| ObjectiveTo establish an idealized mouse model of fungal keratitis that mimics clinical human corneal fungal infection and permits the evaluation of the clinical manifestation and histopathology of corneal damages. On the basis, meanwhile, applying technique of molecular biology to investigate the expression, regulation of predominant inflammatory cytokines that play a crucial role in the inflammation process of fungal keratitis, which is beneficial to promote the investigation of the pathogenesis and development of more effective therapeutic means of fungal keratitis.Methods1. Establishment of animal modelFusarium solani, Aspergillus fumigatus, and Candida albicans as the common corneal pathogenic species were respectively selected to induce fungal keratitis in inbred BALB/c mice aged 6～8 weeks old using "epikeratophakia with the aid of corneal epithelium erasion". The concentration of fungal suspension was 10~6 colony-forming units (CFU) per ml. Mouse palpebral fissure was sutured after the infection surgery and opened 24 hours later. All mice were observed daily for corneal involvement under slit-lamp biomicroscope and taken photographs using digital camera. At 1, 3, 5, 7 and 12d, infectious corneas were given semiquantitative clinical scoring and corneal scrapings were obtained from experimental eyes to perform etiologic examination.2. Histopathology examinationAt 1, 3, 5, 7, and 12d after infection, several mouse eyes in experimental groups were enucleated, immersed in 10% formalin for 24 hours, made into routine paraffinsections, then applied hematoxylin-eosin(HE) and periodic acid-Schiff (PAS) staining respectively. The stained sections were analyzed under light microscope, including inflammatory reaction, fungal burden and hyphal growth pattern. The degree of inflammation and fungal burden was categorized to mild, moderate, and severe grades and was given accordingly scoring. According to the angulation degrees of hyphal direction to the plane of stromal lamellae, we defined the hyphal growth patterns of fungal pathogens as vertical when hyphae grew at an angle >45 degrees, parallel when <45 degrees.3. Expression of predominant inflammatory cytokinesAt 1, 3, 5, 7, and 12 d after infection, four inflammatory cytokines in infectious corneas, including macrophage inflammatory protein (MIP)-2, cytokine-induced neutrophil chemoattractant (KC), interleukin (IL)-1β, IL-6, were detected respectively for expression of mRNA and protein levels, using RT-PCR and ELISA. Results of RT-PCR were made for semiquantitative analysis by Image J software for image analysis.4. Intervention experiment of polyclonal antibodies of inflammatory cytokines Inbred 6～8weeks old BALB/c mice were selected to 3 groups with 3 subgroups ineach and were inoculated 3 common corneal pathogenic species respectively. Group 1 was infection-control group, with subconjunctival injection of 20μl sterilized saline. Mice in group 2 and 3 were injected with 20μl(2ng/μl) MIP-2 polyclonal antibodies and 20 μl (2ng/μl) IL-1β polyclonal antibodies subconjunctivally respectively 1 h before and 24 h after surgery. Models of fungal keratitis were established in mice of 3 groups using "epikeratophakia with the aid of corneal epithelium erasion" with the concentration of 10~6CFU/ml in fungal suspension. 24 h after infection, eyelids were opened All mice were observed daily for corneal involvement under slit-lamp biomicroscope and given clinical scoring. At 1, 3, 5, 7 and 12 d, whole eyes were enucleated from several mice in each group and made into routine paraffin sections, observing tissue inflammatory reaction, fungal burden, and growth patterns of fungal hyphae by HE and PAS staining. Corneas of experimental eyes in group 2 and group 3were incised at different time points, MIP-2 and K.C protein levels in which were measured by ELISA assay. Results1. Features of infectious miceAll mice were successful to challenge the fungi inoculated by etiologic assay. The infectious process of 3 fungi had certain variances, except that the peak time of disease was at 3～7d after infection with involvement on 5 d most severe and typical and relieving gradually after 7 d. The involvement duration of Aspergillus fumigatus and Candida albicans was about 10 days, 12 days of Fusarium solani. The involvement of Aspergillus fumigatus was most severe with conspicuous central ulcer in most corneas in 3～5 d after infection, followed by Candida albican with tofukasu anabrosis in involvement region in the peak time. Fusarium solani had steady process, typical musci appearance and longer duration. At 1, 3, 5, and 7 d after infection, the difference of clinical scorings was statistical significance among each other (P=0.000).2. Histopathology changesThe inflammatory reaction in HE staining displayed infiltrated inflammatory cells at earlier period. With the process going on, inflammatory cells increased gradually and corneal tissues demolished more severely, which