Study Of The Dynamic Changes Of The Hypha And Main Inflammatory Cells During The Course Of Fungal Corneal Infections In Mice

PurposeThe specific pathogenesis of fungal keratitis is not clear. Despite the continuously updated laboratory diagnostic methods and a large number of fungal keratitis susceptibility data for clinical trials have provided relevant information, but the clinical treatment of fungal keratitis still relies on the most common experience. The ultimate outcome of fungal keratitis in most patients is the loss of vision, and even the removal of the eye. Fungal keratitis has become a kind of poor therapeutic efficacy, high blinding rate common disease for eyes. For this reason, this experiment is to establish a C57BL/6 mice animal model which simulation of clinical human corneal filamentous fungal infection. The clinical manifestations of comeal tissue injury after fungal cornal infection were observed by Slit-lamp. The pathological morphological changes of corneal tissue were checked on routine paraffin sections. The imaging on the histopathologic changes of animal models were dynamic observed by Corneal in vivo confocal microscopy; The hyphae and the major inflammatory cells during the course of fungal corneal infections in mice were studied by Confocal laser scanning microscope. This promotes the research of the pathogenesis of fungal keratitis, and provides theoretical and experimental basis in more active and effective clinical prevention and treatment of fungal keratitis.Methods1. Animal model buildingTwo kinds of standard strain as the common pathogenic species were chosen for innoculation, FS (Fusarium Solaui) and AF(Aspergillus flavus). The right eye of C57BL/6 mouse was selected to inoculated fungi, the left eye for the surgical control group. Under the ophthalmic surgical microscope after conventional disinfection, the method of corneal scarification was used for fungal inoculation, the depth of destruction of the corneal epithelium to reach shallow substrate layer, and the surgical control eye inoculated with normal saline in the same way. The clinical manifestations of corneal tissue injury after fungal corneal infection were observed by Slit-lamp and recorded by digital cameras. Respectively on 1,3,5,7,14d after inoculation, the corneal tissue changes were conducted to clinical score. At the same time, corneal scrapings from the model and the control eyes were carried on dyeing and fungal culture, and a variety of pathogenic test results were comparative analysis. 2. Histopathology detectionOn 1,3,5,7,14d after inoculation, the mice eyes were obtained, placed in formalin, fixed for 24h, and made by routine paraffin sections and PAS staining. The corneal tissue destruction and hyphae morphological characteristics of the model eyes were observed under light microscope.3. In vivo corneal confocal microscopy researchThe central and peripheral corneal infected area were observed by corneal confocal microscopy in the early stage (6h,8h,12h, 1d), the medium-term (2d,3d, 5d), and the last stage(7d,14d) from the model and control corneas in mice. Five sites of invasive lesions of the central corneal lesion area and surrounding areas were selected for three-dimensional scanning. On 1,3d, twelve mice in each group were randomly selected for conventional corneal scrapings staining and fungal culture.4. Corneal wholemount research by laser scanning confocal microscopeOn 12h,1,3,5,7d, the entire anterior segment with limbus were cut from eyeball equator. The iris was removed in fresh PBS solution. Fixed in 1% paraformaldehyde-PBS for 30min, then put in 0.2% Triton X-100 with 2% BSA for 15min, CFW fluorescent dyes, marked neutrophil fluorescent antibodies, marked lymphocyte antibodies, incubated in 4℃circumstance for whole night. Radial cuts were made in the cornea on the slide, and fixed by the DAPI glue overnight. Fungal growth patterns, quantitative analysis of fungi, and inflammatory cells in the process of fungal corneal infection were observed by laser scanning confocal microscope.5. Applied research of clinical early diagnosis of fungal keratitis165 consecutive patients with clinically suspected fungal keratitis in central China from January to February 2009 were included in this study. Corneal scrapings from these patients were used