave the way for the development of a new generation of pharmaceuticals to benefit the patients suffering from skin wounds and their sequelae.

Epithelial dysplasia in pterygium postoperative granuloma.

Pterygium postoperative granuloma (PPG) is one of the common complications of pterygium surgery. In order to provide the structural features of PPG, and to further explore its pathogenetic mechanism, we analyzed clinical and pathological characteristics of 12 PPG cases. New blood vessels were observed under a slit lamp in PPG and peripheral conjunctival tissues. In vivo confocal imaging showed that there was extensive neovascularization in the stroma, accompanied by infiltration of dendritic cells and inflammatory cells. Dense fibrous structures were observed in some PPG tissues. H&E staining results confirmed neovascularization and inflammatory cells in PPG tissues. In addition, H&E staining exhibited epithelioid tissue covering some PPG tissues. The immunofluorescence results demonstrated that the PPG epithelium was negative for K19, K10 and Muc5AC. Compared with the normal conjunctiva and pterygium, the expression of collagen IV in PPG basement membrane decreased, the expression of pan-cytokeratin (PCK), claudin 4 and E-cadherin in PPG epithelium was significantly lower, while the expression of vimentin, α-SMA and Snail was significantly increased. Therefore, our results suggest that the expression of epithelial keratin markers and goblet cell specific mucin marker is downregulated in the PPG tissues, and it likely is associated with the occurrence of EMT in granulomatous tissues.

Tissue remodeling after ocular surface reconstruction with denuded amniotic membrane.

Amniotic membrane (AM) has been widely used as a temporary or permanent graft in the treatment of various ocular surface diseases. In this study, we compared the epithelial wound healing and tissue remodeling after ocular surface reconstruction with intact amniotic membrane (iAM) or denuded amniotic membrane (dAM). Partial limbal and bulbar conjunctival removal was performed on New Zealand rabbits followed by transplantation of cryo-preserved human iAM or dAM. In vivo observation showed that the epithelial ingrowth was faster on dAM compared to iAM after AM transplantation. Histological observation showed prominent epithelial stratification and increased goblet cell number on dAM after 2 weeks of follow up. Collagen VII degraded in dAM within 2 weeks, while remained in iAM even after 3 weeks. The number of macrophages and α-SMA positive cells in the stroma of remodelized conjunctiva in the dAM transplantation group was considerably less. In conclusion, dAM facilitates epithelial repopulation and goblet cell differentiation, further reduces inflammation and scar formation during conjunctival and corneal limbal reconstruction.

Characteristics of IL-9 induced by Schistosoma japonicum infection in C57BL/6 mouse liver.

Liver granulomatous inflammation and fibrosis were the primary pathological changes observed during Schistosoma japonicum (S. japonicum) infection. In the present study, the characteristics of IL-9 were investigated in the liver of S. japonicum infection C57BL/6 mice. Immunofluorescence, qRT-PCR, and ELISA results demonstrated that the expression of IL-9 significantly increased after infection (P < 0.01). FACS results indicated that the peak of IL-9+ Th9 cells in the liver mononuclear cells appeared at the early phase of infection (week 5), except that Th9 cells, CD8+ Tc cells, NKT and γδT cells could secrete IL-9 in this model. Although IL-9 neutralization has a limited effect on liver granulomatous inflammation, it could decrease the level of fibrosis-associated factor, PC-III, in the serum of infected mice (P < 0.05). Taken together, our results indicated that IL-9 was an important type of cytokine involved in the progression of S. japonicum infection-induced hepatic damage.

Differential pulmonic NK and NKT cell responses in Schistosoma japonicum-infected mice.