was most obvious in 5 d after infection. Then inflammatory cells diminished obviously and corneal tissues repaired after 7 d. Aspergillus fumigatus possessed most severe inflammatory reaction followed by Candida albicans and Fusarium solani was mildest. Fungal hyphae were exhibited in PAS staining in all 3 groups on 1 d and 3 d and only 2 cases in Candida albicans group were exhibited on 5 d after infection. The features tended to be that hyphae is multitude in earlier period and diminish gradually. As to fungus burden, fungal hyphae of Candida albicans possessed most in quantity and density. The statistical analysis of inflammation and fungus burden indicated inverse correlation (r= -0.806, P=0.029) between inflammation and fungus burden but positive correlation (r=0.961, P=0.000) between corneal inflammation and its manifestation induced by different strains. On the growth patterns, the hyphae of Fusarium solani lay parralel to the corneal lamellae; whereas thehyphea of Aspergillus fumigatus and Candida albicans were observed growing perpendicularly to the stromal lamellae.3. Expression of predominant inflammatory cytokinesMIP-2, KC, IL-1, and IL-6 were expressed at mRNA and protein levels in corneas of all mice including infectious group, injury control group and normal group with various expression amount and peak time point between the groups and different time points in the same group. The expression regularity of MIP-2, KC, and IL-1β was in coincidence and the expression amount sequence was: Aspergillus fumigatus groups > Candida albicans groups > Fusarium solani groups > injury control groups > normal control groups. IL-6 sequence was: Fusarium solani groups > Candida albicans groups > Aspergillus fumigatus groups > injury control groups > normal control groups. The expression peak time points of MIP-2, KC, and IL-1β were 5 d in Fusarium solani groups, 1 d in Aspergillus fumigatus groups and 3 d in Candida albicans groups after infection. The time points of high-levels expression of IL-6 were 1 d and 7 d in Fusarium solani groups and Aspergillus fumigatus groups but 1 d and 5 d in Candida albicans groups.4. Neutralization of MIP-2 and IL-1β activitiesAfter intervened by MIP-2 polyclonal antibodies, clinical scorings of all infectious groups degraded in certain extent with obvious extent of Aspergillus fumigatus groups (P<0.05), obvious extent (P<0.05) at earlier period (1～3 d) and mild (P>0.05) at midanaphase (5～12 d) in Candida albicans groups, and mild extent at earlier periods but not obvious at midanaphase in Fusarium solani groups. As to IL-1β polyclonal antibodies, the degrading extent was obvious at earlier periods (P<0.05) but not at midanaphase in all 3 groups. After neutralization antibodies were applied, the inflammatory reaction extent and the manifestation at corresponding time points were in coincidence of all groups with no difference of fundus burden and hyphae growth patterns. Protein levels of MIP-2 and KC in corneas intervened by IL-1β polyclonal antibodies descended obviously at earlier periods (P<0.05) but had no difference at midanaphase (P>0.05).Conclusions1. Stable and reliable fungal keratitis that mimics clinical keratopathy can be established by "epikeratophakia with the aid of corneal epithelium erasion", which is a substantial foundation for further reseach such as pathogenesis of fungal keratitis.2. The process of fungal keratitis is devided to 3 phases: early phases, intermediate phases, and later phases with infiltration and extending, suppuration and necrosis, scarring and recover as the key features of manifestation respectively. The severity of involvements by each strain is diverse and inflammatory reaction is predominant factors of corneal impairment, which indicates a new direction for basic and clinical reseach of fungal keratitis.3. The different corneal pathogenic species of fungi display various growth patterns in cornea, which have referred roles in the choice of modus and opportunity of operation for clinical patients with fungal keratitis.4. The chemokines and cytokines such as MIP-2, KC, IL-1β, and IL-6 are expressed in mice corneal tissues of fungal keratitis.. MIP-2 is crucial chemokine to fungal keratitis in mice and IL-1β is the predominant cytokine which regulate the expression of MIP-2 and KC. The persistent high-level expression of MIP-2 and KC is the important factors even being predominant factors in corneal impairment, but the expression of IL-6 is the defensive and protective factors in fungal keratitis of mouse.5. Specific polyclonal neutralizing antibodies can be adopted to inhibit predominant chemokines and cytokines and effectivly relieve the corneal damage by fungal keratitis. This result is encouraging, which is a beneficial explore and significant experiment basis for new drugs aimed with predominant inflammatory cytokines as targets for fungal keratitis.
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