for culture with two smears. Potassium Hydroxide was randomly added on one smear then by Calcofluor White. The other smear was firstly stained by Giemsa then by Calcofluor White.6. Statistical analysisAll data were analyzed by SPSS 13.0 statistical package. Kraskal-willis test, Single-factor LSD t test, Mann-Whitney U test, Chi-square test, and Trend chi-square test were used. Less than 0.05 was considered statistical significance.Results1. The model cornea scrapings staining and fungal cultureOn 1,3,5,7,14d after inoculation, the cornea scrapings in mouse model eye was carried on KOH wet mounts, Giemsa staining, CFW staining and fungal culture. CFW staining positive rate was significantly higher than that in KOH wet mounts and Giemsa staining in the early stage(ld) and medium-term(3d) within two kinds of the animal model of fungal keratitis. There was a statistically significant difference (FS 1d CFW vs KOH, P=0.047; 1d CFW vs Giemsa, P=0.047; 3d CFW vs KOH, P=0.007; 3d CFW vs Giemsa, P=0.001\AF 1d CFW vs KOH, P=0.047;1d CFW vs Giemsa, P=0.007; 3d CFW vs KOH, P=0.047; 3d CFW vs Giemsa, P=0.001)2. The pathological characteristics of fungal corneal infectionsBoth of two kinds of fungal keratitis showed the course is about 2w; 2-5d is the peak of disease; after 7d, the lesions reduce step by step. However, there was some difference in the process of two animal model of fungal keratitis. The disease process of Fusarium Solaui group is relatively smooth, but Aspergillus flavus group is fast and serious. On 1,3,5,7,14d, there is significant difference of clinical score among each other (P<0.05). On 3,4d, clinical score of Aspergillus flavus group was significantly higher than Fusarium Solaui, the difference was statistically significant (P<0.05),3. Histopathologic examination of corneaOn 1d after inoculation, scratched partial or complete defect can be seen in corneal epithelium. There is severe edema of cells showing vesicular degeneration and Stromal edema. The hyphae of two kinds of fungi are mainly distributed in shallow and middle layer of stroma. On 3d after inoculation, Stroma collagen fibers were irregularly arranged; serious stromal edema and hyphae were found in whole cornea. The hyphae of FS are thick and long; but the hyphe of AF are short. Before corneal perforation, few hyphae have penetrated corneal endothelium into the anterior chamber. On 5d after inoculation, some mouse corneal epithelium have got proliferative repair. Some are still visible ulcer lesions. Hyphae can occasionally be seen, but the number of hyphae was less than that on 3d, and the hyphae become smaller. There is neovascularization. On 7d after inoculation, no hyphae but more corneal neovascularization can be found.4. The sensitivity of corneal confocal microscopy diagnosisOn 1,3d after inoculation, taking fungal culture-positive as the gold standard for diagnosis of fungal keratitis, there is no significant difference in the positive rate between corneal confocal microscope and CFW staining (P>0.05). On 5d after inoculation, there is lower detection rate of hyphae; and on 7d, there is no detection rate of hyphae by corneal confocal microscope.5. The imaging features of hyphaeConfocal laser microscope image shows that in the early stage of infection, FS appeared as the short line or worm-like structure, and then became long and strong linear structure which had few branches; the shape and structure of the cornea were damaged obviously in pathological change spot. In the early stage of infection, AF appeared as short, small curved worm-like structure and then became more curved dendritic or clustered structure which had more branches, the shape and structure of the cornea disappeared in pathological change spot. Fungal structures were not found in the late period.6. The dynamic changes of mycelium in the corneaFusarium mycelium is thick, relatively slow growth and reproduction; its growth pattern has obvious hierarchy. Fusarium mycelium first lesion adhesion, On 12h, the level of fungal invasion limited to superficial and middle substantia propria layer. On 1d, and the mycelia have distributed into full thickness of cornea. Mycelia reach to the peak on 3d and disappear on 5d. The characteristics of Aspergillus flavus is smaller, denser, rapid growth and reproduction, and