真菌感染小鼠角膜过程中菌丝和主要炎症细胞动态变化的研究
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摘要
目的
由于真菌性角膜炎的具体发病机制尚不清楚,尽管不断更新的实验室诊断方法和大量真菌性角膜炎药敏实验的资料为临床提供了相关信息,但真菌性角膜炎的临床治疗仍然靠最常见的经验进行,以致较多的患者最终丧失视力,甚至眼球的摘除。真菌性角膜炎现已成为治疗效果差、致盲率高的眼科常见病。为此,本实验建立一种模拟临床上丝状真菌感染人角膜模式的C57BL/6纯系小鼠真菌性角膜炎模型,裂隙灯观察真菌感染角膜后角膜组织损伤的临床表现,常规石蜡切片观察角膜组织的病理形态学改变,角膜活体共焦显微镜动态观察真菌性角膜炎动物模型影像学上的组织病理学变化,激光共聚焦扫描显微镜对真菌感染小鼠角膜过程中菌丝和主要炎症细胞的动态变化进行研究,进而推动真菌性角膜炎发病机制研究的步伐,为临床上更加积极有效地防治真菌性角膜炎提供理论和实验基础。
方法
1、动物模型建立
选择国内最常见的致病菌种腐皮镰刀菌和黄曲霉菌,采用C57BL/6纯系小鼠右眼行真菌接种,左眼为手术对照组。常规消毒后眼科手术显微镜下行角膜划痕法接种真菌,深度为破坏角膜上皮到达浅基质层,对照眼同样方式划痕接种生理盐水。裂隙灯显微镜下观察角膜感染情况并拍照记录,分别于接种后第1,3,5,7,14d各时相对角膜组织病变进行临床评分,同时行模型眼和对照眼角膜刮片后涂片染色和真菌培养,并对各种病原学检测结果进行对比分析。
2、组织病理学检测
分别于接种后第1,3,5,7,14d各时相摘取小鼠眼球,置于福尔马林固定24h,常规石蜡切片,行PAS染色,光学显微镜下对不同时间点角膜组织的破坏和菌丝的形态特点进行分析。
3、活体角膜共焦显微镜研究
真菌感染小鼠模型眼和对照眼角膜早期(造模后6h、8h、12h、1d)、中期(2d、3d、5d)、后期(7d、14d),分别行角膜中央感染区及病灶周边部的角膜共焦显微镜检查,对病变角膜浸润病灶的中心区及周边区选择5个位点进行三维扫描。分别于接种后第1,3d时间点,每组随机取12只小鼠常规角膜刮片后染色镜检和真菌培养。
4、全角膜平铺片的激光共聚焦扫描显微镜研究
分别于接种后第12h,1,3,5,7d各时相眼球赤道穿透性切取带角膜缘的整个眼前节,于新鲜配置的PBS液中剔除虹膜,置1%多聚甲醛-PBS中固定30min,0.2%Triton X-100的2%BSA液透膜15min;分别使用CFW荧光染料、标记中性粒细胞荧光抗体、标记淋巴细胞抗体于4℃避光孵育过夜,放射状切开平铺于载玻片上,使用溶有DAPI染料的胶水固定过夜。在激光共聚焦扫描显微镜下观察真菌的生长方式以及对真菌进行定量分析,观察真菌感染角膜过程中的炎症细胞表达的变化。
5、真菌性角膜炎临床早期诊断的应用研究
临床疑似真菌性角膜炎患者165例行角膜刮片,获取的角膜刮取物用于角膜涂片镜检和真菌培养。同一病人一部分角膜刮取物均匀地涂在两张已处理过的载玻片中央,其中一张角膜涂片先行KOH湿片,在光镜下观察后行CFW荧光染色；同一病人的另外一张角膜涂片,先行Giemsa染液染色,光镜下观察后行CFW荧光染色。
6、统计学处理
实验数据采用SPSS13.0软件包进行统计学处理,分别采用Kraskal-willis H检验、单因素LSD t检验、Mann-Whitney U检验、卡方(chi-square test)检验、趋势卡方检验等统计学方法分析,P<0.05为差异有统计学意义。
结果
1、模型眼角膜刮片染色与真菌培养
两种真菌性角膜炎病变过程的早期(1d),中期(3d)CFW荧光染色的阳性率显著高于KOH湿片和Giemsa染色的阳性率,差异有统计学差异(镰刀组1dCFW vs KOH, P=0.047;ldCFW vs Giemsa, P=0.047; 3d CFW vs KOH, P=0.007; 3dCFW vs Giemsa, P=0.001;黄曲组1dCFW vs KOH, P=0.047; 1dCFW vs Giemsa, P=0.007; 3d CFW vs KOH, P=0.047; 3dCFW vs Giemsa, P=0.001),但与真菌培养的阳性率相比,无统计学差异(p>0.05)。在病变的后期,各种检查方法很少能探测到真菌的存在。
2、真菌感染角膜病变特征
