TLR2 activation in corneal stromal cells by Staphylococcus aureus‐induced keratitis

The Toll‐Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus‐induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air‐interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub‐groups exposed to UV‐killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL‐8 mRNA expressions were analyzed by quantitative real‐time RT‐PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL‐8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial‐corneal tissue interactions and their inflammatory consequences.     

Acanthamoeba pathogenicity for corneal cells

OBJECTIVES:Comparison of the relative susceptibility of human keratocyte and corneal epithelial cells to Acanthamoeba castellanii. METHODS: Primary cultures of the mammalian cells were utilised at equivalent concentrations throughout the co-incubations. Preliminary experiments involved visual estimation of the effects of 10-fold dilutions of Acanthamoeba trophozoites after various time intervals. Formal quantative assessment was performed by image analysis of the respective effects of the Acanthamoeba at a concentration of 10(6)/ml/well following 3, 6, 9 and 24 h of co-incubation. RESULTS: Epithelial cells were relatively resistant to the cytopathic effects of Acanthamoeba throughout the experiments course at all concentrations tested: the cut-off point, below which no effect was observed, was also greater. Formal assessment by image analysis confirmed these impressions while revealing that the relative resistance of epithelial cells was confined to the initial phases of co-incubation. For both mammalian cell types the first observable sign of cell damage was cell shrinkage with the formation of retraction fibres and gaps. CONCLUSIONS: The relative resistance of epithelial cells to Acanthamoeba may be due to difficulty in initiating attack of a monolayer with tight junctions between cells. These results provide circumstantial evidence of the importance of the corneal epithelium in the prevention of amoebic invasion. SUMMARY: The relative susceptibility of cultured corneal epithelial cells and keratocytes were compared as regards both the concentration of amoebae required to produce obvious cell damage and the area of cells destroyed for a given concentration at various time intervals. The epithelial cells were more resistant than keratocytes with respect to both factors, at least early in the co-incubation period. This may be related to the different growth patterns of the cell types as epithelial cells form a monolayer connected by tight junctions and indirectly supports the idea that the epithelium forms an important barrier function against invasion in vivo.     

Prostaglandin F2α can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro

The effects of PGF_2α on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF_2α at concentrations equal to or lower than 10⁻⁶ M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10⁻⁵ M PGF_2α induced a decrease in cell growth under all culturing conditions. PGF_2α did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF_2α induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF_2α and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE₂.     

Intrinsic cytotoxic effects of fluoroquinolones on human corneal keratocytes and endothelial cells

ABSTRACT

Objective: To determine the intrinsic cytotoxicity of five fluoroquinolones (ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, ofloxacin) on human corneal keratocytes (HCK) and human corneal endothelial cells (HCE).

Research design and methods: Cultures of replicating HCK and HCE were exposed to ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, or ofloxacin concentrations of 1?mg/mL, 100?μg/mL, 10?μg/mL, 1?μg/mL, 100?ng/mL, or 10?ng/mL for 15, 30, 60, or 240?min. Each of the 24 fluoroquinolone concentration–time exposures was tested against its own serum-free minimal essential medium (MEM) control. Cell number was quantified with a fluorescence bioassay.

Main outcome measure: Cytotoxicity was defined as a significant (?p < 0.05) difference in cell number measured as mean calcein fluorescence product versus control for each fluoroquinolone concentration–time exposure.

Results: Fluoroquinolone-induced cytotoxicity was concentration- and time-dependent in HCK and HCE cultures. The number of cytotoxic concentration–time exposures was highest with ciprofloxacin (23 of 24 exposures in HCK and 24 of 24 exposures in HCE) and lowest with levofloxacin (10 of 24 exposures in both HCK and HCE).

Conclusions: In vitro cell cultures are useful for evaluating cell response to potentially toxic insults, although cell cultures may lack tissue components that may prevent or ameliorate damage in vivo. In this assay, fluoroquinolones displayed the potential to be cytotoxic to human corneal keratocytes and endothelial cells, depending on drug concentration and duration of exposure. The potential for cytotoxicity may differ among fluoroquinolones.     

酮咯酸氨丁三醇对兔角膜细胞及纤连蛋白的影响

目的探讨酮咯酸氨丁三醇（Ketorolac tromethamine 0.5%, Acular,安贺拉）对培养兔角膜细胞及细胞外基质纤连蛋白（Fibronectin,FN）的影响。方法选用原代及传代兔角膜细胞,实验组加入含不同浓度酮咯酸氨丁三醇的培养液,对照组加入等量空白培养液,采用四甲基偶氮唑蓝（MTT）比色法检测其对细胞增殖的抑制,倒置相差显微镜观察其对细胞的形态学改变,并进一步采用免疫细胞化学和ELISA研究其对角膜细胞分泌FN的影响。结果酮咯酸氨丁三醇浓度为50-175μg/ml时,作用24、48、72h均抑制角膜细胞的增殖（P均〈0.05）,并呈剂量依赖性。免疫细胞化学显示对照组可见胞浆着色及胞间丝状着色,而加药组仅见胞浆少量着色。ELISA结果显示低浓度时FN含量增加,而高浓度时其含量明显减少。结论酮咯酸氨丁三醇对角膜细胞的增殖有抑制效应,呈剂量依赖性,并且高浓度时可明显减少FN的含量。酮咯酸氨丁三醇可能通过抑制角膜细胞的增殖和减少细胞外基质,从而防止屈光术后的屈光回退。     

Morphological characteristics and proliferation of keratocytes cultured under simulated microgravity