Natural killer cells (NK cells) and natural killer T cells (NKT cells) play a role in anti-infection, anti-tumor, transplantation immunity, and autoimmune regulation. However, the role of NK and NKT cells during Schistosoma japonicum (S. japonicum) infection has not been widely reported, especially regarding lung infections. The aim of this study was to research the NK and NKT cell response to S. japonicum infection in the lungs of mice. Using immunofluorescent histological analysis, NK and NKT cells were found near pulmonary granulomas. Moreover, flow cytometry revealed that the percentage and number of pulmonic NK cells in S. japonicum-infected mice were significantly increased (P < 0.05). However, the percentage and cell number of NKT cells were decreased compared to those of normal mice (P < 0.05). The expression of CD69 on pulmonic NK and NKT cells was increased after infection (P < 0.05), and CD25 expression increased only on NKT cells (P < 0.05). Intracellular cytokine staining showed a higher percentage of IFN-γ+ and lower percentage of IL-5+ pulmonic NK cells (P < 0.05) compared to controls. However, the percentage of IL-17+, IL-10+, and IL-5+ pulmonic NKT cells significantly increased (P < 0.05). Additionally, there was a significant decrease in NKG2A/C/E (CD94) expression and an increase of NKG2D (CD314) expression on pulmonic NKT cells (P < 0.05), which might serve as a mechanism for NKT cell activation during S. japonicum infection.

APR-246/PRIMA-1Met Inhibits and Reverses Squamous Metaplasia in Human Conjunctival Epithelium.

PURPOSE: Squamous metaplasia is a common pathologic condition in ocular surface diseases for which there is no therapeutic medication in clinic. In this study, we investigated the effect of a small molecule, APR-246/PRIMA-1(Met), on squamous metaplasia in human conjunctival epithelium. METHODS: Human conjunctival explants were cultured for up to 12 days under airlifting conditions. Epithelial cell differentiation and proliferation were assessed by Cytokeratin 10 (K10), K14, K19, Pax6, MUC5AC, and p63 immunostaining patterns. ß-catenin and TCF-4 immunofluorescent staining and real-time PCR characterized Wnt signaling pathway involvement. Pterygium clinical samples were cultured under airlifting conditions with or without APR-246 for 4 days. p63, K10, ß-catenin, and TCF-4 expression in pterygial epithelium was determined by immunofluorescent staining and real-time PCR. RESULTS: Airlift conjunctival explants resulted in increased stratification and intrastromal epithelial invagination. Such pathology was accompanied by increases in K10, K14, and p63 expression, whereas K19 and Pax6 levels declined when compared to those in freshly isolated tissue. On the other hand, APR-246 reversed all of these declines in K10, K14, and p63 expression. Furthermore, K19 and Pax6 increased along with rises in goblet cell density. These effects of APR-246 were accompanied by near restoration of normal conjunctival epithelial histology. APR-246 also reversed squamous metaplasia in pterygial epithelium that had developed after 4 days in ex vivo culture. CONCLUSIONS: Reductions in squamous metaplasia induced by APR-246 suggest it may provide a novel therapeutic approach in different squamous metaplasia-associated ocular surface diseases.

[Transepithelial iontophoresis corneal collagen cross-linking for progressive keratoconus: one year results].