两种真菌感染角膜后病变的发展过程具有一定差异,在2-5d是病变的高峰期,7d后病变逐步减轻。两种真菌感染角膜后病程约2w,腐皮镰刀菌组病变过程相对较平稳,黄曲霉菌组病变发展快且严重。黄曲模型眼在1,3,5,7,14d时间点病变的临床评分组内比较,差异有统计学意义(P<0.05),黄曲霉菌角膜炎第3天及第4天临床评分明显高于腐皮镰刀菌角膜炎,差异有统计学意义(p<0.05)。
3、角膜组织病理学检查
接种后第1 d可见划痕处角膜上皮层部分或完全缺损,缺损面细胞水肿严重,呈水泡样变,基质水肿,两种真菌菌丝主要分布于浅、中基质层；第3d可见上皮缺损面积缩小,角膜基质水肿严重,基质层胶原纤维排列紊乱,菌丝分布于角膜全层,镰刀菌丝体较粗长,曲霉菌丝体短小,部分病变角膜在未穿孔前已有菌丝或孢子穿透角膜内皮进入前房；第5天可见部分小鼠角膜上皮层局限性增生修复,部分仍可见溃疡灶,基质严重水肿,偶见菌丝生长,菌丝量明显少于第3天,并见菌丝体缩小,新生血管长入。接种后第7天,角膜全层未见菌丝,较多新生血管长入角膜基质。
4、角膜共焦显微镜诊断的敏感性
两种真菌性角膜炎动物模型在造模后的第1,3d时间点,以真菌培养为标准,共焦显微镜诊断的阳性率与CFW荧光染色的阳性率相比较,无明显的统计学差异(p>0.05);在第5d时间点,真菌菌丝的检出率较低；第7d后真菌菌丝检出率为0。
5、菌丝的影像学特征
激光共焦显微镜图像显示：在感染早期,腐皮镰刀菌表现为短线状或蚯蚓状结构,中期为长而支挺、分支极少的长线状结构,病变部位角膜形态结构破坏明显；黄曲霉菌在早期为短小、弯曲的蠕虫状结构,中期为走行更加弯曲,分支较多呈树枝状或簇状结构,病变部位角膜形态结构消失。后期均未真菌结构。
6、菌丝在模型眼角膜中的动态变化
镰刀菌首先在病变部位粘附,然后生长繁殖,真菌感染后12h,真菌侵入的层次局限为浅、中基质层,在1d时,真菌已分布角膜全层。在接种点周围,菌丝在浅、中、深基质生长方式有明显的层次性。真菌的菌丝量在3d达到峰值。5d后突然减少消失。黄曲霉菌在病变部位粘附的时间相对较晚,但其生长繁殖速度较快,真菌感染后12h,真菌侵入的层次已有菌丝在深基质层,在1d时,真菌已分布角膜全层,其生长方式丝纵横交错,交织成网。真菌的菌丝量在2d达到峰值。在3d时,真菌的菌丝已有所减少,5d后真菌菌丝较少或已消失。黄曲霉菌第1天及第2天菌丝的含量明显高于镰刀菌,但在第3天低于镰刀菌,差异均有统计学意义(1d,t=-8.657,p=0.000；2d,t=-16.952,p=0.000；3d,t=4.543,p=0.000),在12h与5d时间点,两组间差异均无统计学意义(p>0.05)。
7、模型眼角膜组织中性粒细胞表达的变化
正常小鼠角膜巩膜缘区域有少量中性粒细胞的散在分布。模型对照组鼠眼术后12h角膜上9个区域里中性粒细胞的数量达到峰值,随后中性粒细胞的数量逐渐减少。。两种真菌性角膜炎模型眼12h角膜上各个区域里中性粒细胞的数量都达到第一个峰值,3d时角膜里中性粒细胞的数量达到了第二个峰值,5d后中性粒细胞数量逐步减少(图3-22,3-23)。两组模型眼组内不同时间点中性粒细胞的数量相比有明显统计学差异(镰刀组F=311.858,p=0.000；黄曲组F=570.811,p=0.000)。在12h,1d,3d,7d时间点两组中性粒细胞的数量相比有明显的统计学差异(12h,t=-3.519,p=0.006；1d,t=-4.659,p=0.001；3d,t=-5.338,p=0.000；7d,t=4.335,p=0.001)。
8、模型眼角膜组织淋巴细胞表达的变化
正常小鼠角膜巩膜缘区域有少量淋巴细胞的散在分布。模型对照组鼠眼术后12h角膜上淋巴细胞的数量开始增多,24h淋巴细胞的数量到达峰值,随后逐渐减少。镰刀菌模型5d眼角膜里淋巴细胞的数量达到峰值,随后其数量逐步减少。在病程的中期和后期,黄曲霉菌模型眼角膜里淋巴细胞的数量与镰刀菌淋巴细胞数量相比,有明显的统计学差异(3d,t=-10.217,p=0.000；5d,t=-22.704,p=0.000；7d,t=-7.444,p=0.000)。
9、临床角膜刮片染色与真菌培养
以真菌培养阳性作为诊断真菌性角膜的金标准,在同一张角膜涂片KOH和CFW染色的敏感性分别为81.0%和96.6%,在同一张角膜涂片Giemsa和CFW染色的敏感性为39.7%和98.3%,此外,Giemsa检测到23例细菌感染,其中6例为混合感染。CFW染色的敏感性显著高于KOH和Giemsa染色。