This study probed the changes of keratocytes cultured under simulated microgravity. Keratocytes were isolated from rabbit corneas using collagenase digestion method. Cells were seeded in a 55-mL capacity high-aspect-ratio vessel (HARV) of rotary cell culture system (RCCS) at a density of 1 x 10(4) cells/mL. Dehydrated bovine acellular corneal stroma (5 x 5 x 1 mm, n = 30) was used as a carrier for keratocyte culture. Rotational speed was set at 15, 20, and 30 rpm in the first, second, and third week of culture, respectively. Histological evaluation showed that keratocytes in simulated microgravity culture grew into carriers, but those under conventional gravity grew on the surface of carriers. Scanning electron microscopic evaluation showed that after 19 days in culture, keratocytes on the carriers were spherical and spread in the spaces among the collagen fibers. Cells were dendritic or spindle shaped, and they developed many foot processes linked with surrounding cells. The absorbance values of the simulated microgravity group were significantly higher (P < 0.01) than that of the conventional group from 10 to 19 days of culture. The RCCS obviously enhanced the proliferation of rabbit keratocytes and facilitated the cells' growth into or on the dehydrated bovine acellular corneal stroma. Cells showed more natural morphology.     

Modulation of cell-phenotype during in vitro aging. Glycosaminoglycan biosynthesis by skin fibroblasts and corneal keratocytes

The aim of this study was to compare keratocyte and fibroblast phenotypes during in vitro aging by comparing their biosynthesis of glycosaminoglycans using explant and cell cultures. Human skin and corneal explant cultures were realised with Dulbecco Modified Eagle's medium containing ³H glucosamine. Sequential cell cultures were studied at different passages for GAGs biosynthesis by ³H glucosamine incorporation followed by selective degradation with specific hydrolases. Radioactivity was determined and each GAG fraction evaluated. KS and DS are the major components synthesised by corneal explant culture. During in vitro aging, keratocytes synthesised 41% less KS between passages 4–9 with a decrease by 26% of the proportion of DS observed in the same conditions. In skin explant cultures, as expected the major components are CS and hyaluronan (HA). In the first cell passage studied compared with skin organ cultures we could notice a strong decrease of the proportions of DS and KS compensated by an increase of the proportion of HA. During the successive passages of fibroblasts, the proportions of DS and HS decreased (−30 and −62%, respectively) and those of KS increased (+90%).These results indicate that there remain measurable differences between keratocyte and fibroblast phenotypes as far as GAG-synthesis is concerned all though the successive passages, starting from explant cultures and up to the limits of in vitro cell passages.     

The three-dimensional microanatomy of the rabbit and human cornea. A chemical and mechanical microdissection-SEM approach

The three-dimensional (3D) microanatomy of the cornea is the major determinant of its optical and mechanical properties. Scanning electron microscopy (SEM) is the most commonly used method to obtain information on the overall 3D microanatomy of organs. However, SEM has not been successful in revealing the 3D microanatomy of the cornea, because the interior of the cornea is too compact to be explored by the electron beam. In this study, the 3D organisation of the cells and extracellular materials of human and rabbit corneas was examined after exposure by HCl and NaOH digestion, and by microdissection by the adhesive tape method. In the cornea of both species, all epithelial cells exhibited microplicae regardless of their location. This raises doubts about the tear film-holding role assigned to the microplicae of the superficial cells. Human and rabbit corneas differed in the collagen fibre patterns of the epithelial basement membranes. The 3D organisation of the stromal lamellae was similar in both species. In humans and rabbits, the keratocytes showed similar 3D features. However, the surface of human keratocytes located near Descemet's membrane exhibited small fenestrations that were not present in the rabbit keratocytes. The pattern of keratocyte innervation by the stromal neural plexus and 3D keratocyte microanatomy confirms that keratocytes form a large intercommunicating network within the corneal stroma. Two morphologically discrete subpopulations of keratocytes located at different stromal levels were identified in both human and rabbit corneas, suggesting that keratocytes are not functionally homogeneous. In addition, the density of the stromal neural plexus appeared to be greater in rabbits than in humans. Clear differences between human and rabbit corneas were observed in the collagen arrangement in Descemet's membrane, which may reflect their different biomechanical requirements.     

角膜基质细胞的表型转化及其分子机制

角膜瘢痕是继发于多种角膜疾病的病理性改变,是许多角膜疾病造成视力不同程度损害甚至丧失的直接原因,也是影响角膜屈光手术效果的重要因素.对于角膜损伤愈合后的角膜瘢痕,目前临床上主要治疗方法是穿透性或者板层角膜移植术,但因为角膜供体材料缺乏和经济原因,大量的患者无法得到手术治疗而最终丧失视力.     

Corneal stromal repair and regeneration

The cornea is a specialized, transparent, avascular, immune-privileged, and heavily innervated tissue that affords 2/3rd of refraction to the eye. Ocular injuries, infections, and genetic factors affect corneal function and cause vision impairment. Presently, a variety of laser/non-laser surgeries, immunosuppressants, and/or corneal transplants are predominantly used to revive sight in human patients. The development of novel, precision-guided, and tissue-targeted non-surgical therapies promoting corneal repair and regeneration based on mechanistic understanding is of paramount importance to reduce the impact of global blindness. Research over the past decade revealed that modulation of pathological signaling pathways and factors by a variety of therapeutic delivery methods effectively treats corneal disorders including corneal scar/haze, inflammation, and angiogenesis in various pre-clinical animal models and are primed for human translation. This review discusses recent advances in the areas of corneal repair, restoration, and regeneration. Herein, we provide an overview of evolving approaches and therapeutic modalities that have shown great promise in reviving corneal transparency and function through the use of small drug molecules, gene therapy, nanomedicine, stem cells, trophic factors, exosomes, stromal equivalents, bioengineered stromal scaffolds, tissue adhesives, and 3D bioprinting.