Expression Of Chemokines In Experimental Fungal Keratitis

ObjectivesFirstly, C57BL/6 mice model of fungal keratitis were built and evaluated, and then C3-deficient (C3-/-) mice were used to explore the role of the complement system in the pathogenesis of fungal keratitis. Finally, chemokines was studied for the expression and potential role in fungal keratitis.MethodsPart 1:The Role of complement system in experimental fungal keratitisA mouse model with fungal keratititis was built by "Epikeratophakia". Briefly, the corneal epithelium of C57BL/6 mice was removed, a full-thickness rat corneal graft was covered on the recipient bed, and a standardized inoculum (108CFU/ml) of Fusarium solani, Aspergillus fumigatus or Candida albicans was injected between graft and recipient bed. The corneal graft was removed until 24h after lip being sutured. At 1,3,5,7 days after inoculation, the appearance of corneal ulcer was observed and recorded with clinical scores. Experimental animals were divided into normal control group and C3-deficient group (C3-/- group). Mice models were constructed by infection of Fusarium solani using "Epikeratophakia". At 1,3,5, and 7 days after inoculation, the appearance of corneal ulcer was observed by slit lamp microscope, the severity of keratomycosis was scored, and then eyes were excised. Infected cornea was examined about histopathology.Part 2:Expression of Chemokines in Experimental Fungal KeratitisMice models were constructed by infection of Aspergillus fumigatus and Candida albicans using intrastromal injection. Physiological saline was regarded blank control group. At 6h、12h、24h and 48h after inoculation, clinical score, fungal burden, and histopathology were analyzed. RT-Q-PCR were detected for the level of mRNAof CCL1、CCL2、CCL5、CCL11、CCL27、CXCL10、CXCL12�'�CCR7.ResultsPart1 The clinical features manifested most evidently at 2 and 3 days after inoculation and varied with fungal species (F=3.065, P=0.103). The infected cornea was edematous with dense inflammatory cell infiltrates. Most of the inflammatory cells were polymorphonuclear leukocytes. Clinical score showed that the C3-/- group was characterized by mild inflammation at 1 days after inoculation by Fusarium solani, moderate inflammation at 3 days, focal ulceration, necrosis, and resulting in corneal perforation mostly at 5 and 7 days after inoculation..Part 2Clinical score of two fungus after inoculation increased. In candida albicans keratitis, fungal burden increased during primary 12 h, then decreasd at 24h,but increased at 48h. In Aspergillus fumigatus keratitis, fungal burden decreasd progressively. Level of CCL2、CCL11、CCL27、CXCL10 and CXCL12 increased in mRNA. Among these chemokines, CCL2 increased most obviously, followed by CXCL10. In candida albicans keratitis, CCL2 reached to crest value at 12h, and CXCL10 reached the one at 6h. In Aspergillus fumigatus keratitis, CCL2 reached to crest value at 24h, and CXCL10 reached the one at 12 h. CXCL12 increased, but lower than CXCL10.ConclusionA C57BL/6 mouse model of experimental fungal keratitis can be established by the method named "epikeratophakia". C3 deficiency had a profound effect on the clinical features of keratitis. The dysfunction of the complement system can impair the defense function of the early innate immune significantly, which results in the perforation of the cornea, but the corneal inflammatory response was lighter than normal cornea in the early phase of infection. CCL2 and CXCL10 may play a potential role in early phase of fungal keratitis. CXCL10 and CXCL12 could effect the form and development of corneal neogenesis pannus.