结论
1、我们在宿主正常免疫状态下,采用角膜划痕法成功建立了模拟临床真菌性角膜炎自然感染过程的动物模型。为推动真菌性角膜炎的基础和临床应用研究奠定了基础。
2、真菌感染角膜的病变过程历经发生、发展、转归三个阶段,即早、中、晚三期。不同菌种感染角膜的病变严重程度各有差异,中期是决定真菌性角膜炎预后的关键时间窗。
3、CFW荧光染色简单、快捷、敏感性高、特异性强。活体角膜共焦显微镜能够实时动态监控真菌的变化,且敏感性高、特异性强。两者均是实验室判定真菌性角膜炎动物模型的好方法。
4、不同菌种感染所致真菌性角膜炎激光共焦显微镜下菌丝有不同的影像学特点,且同一菌种不同时期在角膜组织中菌丝形态也有较大差异。为角膜共焦显微镜用于真菌性角膜炎的基础研究奠定了坚实基础,为临床上真菌性角膜炎的共焦显微镜鉴别诊断提供影像学资料。
5、应用激光共聚焦扫描显微镜能够对全角膜真菌信息准确的定性定量观察。黄曲霉菌菌丝较细,多密集分布,生长繁殖较快,生长方式杂乱无章。镰刀菌菌丝较粗,生长繁殖相对较慢,其生长方式有明显的层次性。
6、应用激光共聚焦扫描显微镜能够对全角膜炎症细胞的信息进行准确定性定量观察。早期和中前期浸润、募集的中性粒细胞对真菌的裂解、吞噬起主要作用,中后期浸润、募集的中性粒细胞对角膜组织则主要起到破坏作用。淋巴细胞的浸润募集较中性粒细胞出现晚,其峰值出现在中后期,因此,淋巴细胞在后期角膜病变中也起到重要作用。
7、Giemsa与CFW联合染色是临床诊断疑似真菌性角膜炎的较理想的方法,该联合染色不但灵敏度高,而且还可以识别细菌感染或混合感染,并能提供细胞病理学信息。是一个值得推广的临床早期诊断方法。
Purpose
The 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.
Methods
1. Animal model building
Two 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 detection
On 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 research
The 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 microscope
On 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 keratitis
165 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 analysis
All 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.
Results
1. The model cornea scrapings staining and fungal culture
On 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 infections
Both 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 cornea
On 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 diagnosis
On 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 hyphae
Confocal 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 cornea
Fusarium 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 tissue
Cornea 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 culture
As 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.
