Expression Changes Of Healing-Related Molecules During Experimental Candida Albicans Keratitis And Gene Microarray Analysis Of Corneal Neovascularization

Purpose: To investigate the dynamic change of matrix metalloproteinases (MMPs),tissuse inhibitor of metalloproteinases (TIMPs), collagens and other related moleculesduring the course of expremental Candida albicans keratitis in mice.Method: Candida albicans keratitis (CaK) model was established in 6-8 weekC57BL/6 mice by cornea intrastromal injection of 5×10~4 spores. The corneas weremonitored using slit lamp in the following days. At 1, 3, 5, 7, 9, 11, 14 day postinfection (p.i), eyes were enucleated for histological examination. The expression ofMMPs, TIMPs, chemotactic factors (MCP-1 and MIP-2), collagens (Col1a1, Col3a1and Col4a1), MXRA-7and ECM-1 was evaluated using Real-time quantitative PCR(RT-qPCR) in normal and infected corneas. Some eyes were stored in OCT and usedfor following cells invation examination by immunofluorescence after 1 day p.i.Result: During the first 10 days after cornea intrastromal injection of fungal spores,mice developed severe Candida albicans keratitis. However, most of corneas couldheal spontaneously about two weeks after infection and only a haze remained at 60day p.i. PAS staining demonstrated that fungi spores and filaments exsited at 1 and 3day p.i. Through HE staining, large number of invaded inflammatory cells andactivated fibrolast were observed at days 3 and 11 p.i respectively. At mRNA level,MIP-2, MCP-1, MMP-13 and TIMP-1 were significant elevated at 1 day p.i. Theexpression of MMP-3, -8, -9, -10, -12 reached peaks at 3 or 5 day p.i and thengradually decreased in the following days while MMP-2, TIMP-2, Col1a1, Col3a1and Col4a1 gradually increased and arrived at peak at 11 day p.i. In contrast,MXRA-7 decreased after Candida infection, and recovered in the late of healing.Immunoflurescence examination further elucidated neutrophilic cells invasion at 1day p.i. Conclution: Different healing-related molecules may derive from different cells andplay different roles during the process of Candida albicans keratitis. The healingmechanism of these molecules needs futher reseach. Purpose: Suture placement and alkali burn to the cornea are often used to induceinflammatory corneal neovascularization (CorNV) models in animals. This studycompares the changes in genome-wide gene expression under these two CorNVconditions in mice.Methods: CorNV were induced in Balb/c mice by three interrupted 10-0 suturesplaced at sites about 1 mm from corneal apex, or by alkali burns 2 mm in size in thecentral area of the cornea. At the time points when neovascularization progressedmost quickly, corneas were examined using slit lamp and harvested, total RNA wereextracted for microarray assay, and some eyes were fixed in formaldehyde forhistology examination. After normalization and filtering, the data were subject tostatistical analysis using Significance Analysis Microarrays (SAM) Software, andinterested genes were annotated using the Database for Annotation, Visualization, andIntegrated Discovery (DAVID) program.Results: Suture placement induced CorNV in the areas between the suture and limbusbut did not affect the transparency still vascularized areas of the corneas, while alkaliburn caused edema and total loss of transparency of the whole cornea. Histologyshowed that sutures only caused localized epithelial loss and inflammatory infiltrationbetween the suture and limbus, but chemical burn depleted the whole epithelial layerof the central cornea and caused heavy cellular infiltration of the whole cornea. At day5 after suture placement, 1,055 differentially expressed probes were identified, out of which 586 probes were upregulated and 469 probes were downregulated. At acomparable time point, namely on day 6 after the alkali burns to the corneas, 472probes were upregulated and 389 probes were downregulated. Among thesedifferentially expressed probes, a significant portion (530 probes in total, including286 upregulated and 244 downregulated probes) showed a similar pattern of change inboth models. Annotation using DAVID of the overlapping differential genes revealedthat the significant enrichment Gene Ontology terms were"chemotaxis"and"immuneresponse"for the upregulated genes, and"oxidation reduction"and"programmed celldeath"for the downregulated genes. Some genes or gene families (e.g., S100A familyorα,βorγcrystallin families) that had not been related to corneal pathogenesis orneovascularization were also revealed to be involved in CorNV.Conclusions: Sutures and alkali burn to the corneas produced types of damage thataffected transparency differentially, but gene profiling revealed similar patterns ofchanges in gene expression in these two CorNV models. Further studies of the genesfirst found to be involved in CorNV will supplement current understanding about thepathogenesis of neovascularization diseases.