OBJECTIVE: To evaluate the early clinical results of keratoconic eyes treated with transepithelial iontophoresis corneal collagen cross-linking (i-CXL) within 1 year. METHODS: Propective nonrandomized study. Twenty-three eyes of 23 patients with progressive keratoconus with minimum corneal thickness from 380 µm to 420 µm (including the epithelium) were included in this prospective, nonrandomized clinical study and treated with i-CXL. Scoring of pain and foreign body sensation, slit lamp examination, uncorrected visual acuity (UCVA) and best corrected distance visual acuity (BCVA), corneal topography, anterior segment optical coherence tomography (AS-OCT), in vivo corneal confocal microscopy and endothelial cell count were assessed before surgery and at 1, 3, 6 and 12 months (m) postoperatively. Paired t test was applied for statistical analysis. RESULTS: Moderate pain and foreign body sensation were reported by most patients on postoperative Day (D) 1, but rapidly decreased and eventually disappeared on D3. Mild epithelial damage was observed on D1, and the epithelium fully recovered on D3. Improvement of UCVA and BCVA were recorded at 3 months and 12 months postoperatively. Orbscan II corneal topography revealed the significant reductions of Kmax and Kmin from 3m to 12m (Kmax, t = 2.912, P < 0.01, Kmin, t = 2.508, P < 0.05) postoperatively while the other parameters remained stable. The Kmax and Kmin at 12m was (52.94 ± 4.87) and (46.78 ± 3.71) respectively, while the preoperative values was (54.37 ± 5.56) and (48.53 ± 3.57) respectively. Within 1m postoperatively, AS-OCT exhibited an increase of reflectance with a white line (demarcation line) in the anterior stroma, in vivo confocal microscopy also showed the significant thickening and increased connections of collagen fibers with maximal depth of about 133 µm. The corneal endothelial cell density remained stable (t = 0.915, P > 0.05). None of the patients showed postoperative complications such as corneal infection, scarring, ulcer, persistent epithelial defect, etc. CONCLUSIONS: Priliminary clinical results within 1 year postoperatively demonstrated the efficacy and safety of i-CXL for the management of progressive keratoconus. This technique was applicable for keratoconic eyes with minimum corneal thickness around 400 µm. i-CXL showed the advantage of short time consuming in surgery, rapid recovery and few complication, and has the potential to become a valid alternative for the treatment of keratoconus.

[The clinical study of corneal cross-linking with hypo-osmolar riboflavin solution in thin keratoconic corneas].

OBJECTIVE: To evaluate the clinical results of keratoconic eyes with thin corneas that were treated by using corneal collagen cross-linking with hypo-osmolar riboflavin solution. METHODS: Retrospective, nonrandomized study. Fifteen eyes of 15 patients with progressive keratoconus and corneal thickness of less than 400 µm (without the epithelium) were included in this study. Application of hypo-osmolar riboflavin solution to the cornea for 30 minutes after its de-epithelialization was followed by ultraviolet A collagen cross-linking. Corneal thickness was measured with anterior segment OCT before surgery, after epithelial removal, and after hypotonic riboflavin solution application. Before the ultraviolet A application was started, we must be sure that the thinnest cornea was equal to or greater than 400 µm. Examinations comprised an evaluation of uncorrected distance visual acuity and best corrected visual acuity, slit-lamp microscopy, corneal topography, and endothelial cell counting after the procedure. RESULTS: Before surgery, the mean corneal thickness (with the epithelium) was (399.27 ± 17.87) µm, and after the removal of epithelium, the thickness of the cornea was reduced to (354.00 ± 18.57) µm. After the application of the hypo-osmolar riboflavin solution, this value increased to (477.73 ± 20.87) µm. The improvements in visual acuity and keratometry readings occurred during the follow-up. No statistically signiflcant differences were found between preoperative and postoperative endothelial cell counts. No complications such as scarring lesions in the stroma and corneal endothelial damage were observed throughout the study period. CONCLUSIONS: The results of our study, using hypo-osmolar riboflavin solution in a cross-linking procedure in thin corneas, show a stability of keratoconus 12 months after cross-linking.

SERPINA3K plays antioxidant roles in cultured pterygial epithelial cells through regulating ROS system.

We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM). The cultured pterygial epithelial cells (PECs) were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR) assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4), which is an important enzyme of ROS generation, and by elevating the levels of key antioxidant factors of ROS: such as NAD(P)H dehydrogenase (quinone 1) (NQO1), NF-E2-related factor-2 (NRF2) and superoxide dismutases (SOD2). Meanwhile, SERPINA3K down-regulated the key effectors of Wnt signaling pathway: ß-catenin, nonphospho-ß-catenin, and low-density lipoprotein receptor-related protein 6 (LRP6). We provided novel evidence that SERPINA3K had inhibitory effects on pterygium and SERPINA3K played antioxidant role via regulating the ROS system and antioxidants.

Fibrin glue-assisted for the treatment of corneal perforations using glycerin-cryopreserved corneal tissue.