Conclusion
1. 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.
由于真菌性角膜炎的具体发病机制尚不清楚,尽管不断更新的实验室诊断方法和大量真菌性角膜炎药敏实验的资料为临床提供了相关信息,但真菌性角膜炎的临床治疗仍然靠最常见的经验进行,以致较多的患者最终丧失视力,甚至眼球的摘除。真菌性角膜炎现已成为治疗效果差、致盲率高的眼科常见病。为此,本实验建立一种模拟临床上丝状真菌感染人角膜模式的C57BL/6纯系小鼠真菌性角膜炎模型,裂隙灯观察真菌感染角膜后角膜组织损伤的临床表现,常规石蜡切片观察角膜组织的病理形态学改变,角膜活体共焦显微镜动态观察真菌性角膜炎动物模型影像学上的组织病理学变化,激光共聚焦扫描显微镜对真菌感染小鼠角膜过程中菌丝和主要炎症细胞的动态变化进行研究,进而推动真菌性角膜炎发病机制研究的步伐,为临床上更加积极有效地防治真菌性角膜炎提供理论和实验基础。
方法
1、动物模型建立
选择国内最常见的致病菌种腐皮镰刀菌和黄曲霉菌,采用C57BL/6纯系小鼠右眼行真菌接种,左眼为手术对照组。常规消毒后眼科手术显微镜下行角膜划痕法接种真菌,深度为破坏角膜上皮到达浅基质层,对照眼同样方式划痕接种生理盐水。裂隙灯显微镜下观察角膜感染情况并拍照记录,分别于接种后第1,3,5,7,14d各时相对角膜组织病变进行临床评分,同时行模型眼和对照眼角膜刮片后涂片染色和真菌培养,并对各种病原学检测结果进行对比分析。
2、组织病理学检测
分别于接种后第1,3,5,7,14d各时相摘取小鼠眼球,置于福尔马林固定24h,常规石蜡切片,行PAS染色,光学显微镜下对不同时间点角膜组织的破坏和菌丝的形态特点进行分析。
3、活体角膜共焦显微镜研究
真菌感染小鼠模型眼和对照眼角膜早期(造模后6h、8h、12h、1d)、中期(2d、3d、5d)、后期(7d、14d),分别行角膜中央感染区及病灶周边部的角膜共焦显微镜检查,对病变角膜浸润病灶的中心区及周边区选择5个位点进行三维扫描。分别于接种后第1,3d时间点,每组随机取12只小鼠常规角膜刮片后染色镜检和真菌培养。
4、全角膜平铺片的激光共聚焦扫描显微镜研究
分别于接种后第12h,1,3,5,7d各时相眼球赤道穿透性切取带角膜缘的整个眼前节,于新鲜配置的PBS液中剔除虹膜,置1%多聚甲醛-PBS中固定30min,0.2%Triton X-100的2%BSA液透膜15min;分别使用CFW荧光染料、标记中性粒细胞荧光抗体、标记淋巴细胞抗体于4℃避光孵育过夜,放射状切开平铺于载玻片上,使用溶有DAPI染料的胶水固定过夜。在激光共聚焦扫描显微镜下观察真菌的生长方式以及对真菌进行定量分析,观察真菌感染角膜过程中的炎症细胞表达的变化。
5、真菌性角膜炎临床早期诊断的应用研究
临床疑似真菌性角膜炎患者165例行角膜刮片,获取的角膜刮取物用于角膜涂片镜检和真菌培养。同一病人一部分角膜刮取物均匀地涂在两张已处理过的载玻片中央,其中一张角膜涂片先行KOH湿片,在光镜下观察后行CFW荧光染色；同一病人的另外一张角膜涂片,先行Giemsa染液染色,光镜下观察后行CFW荧光染色。
6、统计学处理
实验数据采用SPSS13.0软件包进行统计学处理,分别采用Kraskal-willis H检验、单因素LSD t检验、Mann-Whitney U检验、卡方(chi-square test)检验、趋势卡方检验等统计学方法分析,P<0.05为差异有统计学意义。
结果
1、模型眼角膜刮片染色与真菌培养
两种真菌性角膜炎病变过程的早期(1d),中期(3d)CFW荧光染色的阳性率显著高于KOH湿片和Giemsa染色的阳性率,差异有统计学差异(镰刀组1dCFW vs KOH, P=0.047;ldCFW vs Giemsa, P=0.047; 3d CFW vs KOH, P=0.007; 3dCFW vs Giemsa, P=0.001;黄曲组1dCFW vs KOH, P=0.047; 1dCFW vs Giemsa, P=0.007; 3d CFW vs KOH, P=0.047; 3dCFW vs Giemsa, P=0.001),但与真菌培养的阳性率相比,无统计学差异(p>0.05)。在病变的后期,各种检查方法很少能探测到真菌的存在。
2、真菌感染角膜病变特征