disorganized of growth modes. On 12h, the level of invasive fungal mycelium has been in the deep stroma. On 1 d, the fungus has been distributed full thickness corneal. Mycelia reach to the peak on 2d, on 3d, the fungal mycelium has been reduced, and disappeared on 5d. On 1d,2d, Content of mycelium of AF were significantly higher than the mycelium of FS, the differences were statistically significant (1d, t=-8.657, p=0.000; 2d, t=-16.952, p= 0.000; 3d, t=4.543,p=0.000)7. Neutrophil changes in the corneal tissueCornea edge region scattered a small amount of neutrophils in normal mouse. The number of neutrophils peaked at 12h in control group, and then begins to decrease. The number of neutrophils reached the first peak at 12h in FS group and AF group, the second peak on 3d, and then gradually decreased. There is significant statistical difference of the number of neutrophils between FS group and AF group at different time points (FSF=311.858,P=0.000; AFF=570.811,p=0.000). On 12h, 1d, 3d,7d, there were significant statistical difference of the number of neutrophils in both groups (12h, t=-3.519, p= 0.006; Id, t=-4.659,P=0.001; 3d, t=-5.338,P= 0.000; 7d,t=4.335,P=0.007)8. Lymphocytes changes in the corneal tissue Cornea edge region scattered small lymphocytes in normal mouse. At 12h, the number of lymphocytes began to increase in control group, reached peak at 24h, and then gradually decreased. The number of lymphocytes in both groups reached peak on 5d, then gradually decreased, but on 3d,5d,7d, the number of lymphocytes in Aspergillus flavus model compared with which in Fusarium Solaui group, has significant statistical difference (3d, t=-10.217,P=0.000; 5d, t=-22.704,p= 0.000; 7d, t=-7.444,P=0.000)9. Clinical corneal smea rs staining and fungal cultureAs compared to culture, the sensitivity of Potassium Hydroxide wet mounts and Calcofluor White stain were 81.0% and 96.6% in one smear; the sensitivity of Giemsa and Calcofluor White stain were 39.7% and 98.3% in the other smear in the diagnosis of fungal keratitis. Besides, Giemsa stain detected 23 cases of bacterial infection, in which 6 cases were mixed fungal and bacterial infections.Conclusion1. The adopted method of corneal scarification can successfully establish the animal model on fungal keratitis in mouse that is under a normal immune status, which is a stable foundation for basic and clinical applied research on fungal keratitis.2. There are early stage, middle stage and late stage during the pathological process of corneal fungal infection. The severity of the disease is varies from corneal infection with different strains. The key time window is on the middle stage which is to determine the fungal keratitis prognosis.3. Calcofluor White stain and corneal confocal microscopy are good methods in the diagnosis of animal model of fungal keratitis. Calcofluor White stain determines the fungal infections with simple, quick, high sensitivity and specificity. Corneal confocal microscopy can monitor the real-time dynamic changes in fungi with high sensitivity and specificity.4. The hypha appeared different configuration in different kinds of fungal keratitis, as well as in different course in vivo confocal microscopy. It put a solid foundation for basic research of fungal kertitis by corneal confocal microscope, and provided imaging information on differential diagnosis of fungal keratitis by corneal confocal microscope.5. Accurate information on the total corneal inflammatory cells and fungi of qualitative and quantitative observations can be through Confocal laser scanning microscopy. The infiltration and recruitment of neutrophils during the course of early and pre-middle periods play a major role in the fungi lysis and phagocytosis. But the infiltration and recruitment of neutrophils during the course of late periods are mainly on the corneal tissue destructive effect. Lymphocyte infiltration appears more lately than neutrophils and its peak in late periods. Therefore, the lymphocytes also play an important role in the latter part of the Keratopathy.6. Coupled Giemsa with Calcofluor White stain is a kind method in the diagnosis of patients with clinically suspected fungal keratitis, which can not only determine the fungal infections with high sensitivity, but also identify bacterial or mixed infection.