AIM: To evaluate the outcomes and safety of lamellar keratoplasty (LK) assisted by fibrin glue in corneal perforations. METHODS: Six eyes of 6 patients affected by different corneal pathologies (2 posttraumatic corneal scar and 3 bacterial keratitis) underwent LK procedures by using fibrin glue. The mean corneal perforation diameter was 1.35±0.64mm (range, 0.7-2.5mm), and the greatest diameter of the ulcerative stromal defect was 2.47±0.77mm in average (range, 1.5-3.5mm). The donor corneal lamella diameters were 0.20-mm larger and thicker than the recipient to restore a physiologic corneal thickness and shape: mean donor diameter was 8.34±0.28mm (range, 8.2-8.7mm) and mean thickness was 352±40.27mm (range, 220-400mm). Mean follow-up was 7.33±1.97 months (range, 6-11 months). Postoperatively, the graft status, graft clarity, anterior chamber response, the visual prognosis, intraocular pressures, and postoperative complications were recorded. RESULTS: All the corneal perforations were successfully healed after the procedure. The best-corrected visual acuity (BCVA) ranged from 20/1 000 to 20/50 in their initial presentation, and from 20/100 to 20/20 in their last visit, showed increase in all the patients. No major complications such as graft dislocation and graft failure were noted. Neovascularization developed in the superficial stroma of donor graft in 1 case. High intraocular pressure developed on day 2 after surgery, while was remained in normal range after application of anti-glaucomatous eyedrops for 1 week in 1 case. CONCLUSION: Fibrin glue-assisted sutureless LK is valuable for maintaining the ocular integrity in the treatment of corneal perforations.

Air exposure induced characteristics of dry eye in conjunctival tissue culture.

There are several animal models illustrating dry eye pathophysiology. Current study would like to establish an ex vivo tissue culture model for characterizing dry eye. Human conjunctival explants were cultured under airlift or submerged conditions for up to 2 weeks, and only airlifted conjunctival cultures underwent increased epithelial stratification. Starting on day 4, the suprabasal cells displayed decreased K19 expression whereas K10 keratin became evident in airlift group. Pax6 nuclear expression attenuated already at 2 days, while its perinuclear and cytoplasmic expression gradually increased. MUC5AC and MUC19 expression dramatically decreased whereas the full thickness MUC4 and MUC16 expression pattern disappeared soon after initiating the airlift condition. Real time PCR showed K16, K10 and MUC16 gene up-regulated while K19, MUC5AC, MUC19 and MUC4 down-regulated on day 8 and day 14. On day 2 was the appearance of apoptotic epithelial and stromal cells appeared. The Wnt signaling pathway was transiently activated from day 2 to day 10. The inflammatory mediators IL-1ß, TNF-α, and MMP-9 were detected in the conditioned media after 6 to 8 days. In conclusion, airlifted conjunctival tissue cultures demonstrated Wnt signaling pathway activation, coupled with squamous metaplasia, mucin pattern alteration, apoptosis and upregulation of proinflammatory cytokine expression. These changes mimic the pathohistological alterations described in dry eye. This correspondence suggests that insight into the pathophysiology of dry eye may be aided through the use of airlifted conjunctival tissue cultures.

[Abnormal epithelial differentiation and tear film alteration in pterygium].