两种真菌感染角膜后病变的发展过程具有一定差异,在2-5d是病变的高峰期,7d后病变逐步减轻。两种真菌感染角膜后病程约2w,腐皮镰刀菌组病变过程相对较平稳,黄曲霉菌组病变发展快且严重。黄曲模型眼在1,3,5,7,14d时间点病变的临床评分组内比较,差异有统计学意义(P<0.05),黄曲霉菌角膜炎第3天及第4天临床评分明显高于腐皮镰刀菌角膜炎,差异有统计学意义(p<0.05)。
3、角膜组织病理学检查
接种后第1 d可见划痕处角膜上皮层部分或完全缺损,缺损面细胞水肿严重,呈水泡样变,基质水肿,两种真菌菌丝主要分布于浅、中基质层；第3d可见上皮缺损面积缩小,角膜基质水肿严重,基质层胶原纤维排列紊乱,菌丝分布于角膜全层,镰刀菌丝体较粗长,曲霉菌丝体短小,部分病变角膜在未穿孔前已有菌丝或孢子穿透角膜内皮进入前房；第5天可见部分小鼠角膜上皮层局限性增生修复,部分仍可见溃疡灶,基质严重水肿,偶见菌丝生长,菌丝量明显少于第3天,并见菌丝体缩小,新生血管长入。接种后第7天,角膜全层未见菌丝,较多新生血管长入角膜基质。
4、角膜共焦显微镜诊断的敏感性
两种真菌性角膜炎动物模型在造模后的第1,3d时间点,以真菌培养为标准,共焦显微镜诊断的阳性率与CFW荧光染色的阳性率相比较,无明显的统计学差异(p>0.05);在第5d时间点,真菌菌丝的检出率较低；第7d后真菌菌丝检出率为0。
5、菌丝的影像学特征
激光共焦显微镜图像显示：在感染早期,腐皮镰刀菌表现为短线状或蚯蚓状结构,中期为长而支挺、分支极少的长线状结构,病变部位角膜形态结构破坏明显；黄曲霉菌在早期为短小、弯曲的蠕虫状结构,中期为走行更加弯曲,分支较多呈树枝状或簇状结构,病变部位角膜形态结构消失。后期均未真菌结构。
6、菌丝在模型眼角膜中的动态变化
镰刀菌首先在病变部位粘附,然后生长繁殖,真菌感染后12h,真菌侵入的层次局限为浅、中基质层,在1d时,真菌已分布角膜全层。在接种点周围,菌丝在浅、中、深基质生长方式有明显的层次性。真菌的菌丝量在3d达到峰值。5d后突然减少消失。黄曲霉菌在病变部位粘附的时间相对较晚,但其生长繁殖速度较快,真菌感染后12h,真菌侵入的层次已有菌丝在深基质层,在1d时,真菌已分布角膜全层,其生长方式丝纵横交错,交织成网。真菌的菌丝量在2d达到峰值。在3d时,真菌的菌丝已有所减少,5d后真菌菌丝较少或已消失。黄曲霉菌第1天及第2天菌丝的含量明显高于镰刀菌,但在第3天低于镰刀菌,差异均有统计学意义(1d,t=-8.657,p=0.000；2d,t=-16.952,p=0.000；3d,t=4.543,p=0.000),在12h与5d时间点,两组间差异均无统计学意义(p>0.05)。
7、模型眼角膜组织中性粒细胞表达的变化
正常小鼠角膜巩膜缘区域有少量中性粒细胞的散在分布。模型对照组鼠眼术后12h角膜上9个区域里中性粒细胞的数量达到峰值,随后中性粒细胞的数量逐渐减少。。两种真菌性角膜炎模型眼12h角膜上各个区域里中性粒细胞的数量都达到第一个峰值,3d时角膜里中性粒细胞的数量达到了第二个峰值,5d后中性粒细胞数量逐步减少(图3-22,3-23)。两组模型眼组内不同时间点中性粒细胞的数量相比有明显统计学差异(镰刀组F=311.858,p=0.000；黄曲组F=570.811,p=0.000)。在12h,1d,3d,7d时间点两组中性粒细胞的数量相比有明显的统计学差异(12h,t=-3.519,p=0.006；1d,t=-4.659,p=0.001；3d,t=-5.338,p=0.000；7d,t=4.335,p=0.001)。
8、模型眼角膜组织淋巴细胞表达的变化
正常小鼠角膜巩膜缘区域有少量淋巴细胞的散在分布。模型对照组鼠眼术后12h角膜上淋巴细胞的数量开始增多,24h淋巴细胞的数量到达峰值,随后逐渐减少。镰刀菌模型5d眼角膜里淋巴细胞的数量达到峰值,随后其数量逐步减少。在病程的中期和后期,黄曲霉菌模型眼角膜里淋巴细胞的数量与镰刀菌淋巴细胞数量相比,有明显的统计学差异(3d,t=-10.217,p=0.000；5d,t=-22.704,p=0.000；7d,t=-7.444,p=0.000)。
9、临床角膜刮片染色与真菌培养
以真菌培养阳性作为诊断真菌性角膜的金标准,在同一张角膜涂片KOH和CFW染色的敏感性分别为81.0%和96.6%,在同一张角膜涂片Giemsa和CFW染色的敏感性为39.7%和98.3%,此外,Giemsa检测到23例细菌感染,其中6例为混合感染。CFW染色的敏感性显著高于KOH和Giemsa染色。
结论