OBJECTIVE: To investigate the differentiation and proliferation of the conjunctival epithelium and tear film function in pterygia. METHODS: It was a retrospective study. Fifteen patients (15 eyes) who underwent excision for pterygium were enrolled in this study. Immunostaining for K10, K14, K19, MUC5AC, K16, Ki67 and P63 was performed on the pterygial epithelium and normal conjunctival epithelium. Schirmer I test was performed, and the tear film break-up time (BUT) was evaluated just prior to and 6 months after surgery. Multi-factor regression analysis was assessed to observe if there is a correlation between pterygial growth and tear film function. RESULTS: The average absorbency of K19 and MUC5AC immuno-staining all significantly changed (3727.86 ± 2544.73 vs. 25 528.00 ± 12 901.06, 2080.48 ± 2340.17 vs. 7182.51 ± 3069.20, t = 9.261,3.538, P < 0.05), and increased in K10 and K14 in patients with pterygia compared with normal conjunctivae keratin (2017.51 ± 2114.3 vs. 0, 6027.5 ± 1058.32 vs. 2123.28 ± 1249.09, t = -6.151, P < 0.05). Furthermore, pterygial epithelium showed activated proliferation, evidenced by significantly up-regulated expression of K16, P63 and Ki67 compared to normal control. The Schirmer I test did not indicate any significant differences pre- and post-operatively. However, the BUT was significantly prolonged 1 month post surgery compared to pre-surgery (t = -4.222, P < 0.05). CONCLUSION: This study indicates that abnormal epithelial differentiation and proliferation are present in pterygium , which is characterized by squamous metaplasia, accompanied with instability of tear film and normal basic tear secretion.

Hypoxia affects in vitro proliferation and differentiation of mouse corneal epithelial progenitor cell.

This study was to investigate the proliferation and differentiation of mouse corneal epithelial progenitor cell in hypoxic airlift culture. Mouse corneal epithelial progenitor cell line progenitor cells were cultured under airlift with normoxic and hypoxic conditions for various durations up to 2 wk. Under normoxic conditions when exposed to air, the hyperproliferation and abnormal epidermal-like differentiation of mouse corneal epithelium was induced, whereas when exposed to air under hypoxic conditions, although we observed augmented proliferation, the abnormal differentiation was inhibited. The mechanism by which hypoxia prevents abnormal differentiation may involve downregulation of Wnt signaling pathways, which were inhibited in cells cultured with hypoxic airlift technique. In conclusion, hypoxia can prevent abnormal differentiation while enhancing the proliferation of corneal epithelial cells by blocking Wnt/ß-catenin signaling pathway.

A novel method for preservation of human corneal limbal tissue.

PURPOSE: We investigated the efficacy of low-temperature airlift preservation of human corneal limbal tissue for ex vivo expansion and allograft keratolimbal transplantation. METHODS: Human limbal tissue either was submerged or airlifted in Optisol-GS medium and preserved at 4°C for up to eight days. Hematoxylin and eosin, and E-cadherin staining was performed to investigate epithelial structure and cell-cell junction. Epithelial cell differentiation and proliferation were studied using the biomarkers, such as K10, K12, K14, Ki67, and p63. Cell apoptosis was detected with the TUNEL assay. The epithelial progenitor cell pool was evaluated by clonal culture of epithelial cells on 3T3 feeder layers. For clinical application, keratolimbal transplantation was performed in three patients with partial limbal stem cell deficiency, using limbal tissues preserved under the airlift manner. Pre- and postoperative evaluations were conducted by slit-lamp microscopy and fluorescein staining. RESULTS: After eight days, intact epithelia with strong cell-cell junctions were retained only in airlifted tissues. Airlifting maintained a normal corneal differentiation pattern, along with low proliferation activity and increased proliferation potential, but little apoptosis. Epithelial cells harvested from the airlift preservation for up to eight days exhibited stable clonogenicity. Limbal tissues preserved under the airlift manner successfully reconstructed corneal and limbal surfaces in partial limbal stem cell-deficient patients. CONCLUSIONS: Limbal tissues preserved under hypothermic airlift conditions maintain the intact structure, normal phenotype, high viability, and stem cell pool of limbal epithelia. Such a method may be used in eye bank tissue processing and corneal epithelial tissue engineering.

[Corneal collagen crosslinking in the treatment of infectious keratitis].

OBJECTIVE: To investigate the feasibility and clinical efficacy of corneal collagen crosslinking (CXL) for the treatment of infectious keratitis. METHODS: Noncomparative interventional case series.19 patients with infectious keratitis admitted to our hospital between November 2011 and January 2012 were recruited into this study, CXL was performed when medications combined proved poor therapeutic effects. Postoperatively, the graft status, graft clarity, the visual prognosis and postoperative complications were recorded. RESULTS: In 15 cases, there was improvement in symptoms one week after operation.3 cases remained stable, while 1 case reported deteriorated function at the same time.One month after operation, Corneal melting was arrested and complete epithelialization was achieved in 13 cases, 5 cases experienced significant improvement and 1 patient experienced corneal ulcer perforation.2 month after surgery, patients with healed corneal ulcer increased to 17 cases, and 2 cases experienced corneal ulcer perforation. Those 17 cases with healed corneal ulcer were followed up for 6 months, 15 cases had significant improvement in best-corrected visual acuity, 2 cases had no significant change, and no relapse was observed in those 17 cases. CONCLUSION: Our experience based on the above and other cases suggest that CXL could be an effective tool in battling difficult cases of infectious keratitis. This treatment could present many advantages but will need further investigation both by in vitro and in vivo studies.

Localization and expression of zonula occludins-1 in the rabbit corneal epithelium following exposure to benzalkonium chloride.

Preservatives are a major component of the ophthalmic preparations in multi-dose bottles. The purpose of this study was to investigate the acute effect of benzalkonium chloride (BAC), a common preservative used in ophthalmic preparations, on the localization and expression of zonula occludens (ZO)-1 in the rabbit corneal epithelium in vivo. BAC at 0.005%, 0.01%, or 0.02% was topically applied to one eye each of albino rabbits at 5 min intervals for a total of 3 times. The contralateral untreated eyes served as controls. The following clinical indications were evaluated: Schirmer test, tear break-up time (BUT), fluorescein and rose Bengal staining. The structure of central cornea was examined by in vivo confocal microscopy, and the corneal barrier function was evaluated by measurement of corneal transepithelial electrical resistance and permeability to carboxy fluorescein. Whole mount corneas were analyzed by using fluorescence confocal microscopy for the presence of ZO-1, 2, occludin, claudin-1, Ki67 and cell apoptosis in the epithelium. The expression of ZO-1 in the corneal epithelium was also examined by western blot and reverse transcription-polymerase chain reaction analyses. Exposure to BAC resulted in higher rose Bengal staining scores while no significant changes in BUT, Schirmer and corneal florescein scores. It also induced corneal epithelial cell damage, dispersion of ZO-1 and ZO-2 from their normal locus at the superficial layer and disruption of epithelial barrier function. However, the amounts of ZO-1 mRNA and protein in the corneal epithelium were not affected by BAC treatment. Exposure to BAC can quickly impair the corneal epithelium without tear deficiency. BAC disrupts the tight junctions of corneal epithelium between superficial cells in the rabbit corneal epithelium in vivo.

[Fibrin glue-assisted lamellar keratoplasty for the treatment of corneal ulcer].

OBJECTIVE: To investigated the feasibility of fibrin glue-assisted sutureless lamellar keratoplasty (LK) in patients with corneal ulcer. METHODS: Noncomparative interventional case series. In 21 eyes of 21 patients with terriens marginal degeneration (TMD) or corneal ulcer after trauma, sutureless lamellar keratoplasty (LK) using fibrin glue was performed. Postoperatively, the graft status, graft clarity, the visual prognosis and postoperative complications were recorded. RESULTS: There were 16 cases suffered with Terriens marginal degeneration (TMD) and 5 cases with corneal ulcer after foreign body removed. All the corneal ulcers were successfully healed after the procedure, the operative complications included corneal perforation during operation in 1 case, recurrence of TMD in 1 case and graft opacity in 2 cases. There was no graft dislocation, hydrops between graft and recipient interface, secondary infection and graft rejection after surgery, 19 cases had improved best-corrected visual acuity (BCVA) while 2 cases had poor vision acuity due to graft opacity (t = -2.587, P = 0.018). CONCLUSION: Fibrin glue-assisted sutureless LK showed excellent graft stability in all patients.