1、我们在宿主正常免疫状态下,采用角膜划痕法成功建立了模拟临床真菌性角膜炎自然感染过程的动物模型。为推动真菌性角膜炎的基础和临床应用研究奠定了基础。
2、真菌感染角膜的病变过程历经发生、发展、转归三个阶段,即早、中、晚三期。不同菌种感染角膜的病变严重程度各有差异,中期是决定真菌性角膜炎预后的关键时间窗。
3、CFW荧光染色简单、快捷、敏感性高、特异性强。活体角膜共焦显微镜能够实时动态监控真菌的变化,且敏感性高、特异性强。两者均是实验室判定真菌性角膜炎动物模型的好方法。
4、不同菌种感染所致真菌性角膜炎激光共焦显微镜下菌丝有不同的影像学特点,且同一菌种不同时期在角膜组织中菌丝形态也有较大差异。为角膜共焦显微镜用于真菌性角膜炎的基础研究奠定了坚实基础,为临床上真菌性角膜炎的共焦显微镜鉴别诊断提供影像学资料。
5、应用激光共聚焦扫描显微镜能够对全角膜真菌信息准确的定性定量观察。黄曲霉菌菌丝较细,多密集分布,生长繁殖较快,生长方式杂乱无章。镰刀菌菌丝较粗,生长繁殖相对较慢,其生长方式有明显的层次性。
6、应用激光共聚焦扫描显微镜能够对全角膜炎症细胞的信息进行准确定性定量观察。早期和中前期浸润、募集的中性粒细胞对真菌的裂解、吞噬起主要作用,中后期浸润、募集的中性粒细胞对角膜组织则主要起到破坏作用。淋巴细胞的浸润募集较中性粒细胞出现晚,其峰值出现在中后期,因此,淋巴细胞在后期角膜病变中也起到重要作用。
7、Giemsa与CFW联合染色是临床诊断疑似真菌性角膜炎的较理想的方法,该联合染色不但灵敏度高,而且还可以识别细菌感染或混合感染,并能提供细胞病理学信息。是一个值得推广的临床早期诊断方法。
Purpose
The 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.
Methods
1. Animal model building
Two 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 detection
On 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 research
The 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 microscope
On 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 keratitis
165 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 analysis
All 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.
Results
1. The model cornea scrapings staining and fungal culture
On 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 infections
Both 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 cornea
On 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 diagnosis
On 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 hyphae
Confocal 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 cornea
Fusarium 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 tissue
Cornea 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 culture
As 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.
Conclusion
1. 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.
引文
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